Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity

Recombinant expression of receptor binding... | F1000Research "use strict";function _typeof(t){return(_typeof="function"==typeof Symbol&&"symbol"==typeof Symbol.iterator?function(t){return typeof t}:function(t){return t&&"function"==typeof Symbol&&t.constructor===Symbol&&t!==Symbol.prototype?"symbol":typeof t})(t)}!function(){var t=function(){var t,e,o=[],n=window,r=n;for(;r;){try{if(r.frames.__tcfapiLocator){t=r;break}}catch(t){}if(r===n.top)break;r=r.parent}t||(!function t(){var e=n.document,o=!!n.frames.__tcfapiLocator;if(!o)if(e.body){var r=e.createElement("iframe");r.style.cssText="display:none",r.name="__tcfapiLocator",e.body.appendChild(r)}else setTimeout(t,5);return!o}(),n.__tcfapi=function(){for(var t=arguments.length,n=new Array(t),r=0;r 3&&2===parseInt(n[1],10)&&"boolean"==typeof n[3]&&(e=n[3],"function"==typeof n[2]&&n[2]("set",!0)):"ping"===n[0]?"function"==typeof n[2]&&n[2]({gdprApplies:e,cmpLoaded:!1,cmpStatus:"stub"}):o.push(n)},n.addEventListener("message",(function(t){var e="string"==typeof t.data,o={};if(e)try{o=JSON.parse(t.data)}catch(t){}else o=t.data;var n="object"===_typeof(o)&&null!==o?o.__tcfapiCall:null;n&&window.__tcfapi(n.command,n.version,(function(o,r){var a={__tcfapiReturn:{returnValue:o,success:r,callId:n.callId}};t&&t.source&&t.source.postMessage&&t.source.postMessage(e?JSON.stringify(a):a,"*")}),n.parameter)}),!1))};"undefined"!=typeof module?module.exports=t:t()}(); dataLayer = dataLayer || []; // Standard GTM initialization - Google Consent Mode handles consent automatically (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start': new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0], j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src= 'https://www.googletagmanager.com/gtm.js?id='+i+dl+ '>m_auth=hzk0Vc3qFsQYhCrIoHz68A>m_preview=env-1>m_cookies_win=x';f.parentNode.insertBefore(j,f); })(window,document,'script','dataLayer','GTM-MWFK8L5J'); ;window.NREUM||(NREUM={});NREUM.init={distributed_tracing:{enabled:true},privacy:{cookies_enabled:true},ajax:{deny_list:["bam.nr-data.net"]}}; ;NREUM.loader_config={accountID:"438030",trustKey:"438030",agentID:"772317073",licenseKey:"97f8f67f26",applicationID:"772317073"} ;NREUM.info={beacon:"bam.nr-data.net",errorBeacon:"bam.nr-data.net",licenseKey:"97f8f67f26",applicationID:"772317073",sa:1} ;/*! For license information please see nr-loader-spa-1.236.0.min.js.LICENSE.txt */ (()=>{"use strict";var e,t,r={5763:(e,t,r)=>{r.d(t,{P_:()=>l,Mt:()=>g,C5:()=>s,DL:()=>v,OP:()=>T,lF:()=>D,Yu:()=>y,Dg:()=>h,CX:()=>c,GE:()=>b,sU:()=>_});var n=r(8632),i=r(9567);const o={beacon:n.ce.beacon,errorBeacon:n.ce.errorBeacon,licenseKey:void 0,applicationID:void 0,sa:void 0,queueTime:void 0,applicationTime:void 0,ttGuid:void 0,user:void 0,account:void 0,product:void 0,extra:void 0,jsAttributes:{},userAttributes:void 0,atts:void 0,transactionName:void 0,tNamePlain:void 0},a={};function s(e){if(!e)throw new Error("All info objects require an agent identifier!");if(!a[e])throw new Error("Info for ".concat(e," was never set"));return a[e]}function c(e,t){if(!e)throw new Error("All info objects require an agent identifier!");a[e]=(0,i.D)(t,o),(0,n.Qy)(e,a[e],"info")}var u=r(7056);const d=()=>{const e={blockSelector:"[data-nr-block]",maskInputOptions:{password:!0}};return{allow_bfcache:!0,privacy:{cookies_enabled:!0},ajax:{deny_list:void 0,enabled:!0,harvestTimeSeconds:10},distributed_tracing:{enabled:void 0,exclude_newrelic_header:void 0,cors_use_newrelic_header:void 0,cors_use_tracecontext_headers:void 0,allowed_origins:void 0},session:{domain:void 0,expiresMs:u.oD,inactiveMs:u.Hb},ssl:void 0,obfuscate:void 0,jserrors:{enabled:!0,harvestTimeSeconds:10},metrics:{enabled:!0},page_action:{enabled:!0,harvestTimeSeconds:30},page_view_event:{enabled:!0},page_view_timing:{enabled:!0,harvestTimeSeconds:30,long_task:!1},session_trace:{enabled:!0,harvestTimeSeconds:10},harvest:{tooManyRequestsDelay:60},session_replay:{enabled:!1,harvestTimeSeconds:60,sampleRate:.1,errorSampleRate:.1,maskTextSelector:"*",maskAllInputs:!0,get blockClass(){return"nr-block"},get ignoreClass(){return"nr-ignore"},get maskTextClass(){return"nr-mask"},get blockSelector(){return e.blockSelector},set blockSelector(t){e.blockSelector+=",".concat(t)},get maskInputOptions(){return e.maskInputOptions},set maskInputOptions(t){e.maskInputOptions={...t,password:!0}}},spa:{enabled:!0,harvestTimeSeconds:10}}},f={};function l(e){if(!e)throw new Error("All configuration objects require an agent identifier!");if(!f[e])throw new Error("Configuration for ".concat(e," was never set"));return f[e]}function h(e,t){if(!e)throw new Error("All configuration objects require an agent identifier!");f[e]=(0,i.D)(t,d()),(0,n.Qy)(e,f[e],"config")}function g(e,t){if(!e)throw new Error("All configuration objects require an agent identifier!");var r=l(e);if(r){for(var n=t.split("."),i=0;i {r.d(t,{D:()=>i});var n=r(50);function i(e,t){try{if(!e||"object"!=typeof e)return(0,n.Z)("Setting a Configurable requires an object as input");if(!t||"object"!=typeof t)return(0,n.Z)("Setting a Configurable requires a model to set its initial properties");const r=Object.create(Object.getPrototypeOf(t),Object.getOwnPropertyDescriptors(t)),o=0===Object.keys(r).length?e:r;for(let a in o)if(void 0!==e[a])try{"object"==typeof e[a]&&"object"==typeof t[a]?r[a]=i(e[a],t[a]):r[a]=e[a]}catch(e){(0,n.Z)("An error occurred while setting a property of a Configurable",e)}return r}catch(e){(0,n.Z)("An error occured while setting a Configurable",e)}}},6818:(e,t,r)=>{r.d(t,{Re:()=>i,gF:()=>o,q4:()=>n});const n="1.236.0",i="PROD",o="CDN"},385:(e,t,r)=>{r.d(t,{FN:()=>a,IF:()=>u,Nk:()=>f,Tt:()=>s,_A:()=>o,il:()=>n,pL:()=>c,v6:()=>i,w1:()=>d});const n="undefined"!=typeof window&&!!window.document,i="undefined"!=typeof WorkerGlobalScope&&("undefined"!=typeof self&&self instanceof WorkerGlobalScope&&self.navigator instanceof WorkerNavigator||"undefined"!=typeof globalThis&&globalThis instanceof WorkerGlobalScope&&globalThis.navigator instanceof WorkerNavigator),o=n?window:"undefined"!=typeof WorkerGlobalScope&&("undefined"!=typeof self&&self instanceof WorkerGlobalScope&&self||"undefined"!=typeof globalThis&&globalThis instanceof WorkerGlobalScope&&globalThis),a=""+o?.location,s=/iPad|iPhone|iPod/.test(navigator.userAgent),c=s&&"undefined"==typeof SharedWorker,u=(()=>{const e=navigator.userAgent.match(/Firefox[/\s](\d+\.\d+)/);return Array.isArray(e)&&e.length>=2?+e[1]:0})(),d=Boolean(n&&window.document.documentMode),f=!!navigator.sendBeacon},1117:(e,t,r)=>{r.d(t,{w:()=>o});var n=r(50);const i={agentIdentifier:"",ee:void 0};class o{constructor(e){try{if("object"!=typeof e)return(0,n.Z)("shared context requires an object as input");this.sharedContext={},Object.assign(this.sharedContext,i),Object.entries(e).forEach((e=>{let[t,r]=e;Object.keys(i).includes(t)&&(this.sharedContext[t]=r)}))}catch(e){(0,n.Z)("An error occured while setting SharedContext",e)}}}},8e3:(e,t,r)=>{r.d(t,{L:()=>d,R:()=>c});var n=r(2177),i=r(1284),o=r(4322),a=r(3325);const s={};function c(e,t){const r={staged:!1,priority:a.p[t]||0};u(e),s[e].get(t)||s[e].set(t,r)}function u(e){e&&(s[e]||(s[e]=new Map))}function d(){let e=arguments.length>0&&void 0!==arguments[0]?arguments[0]:"",t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:"feature";if(u(e),!e||!s[e].get(t))return a(t);s[e].get(t).staged=!0;const r=[...s[e]];function a(t){const r=e?n.ee.get(e):n.ee,a=o.X.handlers;if(r.backlog&&a){var s=r.backlog[t],c=a[t];if(c){for(var u=0;s&&u {let[t,r]=e;return r.staged}))&&(r.sort(((e,t)=>e[1].priority-t[1].priority)),r.forEach((e=>{let[t]=e;a(t)})))}function f(e,t){var r=e[1];(0,i.D)(t[r],(function(t,r){var n=e[0];if(r[0]===n){var i=r[1],o=e[3],a=e[2];i.apply(o,a)}}))}},2177:(e,t,r)=>{r.d(t,{c:()=>f,ee:()=>u});var n=r(8632),i=r(2210),o=r(1284),a=r(5763),s="nr@context";let c=(0,n.fP)();var u;function d(){}function f(e){return(0,i.X)(e,s,l)}function l(){return new d}function h(){u.aborted=!0,u.backlog={}}c.ee?u=c.ee:(u=function e(t,r){var n={},c={},f={},g=!1;try{g=16===r.length&&(0,a.OP)(r).isolatedBacklog}catch(e){}var p={on:b,addEventListener:b,removeEventListener:y,emit:v,get:x,listeners:w,context:m,buffer:A,abort:h,aborted:!1,isBuffering:E,debugId:r,backlog:g?{}:t&&"object"==typeof t.backlog?t.backlog:{}};return p;function m(e){return e&&e instanceof d?e:e?(0,i.X)(e,s,l):l()}function v(e,r,n,i,o){if(!1!==o&&(o=!0),!u.aborted||i){t&&o&&t.emit(e,r,n);for(var a=m(n),s=w(e),d=s.length,f=0;fn,p:()=>i});var n=r(2177).ee.get("handle");function i(e,t,r,i,o){o?(o.buffer([e],i),o.emit(e,t,r)):(n.buffer([e],i),n.emit(e,t,r))}},4322:(e,t,r)=>{r.d(t,{X:()=>o});var n=r(5546);o.on=a;var i=o.handlers={};function o(e,t,r,o){a(o||n.E,i,e,t,r)}function a(e,t,r,i,o){o||(o="feature"),e||(e=n.E);var a=t[o]=t[o]||{};(a[r]=a[r]||[]).push([e,i])}},3239:(e,t,r)=>{r.d(t,{bP:()=>s,iz:()=>c,m$:()=>a});var n=r(385);let i=!1,o=!1;try{const e={get passive(){return i=!0,!1},get signal(){return o=!0,!1}};n._A.addEventListener("test",null,e),n._A.removeEventListener("test",null,e)}catch(e){}function a(e,t){return i||o?{capture:!!e,passive:i,signal:t}:!!e}function s(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2],n=arguments.length>3?arguments[3]:void 0;window.addEventListener(e,t,a(r,n))}function c(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2],n=arguments.length>3?arguments[3]:void 0;document.addEventListener(e,t,a(r,n))}},4402:(e,t,r)=>{r.d(t,{Ht:()=>u,M:()=>c,Rl:()=>a,ky:()=>s});var n=r(385);const i="xxxxxxxx-xxxx-4xxx-yxxx-xxxxxxxxxxxx";function o(e,t){return e?15&e[t]:16*Math.random()|0}function a(){const e=n._A?.crypto||n._A?.msCrypto;let t,r=0;return e&&e.getRandomValues&&(t=e.getRandomValues(new Uint8Array(31))),i.split("").map((e=>"x"===e?o(t,++r).toString(16):"y"===e?(3&o()|8).toString(16):e)).join("")}function s(e){const t=n._A?.crypto||n._A?.msCrypto;let r,i=0;t&&t.getRandomValues&&(r=t.getRandomValues(new Uint8Array(31)));const a=[];for(var s=0;s {r.d(t,{Bq:()=>n,Hb:()=>o,oD:()=>i});const n="NRBA",i=144e5,o=18e5},7894:(e,t,r)=>{function n(){return Math.round(performance.now())}r.d(t,{z:()=>n})},7243:(e,t,r)=>{r.d(t,{e:()=>o});var n=r(385),i={};function o(e){if(e in i)return i[e];if(0===(e||"").indexOf("data:"))return{protocol:"data"};let t;var r=n._A?.location,o={};if(n.il)t=document.createElement("a"),t.href=e;else try{t=new URL(e,r.href)}catch(e){return o}o.port=t.port;var a=t.href.split("://");!o.port&&a[1]&&(o.port=a[1].split("/")[0].split("@").pop().split(":")[1]),o.port&&"0"!==o.port||(o.port="https"===a[0]?"443":"80"),o.hostname=t.hostname||r.hostname,o.pathname=t.pathname,o.protocol=a[0],"/"!==o.pathname.charAt(0)&&(o.pathname="/"+o.pathname);var s=!t.protocol||":"===t.protocol||t.protocol===r.protocol,c=t.hostname===r.hostname&&t.port===r.port;return o.sameOrigin=s&&(!t.hostname||c),"/"===o.pathname&&(i[e]=o),o}},50:(e,t,r)=>{function n(e,t){"function"==typeof console.warn&&(console.warn("New Relic: ".concat(e)),t&&console.warn(t))}r.d(t,{Z:()=>n})},2587:(e,t,r)=>{r.d(t,{N:()=>c,T:()=>u});var n=r(2177),i=r(5546),o=r(8e3),a=r(3325);const s={stn:[a.D.sessionTrace],err:[a.D.jserrors,a.D.metrics],ins:[a.D.pageAction],spa:[a.D.spa],sr:[a.D.sessionReplay,a.D.sessionTrace]};function c(e,t){const r=n.ee.get(t);e&&"object"==typeof e&&(Object.entries(e).forEach((e=>{let[t,n]=e;void 0===u[t]&&(s[t]?s[t].forEach((e=>{n?(0,i.p)("feat-"+t,[],void 0,e,r):(0,i.p)("block-"+t,[],void 0,e,r),(0,i.p)("rumresp-"+t,[Boolean(n)],void 0,e,r)})):n&&(0,i.p)("feat-"+t,[],void 0,void 0,r),u[t]=Boolean(n))})),Object.keys(s).forEach((e=>{void 0===u[e]&&(s[e]?.forEach((t=>(0,i.p)("rumresp-"+e,[!1],void 0,t,r))),u[e]=!1)})),(0,o.L)(t,a.D.pageViewEvent))}const u={}},2210:(e,t,r)=>{r.d(t,{X:()=>i});var n=Object.prototype.hasOwnProperty;function i(e,t,r){if(n.call(e,t))return e[t];var i=r();if(Object.defineProperty&&Object.keys)try{return Object.defineProperty(e,t,{value:i,writable:!0,enumerable:!1}),i}catch(e){}return e[t]=i,i}},1284:(e,t,r)=>{r.d(t,{D:()=>n});const n=(e,t)=>Object.entries(e||{}).map((e=>{let[r,n]=e;return t(r,n)}))},4351:(e,t,r)=>{r.d(t,{P:()=>o});var n=r(2177);const i=()=>{const e=new WeakSet;return(t,r)=>{if("object"==typeof r&&null!==r){if(e.has(r))return;e.add(r)}return r}};function o(e){try{return JSON.stringify(e,i())}catch(e){try{n.ee.emit("internal-error",[e])}catch(e){}}}},3960:(e,t,r)=>{r.d(t,{K:()=>a,b:()=>o});var n=r(3239);function i(){return"undefined"==typeof document||"complete"===document.readyState}function o(e,t){if(i())return e();(0,n.bP)("load",e,t)}function a(e){if(i())return e();(0,n.iz)("DOMContentLoaded",e)}},8632:(e,t,r)=>{r.d(t,{EZ:()=>u,Qy:()=>c,ce:()=>o,fP:()=>a,gG:()=>d,mF:()=>s});var n=r(7894),i=r(385);const o={beacon:"bam.nr-data.net",errorBeacon:"bam.nr-data.net"};function a(){return i._A.NREUM||(i._A.NREUM={}),void 0===i._A.newrelic&&(i._A.newrelic=i._A.NREUM),i._A.NREUM}function s(){let e=a();return e.o||(e.o={ST:i._A.setTimeout,SI:i._A.setImmediate,CT:i._A.clearTimeout,XHR:i._A.XMLHttpRequest,REQ:i._A.Request,EV:i._A.Event,PR:i._A.Promise,MO:i._A.MutationObserver,FETCH:i._A.fetch}),e}function c(e,t,r){let i=a();const o=i.initializedAgents||{},s=o[e]||{};return Object.keys(s).length||(s.initializedAt={ms:(0,n.z)(),date:new Date}),i.initializedAgents={...o,[e]:{...s,[r]:t}},i}function u(e,t){a()[e]=t}function d(){return function(){let e=a();const t=e.info||{};e.info={beacon:o.beacon,errorBeacon:o.errorBeacon,...t}}(),function(){let e=a();const t=e.init||{};e.init={...t}}(),s(),function(){let e=a();const t=e.loader_config||{};e.loader_config={...t}}(),a()}},7956:(e,t,r)=>{r.d(t,{N:()=>i});var n=r(3239);function i(e){let t=arguments.length>1&&void 0!==arguments[1]&&arguments[1],r=arguments.length>2?arguments[2]:void 0,i=arguments.length>3?arguments[3]:void 0;return void(0,n.iz)("visibilitychange",(function(){if(t)return void("hidden"==document.visibilityState&&e());e(document.visibilityState)}),r,i)}},1214:(e,t,r)=>{r.d(t,{em:()=>v,u5:()=>N,QU:()=>S,_L:()=>I,Gm:()=>L,Lg:()=>M,gy:()=>U,BV:()=>Q,Kf:()=>ee});var n=r(2177);const i="nr@original";var o=Object.prototype.hasOwnProperty,a=!1;function s(e,t){return e||(e=n.ee),r.inPlace=function(e,t,n,i,o){n||(n="");var a,s,c,u="-"===n.charAt(0);for(c=0;c 2?n-2:0),o=2;o {r(A[T],e,w),r(E[T],e,w)})),r(l._A,"fetch",y),t.on(y+"end",(function(e,r){var n=this;if(r){var i=r.headers.get("content-length");null!==i&&(n.rxSize=i),t.emit(y+"done",[null,r],n)}else t.emit(y+"done",[e],n)})),t}const O={},j=["pushState","replaceState"];function S(e){const t=function(e){return(e||n.ee).get("history")}(e);return!l.il||O[t.debugId]++||(O[t.debugId]=1,s(t).inPlace(window.history,j,"-")),t}var P=r(3239);const C={},R=["appendChild","insertBefore","replaceChild"];function I(e){const t=function(e){return(e||n.ee).get("jsonp")}(e);if(!l.il||C[t.debugId])return t;C[t.debugId]=!0;var r=s(t),i=/[?&](?:callback|cb)=([^&#]+)/,o=/(.*)\.([^.]+)/,a=/^(\w+)(\.|$)(.*)$/;function c(e,t){var r=e.match(a),n=r[1],i=r[3];return i?c(i,t[n]):t[n]}return r.inPlace(Node.prototype,R,"dom-"),t.on("dom-start",(function(e){!function(e){if(!e||"string"!=typeof e.nodeName||"script"!==e.nodeName.toLowerCase())return;if("function"!=typeof e.addEventListener)return;var n=(a=e.src,s=a.match(i),s?s[1]:null);var a,s;if(!n)return;var u=function(e){var t=e.match(o);if(t&&t.length>=3)return{key:t[2],parent:c(t[1],window)};return{key:e,parent:window}}(n);if("function"!=typeof u.parent[u.key])return;var d={};function f(){t.emit("jsonp-end",[],d),e.removeEventListener("load",f,(0,P.m$)(!1)),e.removeEventListener("error",l,(0,P.m$)(!1))}function l(){t.emit("jsonp-error",[],d),t.emit("jsonp-end",[],d),e.removeEventListener("load",f,(0,P.m$)(!1)),e.removeEventListener("error",l,(0,P.m$)(!1))}r.inPlace(u.parent,[u.key],"cb-",d),e.addEventListener("load",f,(0,P.m$)(!1)),e.addEventListener("error",l,(0,P.m$)(!1)),t.emit("new-jsonp",[e.src],d)}(e[0])})),t}var k=r(5763);const H={};function L(e){const t=function(e){return(e||n.ee).get("mutation")}(e);if(!l.il||H[t.debugId])return t;H[t.debugId]=!0;var r=s(t),i=k.Yu.MO;return i&&(window.MutationObserver=function(e){return this instanceof i?new i(r(e,"fn-")):i.apply(this,arguments)},MutationObserver.prototype=i.prototype),t}const z={};function M(e){const t=function(e){return(e||n.ee).get("promise")}(e);if(z[t.debugId])return t;z[t.debugId]=!0;var r=n.c,o=s(t),a=k.Yu.PR;return a&&function(){function e(r){var n=t.context(),i=o(r,"executor-",n,null,!1);const s=Reflect.construct(a,[i],e);return t.context(s).getCtx=function(){return n},s}l._A.Promise=e,Object.defineProperty(e,"name",{value:"Promise"}),e.toString=function(){return a.toString()},Object.setPrototypeOf(e,a),["all","race"].forEach((function(r){const n=a[r];e[r]=function(e){let i=!1;[...e||[]].forEach((e=>{this.resolve(e).then(a("all"===r),a(!1))}));const o=n.apply(this,arguments);return o;function a(e){return function(){t.emit("propagate",[null,!i],o,!1,!1),i=i||!e}}}})),["resolve","reject"].forEach((function(r){const n=a[r];e[r]=function(e){const r=n.apply(this,arguments);return e!==r&&t.emit("propagate",[e,!0],r,!1,!1),r}})),e.prototype=a.prototype;const n=a.prototype.then;a.prototype.then=function(){var e=this,i=r(e);i.promise=e;for(var a=arguments.length,s=new Array(a),c=0;c e())),t};function m(e,t){i.inPlace(t,["onreadystatechange"],"fn-",E)}function b(){var e=this,t=r.context(e);e.readyState>3&&!t.resolved&&(t.resolved=!0,r.emit("xhr-resolved",[],e)),i.inPlace(e,f,"fn-",E)}if(function(e,t){for(var r in e)t[r]=e[r]}(o,p),p.prototype=o.prototype,i.inPlace(p.prototype,J,"-xhr-",E),r.on("send-xhr-start",(function(e,t){m(e,t),function(e){h.push(e),a&&(y?y.then(A):u?u(A):(w=-w,x.data=w))}(t)})),r.on("open-xhr-start",m),a){var y=c&&c.resolve();if(!u&&!c){var w=1,x=document.createTextNode(w);new a(A).observe(x,{characterData:!0})}}else t.on("fn-end",(function(e){e[0]&&e[0].type===d||A()}));function A(){for(var e=0;e {r.d(t,{t:()=>n});const n=r(3325).D.ajax},6660:(e,t,r)=>{r.d(t,{A:()=>i,t:()=>n});const n=r(3325).D.jserrors,i="nr@seenError"},3081:(e,t,r)=>{r.d(t,{gF:()=>o,mY:()=>i,t9:()=>n,vz:()=>s,xS:()=>a});const n=r(3325).D.metrics,i="sm",o="cm",a="storeSupportabilityMetrics",s="storeEventMetrics"},4649:(e,t,r)=>{r.d(t,{t:()=>n});const n=r(3325).D.pageAction},7633:(e,t,r)=>{r.d(t,{Dz:()=>i,OJ:()=>a,qw:()=>o,t9:()=>n});const n=r(3325).D.pageViewEvent,i="firstbyte",o="domcontent",a="windowload"},9251:(e,t,r)=>{r.d(t,{t:()=>n});const n=r(3325).D.pageViewTiming},3614:(e,t,r)=>{r.d(t,{BST_RESOURCE:()=>i,END:()=>s,FEATURE_NAME:()=>n,FN_END:()=>u,FN_START:()=>c,PUSH_STATE:()=>d,RESOURCE:()=>o,START:()=>a});const n=r(3325).D.sessionTrace,i="bstResource",o="resource",a="-start",s="-end",c="fn"+a,u="fn"+s,d="pushState"},7836:(e,t,r)=>{r.d(t,{BODY:()=>A,CB_END:()=>E,CB_START:()=>u,END:()=>x,FEATURE_NAME:()=>i,FETCH:()=>_,FETCH_BODY:()=>v,FETCH_DONE:()=>m,FETCH_START:()=>p,FN_END:()=>c,FN_START:()=>s,INTERACTION:()=>l,INTERACTION_API:()=>d,INTERACTION_EVENTS:()=>o,JSONP_END:()=>b,JSONP_NODE:()=>g,JS_TIME:()=>T,MAX_TIMER_BUDGET:()=>a,REMAINING:()=>f,SPA_NODE:()=>h,START:()=>w,originalSetTimeout:()=>y});var n=r(5763);const i=r(3325).D.spa,o=["click","submit","keypress","keydown","keyup","change"],a=999,s="fn-start",c="fn-end",u="cb-start",d="api-ixn-",f="remaining",l="interaction",h="spaNode",g="jsonpNode",p="fetch-start",m="fetch-done",v="fetch-body-",b="jsonp-end",y=n.Yu.ST,w="-start",x="-end",A="-body",E="cb"+x,T="jsTime",_="fetch"},5938:(e,t,r)=>{r.d(t,{W:()=>o});var n=r(5763),i=r(2177);class o{constructor(e,t,r){this.agentIdentifier=e,this.aggregator=t,this.ee=i.ee.get(e,(0,n.OP)(this.agentIdentifier).isolatedBacklog),this.featureName=r,this.blocked=!1}}},9144:(e,t,r)=>{r.d(t,{j:()=>m});var n=r(3325),i=r(5763),o=r(5546),a=r(2177),s=r(7894),c=r(8e3),u=r(3960),d=r(385),f=r(50),l=r(3081),h=r(8632);function g(){const e=(0,h.gG)();["setErrorHandler","finished","addToTrace","inlineHit","addRelease","addPageAction","setCurrentRouteName","setPageViewName","setCustomAttribute","interaction","noticeError","setUserId"].forEach((t=>{e[t]=function(){for(var r=arguments.length,n=new Array(r),i=0;i 1?r-1:0),i=1;i {e.exposed&&e.api[t]&&o.push(e.api[t](...n))})),o.length>1?o:o[0]}(t,...n)}}))}var p=r(2587);function m(e){let t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:{},m=arguments.length>2?arguments[2]:void 0,v=arguments.length>3?arguments[3]:void 0,{init:b,info:y,loader_config:w,runtime:x={loaderType:m},exposed:A=!0}=t;const E=(0,h.gG)();y||(b=E.init,y=E.info,w=E.loader_config),(0,i.Dg)(e,b||{}),(0,i.GE)(e,w||{}),(0,i.sU)(e,x),y.jsAttributes??={},d.v6&&(y.jsAttributes.isWorker=!0),(0,i.CX)(e,y),g();const T=function(e,t){t||(0,c.R)(e,"api");const h={};var g=a.ee.get(e),p=g.get("tracer"),m="api-",v=m+"ixn-";function b(t,r,n,o){const a=(0,i.C5)(e);return null===r?delete a.jsAttributes[t]:(0,i.CX)(e,{...a,jsAttributes:{...a.jsAttributes,[t]:r}}),x(m,n,!0,o||null===r?"session":void 0)(t,r)}function y(){}["setErrorHandler","finished","addToTrace","inlineHit","addRelease"].forEach((e=>h[e]=x(m,e,!0,"api"))),h.addPageAction=x(m,"addPageAction",!0,n.D.pageAction),h.setCurrentRouteName=x(m,"routeName",!0,n.D.spa),h.setPageViewName=function(t,r){if("string"==typeof t)return"/"!==t.charAt(0)&&(t="/"+t),(0,i.OP)(e).customTransaction=(r||"http://custom.transaction")+t,x(m,"setPageViewName",!0)()},h.setCustomAttribute=function(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2];if("string"==typeof e){if(["string","number"].includes(typeof t)||null===t)return b(e,t,"setCustomAttribute",r);(0,f.Z)("Failed to execute setCustomAttribute.\nNon-null value must be a string or number type, but a type of was provided."))}else(0,f.Z)("Failed to execute setCustomAttribute.\nName must be a string type, but a type of was provided."))},h.setUserId=function(e){if("string"==typeof e||null===e)return b("enduser.id",e,"setUserId",!0);(0,f.Z)("Failed to execute setUserId.\nNon-null value must be a string type, but a type of was provided."))},h.interaction=function(){return(new y).get()};var w=y.prototype={createTracer:function(e,t){var r={},i=this,a="function"==typeof t;return(0,o.p)(v+"tracer",[(0,s.z)(),e,r],i,n.D.spa,g),function(){if(p.emit((a?"":"no-")+"fn-start",[(0,s.z)(),i,a],r),a)try{return t.apply(this,arguments)}catch(e){throw p.emit("fn-err",[arguments,this,"string"==typeof e?new Error(e):e],r),e}finally{p.emit("fn-end",[(0,s.z)()],r)}}}};function x(e,t,r,i){return function(){return(0,o.p)(l.xS,["API/"+t+"/called"],void 0,n.D.metrics,g),i&&(0,o.p)(e+t,[(0,s.z)(),...arguments],r?null:this,i,g),r?void 0:this}}function A(){r.e(439).then(r.bind(r,7438)).then((t=>{let{setAPI:r}=t;r(e),(0,c.L)(e,"api")})).catch((()=>(0,f.Z)("Downloading runtime APIs failed...")))}return["actionText","setName","setAttribute","save","ignore","onEnd","getContext","end","get"].forEach((e=>{w[e]=x(v,e,void 0,n.D.spa)})),h.noticeError=function(e,t){"string"==typeof e&&(e=new Error(e)),(0,o.p)(l.xS,["API/noticeError/called"],void 0,n.D.metrics,g),(0,o.p)("err",[e,(0,s.z)(),!1,t],void 0,n.D.jserrors,g)},d.il?(0,u.b)((()=>A()),!0):A(),h}(e,v);return(0,h.Qy)(e,T,"api"),(0,h.Qy)(e,A,"exposed"),(0,h.EZ)("activatedFeatures",p.T),T}},3325:(e,t,r)=>{r.d(t,{D:()=>n,p:()=>i});const n={ajax:"ajax",jserrors:"jserrors",metrics:"metrics",pageAction:"page_action",pageViewEvent:"page_view_event",pageViewTiming:"page_view_timing",sessionReplay:"session_replay",sessionTrace:"session_trace",spa:"spa"},i={[n.pageViewEvent]:1,[n.pageViewTiming]:2,[n.metrics]:3,[n.jserrors]:4,[n.ajax]:5,[n.sessionTrace]:6,[n.pageAction]:7,[n.spa]:8,[n.sessionReplay]:9}}},n={};function i(e){var t=n[e];if(void 0!==t)return t.exports;var o=n[e]={exports:{}};return r[e](o,o.exports,i),o.exports}i.m=r,i.d=(e,t)=>{for(var r in t)i.o(t,r)&&!i.o(e,r)&&Object.defineProperty(e,r,{enumerable:!0,get:t[r]})},i.f={},i.e=e=>Promise.all(Object.keys(i.f).reduce(((t,r)=>(i.f[r](e,t),t)),[])),i.u=e=>(({78:"page_action-aggregate",147:"metrics-aggregate",242:"session-manager",317:"jserrors-aggregate",348:"page_view_timing-aggregate",412:"lazy-feature-loader",439:"async-api",538:"recorder",590:"session_replay-aggregate",675:"compressor",733:"session_trace-aggregate",786:"page_view_event-aggregate",873:"spa-aggregate",898:"ajax-aggregate"}[e]||e)+"."+{78:"ac76d497",147:"3dc53903",148:"1a20d5fe",242:"2a64278a",317:"49e41428",348:"bd6de33a",412:"2f55ce66",439:"30bd804e",538:"1b18459f",590:"cf0efb30",675:"ae9f91a8",733:"83105561",786:"06482edd",860:"03a8b7a5",873:"e6b09d52",898:"998ef92b"}[e]+"-1.236.0.min.js"),i.o=(e,t)=>Object.prototype.hasOwnProperty.call(e,t),e={},t="NRBA:",i.l=(r,n,o,a)=>{if(e[r])e[r].push(n);else{var s,c;if(void 0!==o)for(var u=document.getElementsByTagName("script"),d=0;d {s.onerror=s.onload=null,clearTimeout(h);var i=e[r];if(delete e[r],s.parentNode&&s.parentNode.removeChild(s),i&&i.forEach((e=>e(n))),t)return t(n)},h=setTimeout(l.bind(null,void 0,{type:"timeout",target:s}),12e4);s.onerror=l.bind(null,s.onerror),s.onload=l.bind(null,s.onload),c&&document.head.appendChild(s)}},i.r=e=>{"undefined"!=typeof Symbol&&Symbol.toStringTag&&Object.defineProperty(e,Symbol.toStringTag,{value:"Module"}),Object.defineProperty(e,"__esModule",{value:!0})},i.j=364,i.p="https://js-agent.newrelic.com/",(()=>{var e={364:0,953:0};i.f.j=(t,r)=>{var n=i.o(e,t)?e[t]:void 0;if(0!==n)if(n)r.push(n[2]);else{var o=new Promise(((r,i)=>n=e[t]=[r,i]));r.push(n[2]=o);var a=i.p+i.u(t),s=new Error;i.l(a,(r=>{if(i.o(e,t)&&(0!==(n=e[t])&&(e[t]=void 0),n)){var o=r&&("load"===r.type?"missing":r.type),a=r&&r.target&&r.target.src;s.message="Loading chunk "+t+" failed.\n("+o+": "+a+")",s.name="ChunkLoadError",s.type=o,s.request=a,n[1](s)}}),"chunk-"+t,t)}};var t=(t,r)=>{var n,o,[a,s,c]=r,u=0;if(a.some((t=>0!==e[t]))){for(n in s)i.o(s,n)&&(i.m[n]=s[n]);if(c)c(i)}for(t&&t(r);u {i.r(o);var e=i(3325),t=i(5763);const r=Object.values(e.D);function n(e){const n={};return r.forEach((r=>{n[r]=function(e,r){return!1!==(0,t.Mt)(r,"".concat(e,".enabled"))}(r,e)})),n}var a=i(9144);var s=i(5546),c=i(385),u=i(8e3),d=i(5938),f=i(3960),l=i(50);class h extends d.W{constructor(e,t,r){let n=!(arguments.length>3&&void 0!==arguments[3])||arguments[3];super(e,t,r),this.auto=n,this.abortHandler,this.featAggregate,this.onAggregateImported,n&&(0,u.R)(e,r)}importAggregator(){let e=arguments.length>0&&void 0!==arguments[0]?arguments[0]:{};if(this.featAggregate||!this.auto)return;const r=c.il&&!0===(0,t.Mt)(this.agentIdentifier,"privacy.cookies_enabled");let n;this.onAggregateImported=new Promise((e=>{n=e}));const o=async()=>{let t;try{if(r){const{setupAgentSession:e}=await Promise.all([i.e(860),i.e(242)]).then(i.bind(i,3228));t=e(this.agentIdentifier)}}catch(e){(0,l.Z)("A problem occurred when starting up session manager. This page will not start or extend any session.",e)}try{if(!this.shouldImportAgg(this.featureName,t))return void(0,u.L)(this.agentIdentifier,this.featureName);const{lazyFeatureLoader:r}=await i.e(412).then(i.bind(i,8582)),{Aggregate:o}=await r(this.featureName,"aggregate");this.featAggregate=new o(this.agentIdentifier,this.aggregator,e),n(!0)}catch(e){(0,l.Z)("Downloading and initializing ".concat(this.featureName," failed..."),e),this.abortHandler?.(),n(!1)}};c.il?(0,f.b)((()=>o()),!0):o()}shouldImportAgg(r,n){return r!==e.D.sessionReplay||!1!==(0,t.Mt)(this.agentIdentifier,"session_trace.enabled")&&(!!n?.isNew||!!n?.state.sessionReplay)}}var g=i(7633),p=i(7894);class m extends h{static featureName=g.t9;constructor(r,n){let i=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];if(super(r,n,g.t9,i),("undefined"==typeof PerformanceNavigationTiming||c.Tt)&&"undefined"!=typeof PerformanceTiming){const n=(0,t.OP)(r);n[g.Dz]=Math.max(Date.now()-n.offset,0),(0,f.K)((()=>n[g.qw]=Math.max((0,p.z)()-n[g.Dz],0))),(0,f.b)((()=>{const t=(0,p.z)();n[g.OJ]=Math.max(t-n[g.Dz],0),(0,s.p)("timing",["load",t],void 0,e.D.pageViewTiming,this.ee)}))}this.importAggregator()}}var v=i(1117),b=i(1284);class y extends v.w{constructor(e){super(e),this.aggregatedData={}}store(e,t,r,n,i){var o=this.getBucket(e,t,r,i);return o.metrics=function(e,t){t||(t={count:0});return t.count+=1,(0,b.D)(e,(function(e,r){t[e]=w(r,t[e])})),t}(n,o.metrics),o}merge(e,t,r,n,i){var o=this.getBucket(e,t,n,i);if(o.metrics){var a=o.metrics;a.count+=r.count,(0,b.D)(r,(function(e,t){if("count"!==e){var n=a[e],i=r[e];i&&!i.c?a[e]=w(i.t,n):a[e]=function(e,t){if(!t)return e;t.c||(t=x(t.t));return t.min=Math.min(e.min,t.min),t.max=Math.max(e.max,t.max),t.t+=e.t,t.sos+=e.sos,t.c+=e.c,t}(i,a[e])}}))}else o.metrics=r}storeMetric(e,t,r,n){var i=this.getBucket(e,t,r);return i.stats=w(n,i.stats),i}getBucket(e,t,r,n){this.aggregatedData[e]||(this.aggregatedData[e]={});var i=this.aggregatedData[e][t];return i||(i=this.aggregatedData[e][t]={params:r||{}},n&&(i.custom=n)),i}get(e,t){return t?this.aggregatedData[e]&&this.aggregatedData[e][t]:this.aggregatedData[e]}take(e){for(var t={},r="",n=!1,i=0;i t.max&&(t.max=e),e 2&&void 0!==arguments[2])||arguments[2];super(e,r,j.t,n),c.il&&((0,t.OP)(e).initHidden=Boolean("hidden"===document.visibilityState),(0,N.N)((()=>(0,s.p)("docHidden",[(0,p.z)()],void 0,j.t,this.ee)),!0),(0,O.bP)("pagehide",(()=>(0,s.p)("winPagehide",[(0,p.z)()],void 0,j.t,this.ee))),this.importAggregator())}}var P=i(3081);class C extends h{static featureName=P.t9;constructor(e,t){let r=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];super(e,t,P.t9,r),this.importAggregator()}}var R,I=i(2210),k=i(1214),H=i(2177),L={};try{R=localStorage.getItem("__nr_flags").split(","),console&&"function"==typeof console.log&&(L.console=!0,-1!==R.indexOf("dev")&&(L.dev=!0),-1!==R.indexOf("nr_dev")&&(L.nrDev=!0))}catch(e){}function z(e){try{L.console&&z(e)}catch(e){}}L.nrDev&&H.ee.on("internal-error",(function(e){z(e.stack)})),L.dev&&H.ee.on("fn-err",(function(e,t,r){z(r.stack)})),L.dev&&(z("NR AGENT IN DEVELOPMENT MODE"),z("flags: "+(0,b.D)(L,(function(e,t){return e})).join(", ")));var M=i(6660);class B extends h{static featureName=M.t;constructor(r,n){let i=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];super(r,n,M.t,i),this.skipNext=0;try{this.removeOnAbort=new AbortController}catch(e){}const o=this;o.ee.on("fn-start",(function(e,t,r){o.abortHandler&&(o.skipNext+=1)})),o.ee.on("fn-err",(function(t,r,n){o.abortHandler&&!n[M.A]&&((0,I.X)(n,M.A,(function(){return!0})),this.thrown=!0,(0,s.p)("err",[n,(0,p.z)()],void 0,e.D.jserrors,o.ee))})),o.ee.on("fn-end",(function(){o.abortHandler&&!this.thrown&&o.skipNext>0&&(o.skipNext-=1)})),o.ee.on("internal-error",(function(t){(0,s.p)("ierr",[t,(0,p.z)(),!0],void 0,e.D.jserrors,o.ee)})),this.origOnerror=c._A.onerror,c._A.onerror=this.onerrorHandler.bind(this),c._A.addEventListener("unhandledrejection",(t=>{const r=function(e){let t="Unhandled Promise Rejection: ";if(e instanceof Error)try{return e.message=t+e.message,e}catch(t){return e}if(void 0===e)return new Error(t);try{return new Error(t+(0,D.P)(e))}catch(e){return new Error(t)}}(t.reason);(0,s.p)("err",[r,(0,p.z)(),!1,{unhandledPromiseRejection:1}],void 0,e.D.jserrors,this.ee)}),(0,O.m$)(!1,this.removeOnAbort?.signal)),(0,k.gy)(this.ee),(0,k.BV)(this.ee),(0,k.em)(this.ee),(0,t.OP)(r).xhrWrappable&&(0,k.Kf)(this.ee),this.abortHandler=this.#e,this.importAggregator()}#e(){this.removeOnAbort?.abort(),this.abortHandler=void 0}onerrorHandler(t,r,n,i,o){"function"==typeof this.origOnerror&&this.origOnerror(...arguments);try{this.skipNext?this.skipNext-=1:(0,s.p)("err",[o||new F(t,r,n),(0,p.z)()],void 0,e.D.jserrors,this.ee)}catch(t){try{(0,s.p)("ierr",[t,(0,p.z)(),!0],void 0,e.D.jserrors,this.ee)}catch(e){}}return!1}}function F(e,t,r){this.message=e||"Uncaught error with no additional information",this.sourceURL=t,this.line=r}let U=1;const q="nr@id";function G(e){const t=typeof e;return!e||"object"!==t&&"function"!==t?-1:e===c._A?0:(0,I.X)(e,q,(function(){return U++}))}function V(e){if("string"==typeof e&&e.length)return e.length;if("object"==typeof e){if("undefined"!=typeof ArrayBuffer&&e instanceof ArrayBuffer&&e.byteLength)return e.byteLength;if("undefined"!=typeof Blob&&e instanceof Blob&&e.size)return e.size;if(!("undefined"!=typeof FormData&&e instanceof FormData))try{return(0,D.P)(e).length}catch(e){return}}}var X=i(7243);class W{constructor(e){this.agentIdentifier=e,this.generateTracePayload=this.generateTracePayload.bind(this),this.shouldGenerateTrace=this.shouldGenerateTrace.bind(this)}generateTracePayload(e){if(!this.shouldGenerateTrace(e))return null;var r=(0,t.DL)(this.agentIdentifier);if(!r)return null;var n=(r.accountID||"").toString()||null,i=(r.agentID||"").toString()||null,o=(r.trustKey||"").toString()||null;if(!n||!i)return null;var a=(0,_.M)(),s=(0,_.Ht)(),c=Date.now(),u={spanId:a,traceId:s,timestamp:c};return(e.sameOrigin||this.isAllowedOrigin(e)&&this.useTraceContextHeadersForCors())&&(u.traceContextParentHeader=this.generateTraceContextParentHeader(a,s),u.traceContextStateHeader=this.generateTraceContextStateHeader(a,c,n,i,o)),(e.sameOrigin&&!this.excludeNewrelicHeader()||!e.sameOrigin&&this.isAllowedOrigin(e)&&this.useNewrelicHeaderForCors())&&(u.newrelicHeader=this.generateTraceHeader(a,s,c,n,i,o)),u}generateTraceContextParentHeader(e,t){return"00-"+t+"-"+e+"-01"}generateTraceContextStateHeader(e,t,r,n,i){return i+"@nr=0-1-"+r+"-"+n+"-"+e+"----"+t}generateTraceHeader(e,t,r,n,i,o){if(!("function"==typeof c._A?.btoa))return null;var a={v:[0,1],d:{ty:"Browser",ac:n,ap:i,id:e,tr:t,ti:r}};return o&&n!==o&&(a.d.tk=o),btoa((0,D.P)(a))}shouldGenerateTrace(e){return this.isDtEnabled()&&this.isAllowedOrigin(e)}isAllowedOrigin(e){var r=!1,n={};if((0,t.Mt)(this.agentIdentifier,"distributed_tracing")&&(n=(0,t.P_)(this.agentIdentifier).distributed_tracing),e.sameOrigin)r=!0;else if(n.allowed_origins instanceof Array)for(var i=0;i 2&&void 0!==arguments[2])||arguments[2];super(r,n,Z.t,i),(0,t.OP)(r).xhrWrappable&&(this.dt=new W(r),this.handler=(e,t,r,n)=>(0,s.p)(e,t,r,n,this.ee),(0,k.u5)(this.ee),(0,k.Kf)(this.ee),function(r,n,i,o){function a(e){var t=this;t.totalCbs=0,t.called=0,t.cbTime=0,t.end=E,t.ended=!1,t.xhrGuids={},t.lastSize=null,t.loadCaptureCalled=!1,t.params=this.params||{},t.metrics=this.metrics||{},e.addEventListener("load",(function(r){_(t,e)}),(0,O.m$)(!1)),c.IF||e.addEventListener("progress",(function(e){t.lastSize=e.loaded}),(0,O.m$)(!1))}function s(e){this.params={method:e[0]},T(this,e[1]),this.metrics={}}function u(e,n){var i=(0,t.DL)(r);i.xpid&&this.sameOrigin&&n.setRequestHeader("X-NewRelic-ID",i.xpid);var a=o.generateTracePayload(this.parsedOrigin);if(a){var s=!1;a.newrelicHeader&&(n.setRequestHeader("newrelic",a.newrelicHeader),s=!0),a.traceContextParentHeader&&(n.setRequestHeader("traceparent",a.traceContextParentHeader),a.traceContextStateHeader&&n.setRequestHeader("tracestate",a.traceContextStateHeader),s=!0),s&&(this.dt=a)}}function d(e,t){var r=this.metrics,i=e[0],o=this;if(r&&i){var a=V(i);a&&(r.txSize=a)}this.startTime=(0,p.z)(),this.listener=function(e){try{"abort"!==e.type||o.loadCaptureCalled||(o.params.aborted=!0),("load"!==e.type||o.called===o.totalCbs&&(o.onloadCalled||"function"!=typeof t.onload)&&"function"==typeof o.end)&&o.end(t)}catch(e){try{n.emit("internal-error",[e])}catch(e){}}};for(var s=0;s 1?e[1]=i:e.push(i)}else e[0]&&e[0].headers&&s(e[0].headers,n)&&(this.dt=n);function s(e,t){var r=!1;return t.newrelicHeader&&(e.set("newrelic",t.newrelicHeader),r=!0),t.traceContextParentHeader&&(e.set("traceparent",t.traceContextParentHeader),t.traceContextStateHeader&&e.set("tracestate",t.traceContextStateHeader),r=!0),r}}function x(e,t){this.params={},this.metrics={},this.startTime=(0,p.z)(),this.dt=t,e.length>=1&&(this.target=e[0]),e.length>=2&&(this.opts=e[1]);var r,n=this.opts||{},i=this.target;"string"==typeof i?r=i:"object"==typeof i&&i instanceof Y?r=i.url:c._A?.URL&&"object"==typeof i&&i instanceof URL&&(r=i.href),T(this,r);var o=(""+(i&&i instanceof Y&&i.method||n.method||"GET")).toUpperCase();this.params.method=o,this.txSize=V(n.body)||0}function A(t,r){var n;this.endTime=(0,p.z)(),this.params||(this.params={}),this.params.status=r?r.status:0,"string"==typeof this.rxSize&&this.rxSize.length>0&&(n=+this.rxSize);var o={txSize:this.txSize,rxSize:n,duration:(0,p.z)()-this.startTime};i("xhr",[this.params,o,this.startTime,this.endTime,"fetch"],this,e.D.ajax)}function E(t){var r=this.params,n=this.metrics;if(!this.ended){this.ended=!0;for(var o=0;o 2&&void 0!==arguments[2])||arguments[2];super(e,t,we.t,r),this.importAggregator()}}new class{constructor(e){let t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:(0,_.ky)(16);c._A?(this.agentIdentifier=t,this.sharedAggregator=new y({agentIdentifier:this.agentIdentifier}),this.features={},this.desiredFeatures=new Set(e.features||[]),this.desiredFeatures.add(m),Object.assign(this,(0,a.j)(this.agentIdentifier,e,e.loaderType||"agent")),this.start()):(0,l.Z)("Failed to initial the agent. Could not determine the runtime environment.")}get config(){return{info:(0,t.C5)(this.agentIdentifier),init:(0,t.P_)(this.agentIdentifier),loader_config:(0,t.DL)(this.agentIdentifier),runtime:(0,t.OP)(this.agentIdentifier)}}start(){const t="features";try{const r=n(this.agentIdentifier),i=[...this.desiredFeatures];i.sort(((t,r)=>e.p[t.featureName]-e.p[r.featureName])),i.forEach((t=>{if(r[t.featureName]||t.featureName===e.D.pageViewEvent){const n=function(t){switch(t){case e.D.ajax:return[e.D.jserrors];case e.D.sessionTrace:return[e.D.ajax,e.D.pageViewEvent];case e.D.sessionReplay:return[e.D.sessionTrace];case e.D.pageViewTiming:return[e.D.pageViewEvent];default:return[]}}(t.featureName);n.every((e=>r[e]))||(0,l.Z)("".concat(t.featureName," is enabled but one or more dependent features has been disabled (").concat((0,D.P)(n),"). This may cause unintended consequences or missing data...")),this.features[t.featureName]=new t(this.agentIdentifier,this.sharedAggregator)}})),(0,T.Qy)(this.agentIdentifier,this.features,t)}catch(e){(0,l.Z)("Failed to initialize all enabled instrument classes (agent aborted) -",e);for(const e in this.features)this.features[e].abortHandler?.();const r=(0,T.fP)();return delete r.initializedAgents[this.agentIdentifier]?.api,delete r.initializedAgents[this.agentIdentifier]?.[t],delete this.sharedAggregator,r.ee?.abort(),delete r.ee?.get(this.agentIdentifier),!1}}}({features:[J,m,S,class extends h{static featureName=oe;constructor(t,r){if(super(t,r,oe,!(arguments.length>2&&void 0!==arguments[2])||arguments[2]),!c.il)return;const n=this.ee;let i;(0,k.QU)(n),this.eventsEE=(0,k.em)(n),this.eventsEE.on(se,(function(e,t){this.bstStart=(0,p.z)()})),this.eventsEE.on(ae,(function(t,r){(0,s.p)("bst",[t[0],r,this.bstStart,(0,p.z)()],void 0,e.D.sessionTrace,n)})),n.on(ce+ne,(function(e){this.time=(0,p.z)(),this.startPath=location.pathname+location.hash})),n.on(ce+ie,(function(t){(0,s.p)("bstHist",[location.pathname+location.hash,this.startPath,this.time],void 0,e.D.sessionTrace,n)}));try{i=new PerformanceObserver((t=>{const r=t.getEntries();(0,s.p)(te,[r],void 0,e.D.sessionTrace,n)})),i.observe({type:re,buffered:!0})}catch(e){}this.importAggregator({resourceObserver:i})}},C,xe,B,class extends h{static featureName=de;constructor(e,r){if(super(e,r,de,!(arguments.length>2&&void 0!==arguments[2])||arguments[2]),!c.il)return;if(!(0,t.OP)(e).xhrWrappable)return;try{this.removeOnAbort=new AbortController}catch(e){}let n,i=0;const o=this.ee.get("tracer"),a=(0,k._L)(this.ee),s=(0,k.Lg)(this.ee),u=(0,k.BV)(this.ee),d=(0,k.Kf)(this.ee),f=this.ee.get("events"),l=(0,k.u5)(this.ee),h=(0,k.QU)(this.ee),g=(0,k.Gm)(this.ee);function m(e,t){h.emit("newURL",[""+window.location,t])}function v(){i++,n=window.location.hash,this[ve]=(0,p.z)()}function b(){i--,window.location.hash!==n&&m(0,!0);var e=(0,p.z)();this[pe]=~~this[pe]+e-this[ve],this[ye]=e}function y(e,t){e.on(t,(function(){this[t]=(0,p.z)()}))}this.ee.on(ve,v),s.on(be,v),a.on(be,v),this.ee.on(ye,b),s.on(ge,b),a.on(ge,b),this.ee.buffer([ve,ye,"xhr-resolved"],this.featureName),f.buffer([ve],this.featureName),u.buffer(["setTimeout"+le,"clearTimeout"+fe,ve],this.featureName),d.buffer([ve,"new-xhr","send-xhr"+fe],this.featureName),l.buffer([me+fe,me+"-done",me+he+fe,me+he+le],this.featureName),h.buffer(["newURL"],this.featureName),g.buffer([ve],this.featureName),s.buffer(["propagate",be,ge,"executor-err","resolve"+fe],this.featureName),o.buffer([ve,"no-"+ve],this.featureName),a.buffer(["new-jsonp","cb-start","jsonp-error","jsonp-end"],this.featureName),y(l,me+fe),y(l,me+"-done"),y(a,"new-jsonp"),y(a,"jsonp-end"),y(a,"cb-start"),h.on("pushState-end",m),h.on("replaceState-end",m),window.addEventListener("hashchange",m,(0,O.m$)(!0,this.removeOnAbort?.signal)),window.addEventListener("load",m,(0,O.m$)(!0,this.removeOnAbort?.signal)),window.addEventListener("popstate",(function(){m(0,i>1)}),(0,O.m$)(!0,this.removeOnAbort?.signal)),this.abortHandler=this.#e,this.importAggregator()}#e(){this.removeOnAbort?.abort(),this.abortHandler=void 0}}],loaderType:"spa"})})(),window.NRBA=o})(); window.jQuery || document.write(' ') CKEDITOR_BASEPATH='https://f1000research.com/js/vendor/ckeditor/' window.reactTheme = 'research'; window.MathJax = { CommonHTML: { linebreaks: { automatic: true } }, 'HTML-CSS': { linebreaks: { automatic: true } }, SVG: { linebreaks: { automatic: true } }, AuthorInit: function() { MathJax.Hub.Register.MessageHook('End Process', function () { let timeout = false; // holder for timeout id const delay = 250; // delay after event is "complete" to run callback const reflowMath = function() { const dispFormulas = document.querySelectorAll('.disp-formula.panel'); if (!dispFormulas) { return; } for (const dispFormula of dispFormulas) { const child = dispFormula.querySelector('.MathJax_Preview').nextSibling.firstChild; const isMultiline = MathJax.Hub.getAllJax(dispFormula)[0].root.isMultiline; if (dispFormula.offsetWidth < child.offsetWidth || isMultiline) { MathJax.Hub.Queue(['Rerender', MathJax.Hub, dispFormula]); } } }; window.addEventListener('resize', function() { clearTimeout(timeout); // clear the timeout timeout = setTimeout(reflowMath, delay); // start timing for event "completion" }); }); }, }; if (window.location.hash == '#_=_'){ window.location = window.location.href.split('#')[0] } !function(f,b,e,v,n,t,s){if(f.fbq)return;n=f.fbq=function() {n.callMethod? n.callMethod.apply(n,arguments):n.queue.push(arguments)} ;if(!f._fbq)f._fbq=n; n.push=n;n.loaded=!0;n.version='2.0';n.queue=[];t=b.createElement(e);t.async=!0; t.src=v;s=b.getElementsByTagName(e)[0];s.parentNode.insertBefore(t,s)}(window, document,'script','https://connect.facebook.net/en_US/fbevents.js'); fbq('init', '1641728616063202'); fbq('track', "PixelInitialized", {}); (function(h,o,t,j,a,r){ h.hj=h.hj||function(){(h.hj.q=h.hj.q||[]).push(arguments)}; h._hjSettings={hjid:2318163,hjsv:6}; a=o.getElementsByTagName('head')[0]; r=o.createElement('script');r.async=1; r.src=t+h._hjSettings.hjid+j+h._hjSettings.hjsv; a.appendChild(r); })(window,document,'https://static.hotjar.com/c/hotjar-','.js?sv='); search file_upload Submit your research search menu close search Browse Gateways & Collections How to Publish Submit your Research My Submissions Article Guidelines Article Guidelines (New Versions) Open Data, Software and Code Guidelines Open Data and Accessible Source Materials Guidelines (HSS) Open Data, Software and Code Guidelines (PSE) Prepublication Checks Production Process Posters and Slides Guidelines Document Guidelines Article Processing Charges Peer Review Finding Article Reviewers About How it Works For Reviewers Our Advisors Policies Glossary FAQs For Developers Newsroom Contact My Research Submissions Content and Tracking Alerts My Details Sign In file_upload Submit your research { "@context": "https://schema.org", "@type": "ScholarlyArticle", "mainEntityOfPage": { "@type": "WebPage", "@id": "https://f1000research.com/articles/14-163" }, "headline": "Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for...", "datePublished": "2025-02-05T09:16:25", "dateModified": "2025-02-05T09:16:25", "author": [ { "@type": "Person", "name": "Nga Quynh Pham" }, { "@type": "Person", "name": "Tam Trang Mai" }, { "@type": "Person", "name": "Tran Bao Anh Dang" }, { "@type": "Person", "name": "Ly Huong Tran" }, { "@type": "Person", "name": "Quynh Mai Vu" }, { "@type": "Person", "name": "Chien Trong Nguyen" }, { "@type": "Person", "name": "Anh Thi Phuong Tran" }, { "@type": "Person", "name": "Tran Nhat Minh Dang" }, { "@type": "Person", "name": "Van Anh Tran" }, { "@type": "Person", "name": "Thinh Huy Tran" }, { "@type": "Person", "name": "Van Khanh Tran" }, { "@type": "Person", "name": "Hoa Quang Le" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": " Background Botulinum neurotoxin type A (BoNT/A) represents a major threat to global public health because of its most potent toxicity with the longest persistence. Several camelid single-domain antibodies (or VHHs) have been reported to exhibit high neutralizing activity against the receptor binding domain (HC) of the BoNT/A subtype used to generate them. However, it remains unclear if these VHHs can neutralize effectively HC of other BoNT/A subtypes. This study aimed to generate HC domains of all eight BoNT/A subtypes and to screen for VHHs with broad reactivity against these domains. Methods HC domains of BoNT/A1-A8 were recombinantly produced in Escherichia coli. The bont/HCA1 fragment was amplified from sludge sample and cloned into pET45b vector by Gibson assembly. Expression vectors for HC domains of BoNT/A2-A8 were derived from pET45b-HCA1 by site-directed mutagenesis and/or in-house gene synthesis. Similarly, VHHs were synthesized and cloned into pET22b vector. Recombinant protein were purified by Ni-NTA spin columns and analyzed by SDS-PAGE. ELISA was used to confirm the antigenicity of HC domains and to evaluate the reactivity of VHHs to these domains. Results SDS-PAGE analysis and ELISA results with commercial polyclonal antibody demonstrated the HC domains of all eight BoNT/A subtypes were correctly produced. ELISA results using a VHH panel indicated that, apart from ciA-C2, a well-characterized VHH specific for HC of BoNT/A1, two new VHHs were found to recognize the HC domains of all BoNT/A subtypes, of which VHH-A3 displayed EC50 values for these domains close to those of ciA-C2. Conclusion This study provided a resource to comprehensively identify antitoxins conferring broad protection against BoNT/A. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/14-163", "name": "Recombinant expression of receptor binding domains of all eight subtypes..." } } ] } Home Browse Recombinant expression of receptor binding domains of all eight subtypes... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Quynh Pham N, Mai TT, Dang TBA et al. Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.12688/f1000research.160607.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Brief Report Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] Nga Quynh Pham 1 , Tam Trang Mai 2 , Tran Bao Anh Dang 2 , [...] Ly Huong Tran https://orcid.org/0009-0003-6759-6312 1 , Quynh Mai Vu 1 , Chien Trong Nguyen 1 , Anh Thi Phuong Tran 1 , Tran Nhat Minh Dang 3 , Van Anh Tran 3 , Thinh Huy Tran 4 , Van Khanh Tran 4 , Hoa Quang Le https://orcid.org/0000-0002-1853-7397 1 Nga Quynh Pham 1 , Tam Trang Mai 2 , [...] Tran Bao Anh Dang 2 , Ly Huong Tran https://orcid.org/0009-0003-6759-6312 1 , Quynh Mai Vu 1 , Chien Trong Nguyen 1 , Anh Thi Phuong Tran 1 , Tran Nhat Minh Dang 3 , Van Anh Tran 3 , Thinh Huy Tran 4 , Van Khanh Tran 4 , Hoa Quang Le https://orcid.org/0000-0002-1853-7397 1 PUBLISHED 05 Feb 2025 Author details Author details 1 School of Chemistry and Life Sciences, Hanoi University of Science and Technology, Hanoi, Vietnam 2 High School for the Gifted in Natural Sciences, Vietnam National University, Hanoi, 120558, Vietnam 3 Hanoi Medical University, Hanoi, 11521, Vietnam 4 Center for Gene and Protein Research, Hanoi Medical University, Hanoi, 11521, Vietnam Nga Quynh Pham Roles: Data Curation, Formal Analysis, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Tam Trang Mai Roles: Investigation, Writing – Original Draft Preparation Tran Bao Anh Dang Roles: Investigation, Writing – Original Draft Preparation Ly Huong Tran Roles: Data Curation, Formal Analysis, Supervision, Writing – Review & Editing Quynh Mai Vu Roles: Data Curation, Formal Analysis, Supervision, Writing – Review & Editing Chien Trong Nguyen Roles: Data Curation, Formal Analysis, Supervision, Writing – Review & Editing Anh Thi Phuong Tran Roles: Data Curation, Formal Analysis, Supervision, Writing – Review & Editing Tran Nhat Minh Dang Roles: Investigation, Writing – Original Draft Preparation Van Anh Tran Roles: Investigation, Writing – Original Draft Preparation Thinh Huy Tran Roles: Investigation Van Khanh Tran Roles: Conceptualization, Investigation, Supervision Hoa Quang Le Roles: Conceptualization, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Cell & Molecular Biology gateway. Abstract Background Botulinum neurotoxin type A (BoNT/A) represents a major threat to global public health because of its most potent toxicity with the longest persistence. Several camelid single-domain antibodies (or VHHs) have been reported to exhibit high neutralizing activity against the receptor binding domain (HC) of the BoNT/A subtype used to generate them. However, it remains unclear if these VHHs can neutralize effectively HC of other BoNT/A subtypes. This study aimed to generate HC domains of all eight BoNT/A subtypes and to screen for VHHs with broad reactivity against these domains. Methods HC domains of BoNT/A1-A8 were recombinantly produced in Escherichia coli. The bont/HCA1 fragment was amplified from sludge sample and cloned into pET45b vector by Gibson assembly. Expression vectors for HC domains of BoNT/A2-A8 were derived from pET45b-HCA1 by site-directed mutagenesis and/or in-house gene synthesis. Similarly, VHHs were synthesized and cloned into pET22b vector. Recombinant protein were purified by Ni-NTA spin columns and analyzed by SDS-PAGE. ELISA was used to confirm the antigenicity of HC domains and to evaluate the reactivity of VHHs to these domains. Results SDS-PAGE analysis and ELISA results with commercial polyclonal antibody demonstrated the HC domains of all eight BoNT/A subtypes were correctly produced. ELISA results using a VHH panel indicated that, apart from ciA-C2, a well-characterized VHH specific for HC of BoNT/A1, two new VHHs were found to recognize the HC domains of all BoNT/A subtypes, of which VHH-A3 displayed EC50 values for these domains close to those of ciA-C2. Conclusion This study provided a resource to comprehensively identify antitoxins conferring broad protection against BoNT/A. READ ALL READ LESS Keywords botulinum neurotoxins, botulism, receptor-binding domain, HC, recombinant proteins, neutralization, VHH, nanobody, antitoxin Corresponding Author(s) Hoa Quang Le ( [email protected] ) Close Corresponding author: Hoa Quang Le Competing interests: No competing interests were disclosed. Grant information: Research reported in this publication was supported by Hanoi University of Science and Technology [Grant number: T2022-TĐ-002] The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Quynh Pham N et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Quynh Pham N, Mai TT, Dang TBA et al. Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.12688/f1000research.160607.1 ) First published: 05 Feb 2025, 14 :163 ( https://doi.org/10.12688/f1000research.160607.1 ) Latest published: 05 Feb 2025, 14 :163 ( https://doi.org/10.12688/f1000research.160607.1 ) Introduction Botulinum neurotoxins (BoNTs) are the most toxic substances known to the humankind with lethal dose values in the range of nanogram per kilogram body weight scale. 1 Most commonly produced by Clostridium botulinum , these toxins are proteins composed of a 50-kDa light chain (LC) linked to a 100-kDa heavy chain (HC) via a disulfide bond. The LC fragment contains a zinc-protease specific domain, whereas the HC consists of an N-terminal translocation domain (H N ) and a C-terminal receptor-binding domain (H C ). 2 The mode of action of BoNTs includes three steps. In the first place, the H C domain of BoNTs bind specifically to peripheral nerve terminals via polysialoganglioside and synaptic vesicle receptors. Subsequently, BoNTs enter into nerve terminals by endocytosis. Under acidic conditions, the H N domain translocates the LC into the nerve terminal cytosol where the latter cleaves one of three soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) that are involved in neurotransmitter release, thereby causing nerve paralysis. 2 BoNTs are traditionally classified into seven serotypes (BoNT/A–BoNT/G), of which BoNT/A represents a great threat to humans because of its most potent toxicity with the longest duration of paralysis. 3 In addition, BoNT/A is categorized into eight subtypes (BoNT/A1–A8) with significant levels of protein sequence differences (up to 12.3%), 4 which complicates the development of a broadly protective monoclonal antitoxin. Botulinum intoxication is fatal in 5–10% of cases and requires early treatment with antitoxin. Currently, the only available antitoxins for botulism are the heptavalent botulinum antitoxin (HBAT), which contains fragments of immunoglobulins from horses vaccinated with all seven traditionnal serotypes of BoNTs, and BabyBIG, which consists of polyclonal antibodies from human immunized with recombinant botulinum vaccine for serotypes A and B. However, these antitoxin types have limitations due to adverse side effects, limited availability and exorbitant cost. 5 To overcome these drawbacks, neutralizing monoclonal antibodies (mAbs) against BoNT/A, B, E, and F, which cause human botulism, have been generated. 6 – 9 It has been shown that a combination of several mAbs is required to efficiently neutralize subtypes belonging to a BoNT serotype. 8 , 9 Another strategy to combat botulism is to develop camelid single-domain antibodies (sdAbs), also referred to as VHHs or nanobodies, that can neutralize BoNTs via the interactions with the functional domains of the toxins. Several VHHs with high affinity against H C domain of a BoNT/A subtype have been shown to display protective activity when challenged with the same toxin in animal models. 10 – 12 However, it remains unclear if these VHHs can neutralize effectively other subtypes of BoNT/A. This is because of the lack of a comprehensive toxin resource available for all subtypes of BoNT/A. Here, for the first time, the generation of recombinant H C domains of BoNT/A1-A8 was described. These proteins were then used to characterize a panel of VHHs targeting BoNT/A1 with unknown binding sites in order to identify novel VHHs with broad reactivity against all subtypes of BoNT/A. Methods Materials Clostridium botulinum genomic DNA carrying the bont/A1 gene was extracted from an enrichment of a sludge sample from Hanoi, Vietnam. Oligonucleotides were synthesized by Macrogen (Korea). Expression vectors used in this study comprised pET-45b and pET-22b (Novagen, cat. number 71327-3 and 69744-3, respectively). Hosts used for recombinant protein production were E. coli BL21(DE3) and E. coli Rosetta™ 2(DE3) (Novagen, cat. number 69450 and 71397, respectively). All other reagents were from Thermo Scientific™, New England Biolabs, Merck, Qiagen, Tetracore, Immunology Consultants Laboratory, Vazyme, and Himedia unless otherwise stated. Construction of expression vectors for BoNT/A1-A8 production The bont/H C A1 fragment (residues 871-1296 of BoNT/A1) was amplified from extracted C. botulinum DNA and cloned into pET45b vector with an N-terminal His-tag by NEBuilder ® HiFi DNA Assembly Master Mix (New England Biolabs, cat. number E2621L). 13 Expression vectors for H C domains of BoNT/A2-A8 ( Table 1 ) were derived from pET45b-H C A1 by site-directed mutagenesis and/or in-house gene synthesis. 14 – 20 All vectors were sent for sequencing to verify the accuracy of the constructs. Sequencing results can be found in the Sequence Read Archive under accession number PRJNA1206782. Table 1. Accession numbers of BoNT/A subtypes used in this study. BoNT/A subtypes Accession numbers A2 WP_061323842.1 A3 WP_012301031.1 A4 WP_012720356.1 A5 WP_078992015.1 A6 ACW83608 A7 AFV13854 A8 AJA05787 Construction of expression vectors for VHH production Genes encoding VHHs (A1, A3, A16, A17, A18, and ciA-C2) from previously reported studies 10 , 21 were codon-optimized for expression in E. coli and synthesized by Genscript. These genes were inserted into the pET22b expression vector which was modified to carry a FLAG tag (DYKDDDDK) at the C-terminal end for detection. 22 All vectors were sent for sequencing to verify the accuracy of the constructs. Sequencing results can be found in the Sequence Read Archive under accession number PRJNA1206786. Expression and purification of recombinant proteins H C domains of BoNT/A1-A8 and VHHs were produced in E. coli Rosetta 2(DE3) and E. coli BL21(DE3), respectively. Bacteria carrying expression vectors were cultured at 37°C in LB medium (Himedia, cat. number 81254) supplemented with appropriate selecting antibiotics until OD 600 ~ 0.6 – 0.8 and then induced with 0.5 mM IPTG (Thermo Scientific, cat. number R0392) at 20°C for 12 hours. The His tagged recombinant proteins were purified by affinity chromatography using Ni-NTA spin columns (Qiagen, cat. number 31014) under native conditions according to the manufacturer’s instructions and then analyzed by SDS-PAGE. ELISA assays The reactivity of VHHs and commercial Rabbit Anti-Botulinum Toxin A and B IgG (Tetracore, cat. number TC-7007-001) against purified recombinant H C domains of BoNT/A1-A8 were tested by ELISA. High Bind Stripwell™ Microplates (Corning, cat. number 07-200-24) were coated with 2 μg/mL recombinant antigens in carbonate buffer (Thermo Scientific, cat. number CB01100) at 4°C overnight. Plates were then blocked by 1% bovine serum albumin (Sigma-Aldrich, cat. number A2058) for 2 h at 37°C. VHHs in serial dilutions and commercial polyclonal antibody were added to the wells and incubated for 2 h at 37°C. After washing, anti-DYKDDDDK (Flag) Antibody Rabbit - HRP Conjugated (for VHHs) and HRP Conjugated Goat anti-Rabbit IgG h+l Antibody (for polyclonal antibody) (Immunology Consultants Laboratory, cat. number RFLG-45P-Z and GGHL-15P, respectively) were added and incubated for 1 h at 37 °C. Plates were then washed six times with PBST (Sigma-Aldrich, cat. number P3563) and reactions were developed with TMB substrate (Abcam, cat. number AB171523) and read at 450 nm. EC 50 values were calculated via non-linear regression analysis using GraphPad Prism. Results and discussion Due to the extremely high toxicity of BoNTs, the development of antitoxins against them is of major interest for therapeutic applications. The target for antitoxin can be each of the three structural domains of BoNTs: (i) H C responsible for receptor binding; (ii) H N for toxin translocation; and (iii) LC for cleavage of SNARE proteins. Among these domains, the H C fragment is the target of choice for the generation of antidote to BoNT intoxication, 23 as well as for the development of vaccines 24 and for intracellular delivery of cargo molecules specifically to neurons. 25 In the present study, we focused on the production of the H C fragments of all subtypes of BoNT/A because of its extremely high toxicity, long persistence, and high sequence divergence among subtypes. However, only bont/A1 gene was available in our group. Consequently, we opted for an approach that involved both site-directed mutagenesis from H C A1 construct and in-house gene synthesis to generate expression vectors for H C domains of BoNT/A2-A8. 14 – 20 These recombinant proteins were expressed in the E. coli Rosetta 2(DE3) and purified by nickel affinity chromatography. SDS-PAGE analysis showed that only one band was observed for all purified samples at the expected molecular mass of 50 kDa ( Figure 1 ), suggesting that H C fragments of BoNT/A1-A8 were successfully expressed and prepared. The purified proteins were then evaluated by ELISA for antigenicity using the Rabbit Anti-Botulinum Toxin A and B IgG. All samples were recognized by the commercial polyclonal antibody, 26 which indicated that the receptor binding domains of all BoNT/A subtypes were correctly expressed in E. coli Rosetta 2(DE3). Figure 1. SDS-PAGE analysis of purified recombinant BoNT/A H C domains. To our knowledge, only three VHHs neutralizing BoNT/A by binding to the H C domain have been described in the literature, 10 – 12 of which ciA-C2 have been extensively characterized for the inhibition mechanism on the BoNT/A1. 11 In a separate report, 18 VHHs have been identified for their specific recognition of BoNT/A1 21 but their binding sites were unknown. We therefore performed a systematic screening of a panel of five most promising VHHs from this report and ciA-C2, on the H C fragments of BoNT/A1-A8 by ELISA. All the VHHs were successfully expressed and purified. 22 Screening results clearly indicated that apart from ciA-C2, two nanobodies (VHH-A1 and VHH-A3) could recognize the H C domains of all BoNT/A subtypes with VHH-A3 exhibiting significantly higher affinity than VHH-A1. 27 Consequently, only ciA-C2 and VHH-A3 were characterized in subsequent experiments. According to the EC 50 values calculated using ELISA ( Table 2 ), 28 both ciA-C2 and VHH-A3 exhibited the highest affinity for the H C A1 (EC 50 = 11.0 and 24.0 nM respectively). This is not surprising, because these two VHHs were generated based on the selection with BoNT/A1. 11 , 21 VHH-A3 displayed an intermediate affinity for H C A4 (EC 50 = 46.0 nM) and comparably low affinity for the remaining subtypes. In comparison to ciA-C2, VHH-A3 displayed similar EC 50 values for H C domains of BoNT/A2, A3, A5, A6, A7, and A8. Concerning ciA-C2, the binding mechanism of this VHH to H C A1 involves a cation-π interaction and multiple hydrogen bonds between CDR1 and residues K289, N318 and D419 of H C A1. In addition, CDR2, CDR3, FR2, FR3, and FR4 of ciA-C2 also participate in the binding to H C A1 through hydrogen bonds with residues T193, H194, Y242, T276, E423 and a hydrophobic interaction with P425 of the domain. 11 Consistent with these structural observations, the affinity of ciA-C2 was least affected for H C A4 (EC 50 = 26.2 nM) with only a T193 to P193 replacement, 16 whereas it was most affected for H C A2, H C A3 and H C A8 (EC 50 ≥ 80.4 nM) containing three major substitutions T193P, H194R, and P425S. 14 , 15 , 20 These data underline the importance of the structural studies of VHHs in order to generate antitoxins with a broad protection to BoNTs. Furthermore, considering the sequence divergence among H C domains of BoNTs 29 and most studies so far use BoNT/A1 as the selection agent to generate VHHs, it would be of interest to include a divergent H C domain, for instance, H C A2, H C A3 or H C A8, during the selection steps in order to obtain VHHs having high affinity against these domains. Similarly, these recombinant fragments could be combined with H C A1 for the development of vaccines or polyclonal antitoxins with broad potency compared to conventional approach using only one BoNT/A subtype for immunization. Table 2. EC 50 values of ciA-C2 and VHH-A3 against H C domains of BoNT/A1-A8. BoNT/A subtypes ciA-C2 VHH-A3 A1 11.0 24.0 A2 80.4 93.5 A3 83.8 88.9 A4 26.2 46.0 A5 51.4 67.6 A6 77.5 97.1 A7 62.3 73.3 A8 109.3 98.2 In summary, this study provided recombinant H C domains of all BoNT/A subtypes, which could be used for the development of antitoxins and vaccines against BoNTs. This study also identified two new nanobodies, VHH-A1 and VHH-A3, capable of binding to all BoNT/A H C domains. However, one question remains unsolved in this study, whether the VHH-A1, VHH-A3 and ciA-C2 would bind to a distinct, non-overlapping epitope. Further research is on-going to resolve this question and to improve neutralizing activity of ciA-C2 through the generation of heterodimers. Ethics and consent Ethical approval and consent were not required. Data availability Underlying data Figshare: Raw data of ELISA results for reactivity of H C A1-H C A8 to VHHs and Rabbit Anti-Botulinum Toxin A and B IgG; for determination of EC 50 values of ciA-C2 and VHH-A3 against HCA1-HCA8. Doi: https://doi.org/10.6084/m9.figshare.28171994 . 30 This project contains the following underlying data: • ELISA raw data.xlsx Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). Dataset from NCBI Sequence Read Archive: Sequencing results of H C -BoNT/A subtypes. Accession number PRJNA1206782; https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA1206782 . Dataset from NCBI Sequence Read Archive: Sequencing results of VHHs against H C -BoNT/A subtypes. Accession number PRJNA1206786; https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA1206786 . Extended data Figshare: Supplement I for “Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity”. Figshare: Construction of pET45b-HCA1 plasmid. Doi: https://doi.org/10.6084/m9.figshare.28159514.v1 . 13 This project contains the following extended data: • Supplement I.pdf Figshare: Construction of pET45b-HCA2 plasmid. https://doi.org/10.6084/m9.figshare.28159544.v1 . 14 This project contains the following extended data: • Supplement II.pdf Figshare: Construction of pET45b-HCA3 plasmid. https://doi.org/10.6084/m9.figshare.28159553.v1 . 15 This project contains the following extended data: • Supplement III.pdf Figshare: Construction of pET45b-HCA4 plasmid. https://doi.org/10.6084/m9.figshare.28159559.v1 . 16 This project contains the following extended data: • Supplement IV.pdf Figshare: Construction of pET45b-HCA5 plasmid. https://doi.org/10.6084/m9.figshare.28159574.v1 . 17 This project contains the following extended data: • Supplement V.pdf Figshare: Construction of pET45b-HCA6 plasmid. https://doi.org/10.6084/m9.figshare.28159586.v1 . 18 This project contains the following extended data: • Supplement VI.pdf Figshare: Construction of pET45b-HCA7 plasmid. https://doi.org/10.6084/m9.figshare.28159592.v1 . 19 This project contains the following extended data: • Supplement VII.pdf Figshare: Construction of pET45b-HCA8 plasmid. https://doi.org/10.6084/m9.figshare.28159598.v1 . 20 This project contains the following extended data: • Supplement VIII.pdf Figshare: Construction of pET22b-VHH plasmids. https://doi.org/10.6084/m9.figshare.28159622.v1 . 22 This project contains the following extended data: • Supplement IX.pdf Figshare: Reactivity of HCA1-HCA8 to the Rabbit Anti-Botulinum Toxin A and B IgG. https://doi.org/10.6084/m9.figshare.28159754.v1 . 26 This project contains the following extended data: • Supplement X.pdf Figshare: Reactivity of HCA1-HCA8 to VHHs. https://doi.org/10.6084/m9.figshare.28159982.v1 . 27 This project contains the following extended data: • Supplement XI.pdf Figshare: Determination of EC 50 values of ciA-C2 and VHH-A3 against HCA1-HCA8. https://doi.org/10.6084/m9.figshare.28160042.v1 . 28 This project contains the following extended data: • Supplement XII.pdf Figshare: The overall amino acid sequence identity among BoNT/A subtypes using, A1 as the benchmark. https://doi.org/10.6084/m9.figshare.28160051.v1 29 This project contains the following extended data: • Supplement XIII.pdf Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgements We would like to thank Anh Minh Nguyen, Vinh Ba Tran, Anh Thao Nguyen, Duong Thuy Le Nguyen, and Chi Linh Nguyen, School of Chemistry and Life Sciences, Hanoi University of Science and Technology, for their support during the production of recombinant proteins. References 1. Rossetto O, Montecucco C: Tables of toxicity of botulinum and tetanus neurotoxins. Toxins (Basel). 2019; 11 (12). PubMed Abstract | Publisher Full Text | Free Full Text 2. Rossetto O, Pirazzini M, Montecucco C: Botulinum neurotoxins: Genetic, structural and mechanistic insights. Nat. Rev. Microbiol. 2014; 12 (8): 535–549. PubMed Abstract | Publisher Full Text 3. Gregg BM, Matsumura T, Wentz TG, et al. : Botulinum neurotoxin X lacks potency in mice and in human neurons. MBio. 2024; 15 (3): e0310623. PubMed Abstract | Publisher Full Text | Free Full Text 4. Kull S, Schulz KM, Strotmeier JW, et al. : Isolation and functional characterization of the novel clostridium botulinum neurotoxin A8 subtype. PLoS One. 2015; 10 (2): e0116381. PubMed Abstract | Publisher Full Text | Free Full Text 5. Rasetti-Escargueil C, Popoff MR: Antibodies and vaccines against botulinum toxins: Available measures and novel approaches. Toxins (Basel). 2019; 11 (9). PubMed Abstract | Publisher Full Text | Free Full Text 6. Fan Y, Geren IN, Dong J, et al. : Monoclonal antibodies targeting the alpha-exosite of botulinum neurotoxin serotype/a inhibit catalytic activity. PLoS One. 2015; 10 (8): e0135306. PubMed Abstract | Publisher Full Text | Free Full Text 7. Fan Y, Dong J, Lou J, et al. : Monoclonal antibodies that inhibit the proteolytic activity of botulinum neurotoxin serotype/B. Toxins (Basel). 2015; 7 (9): 3405–3423. PubMed Abstract | Publisher Full Text | Free Full Text 8. Garcia-Rodriguez C, Razai A, Geren IN, et al. : A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype E subtypes. Toxins (Basel). 2018; 10 (3). PubMed Abstract | Publisher Full Text | Free Full Text 9. Fan Y, Garcia-Rodriguez C, Lou J, et al. : A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes. PLoS One. 2017; 12 (3): e0174187. PubMed Abstract | Publisher Full Text | Free Full Text 10. Mukherjee J, Tremblay JM, Leysath CE, et al. : A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model. PLoS One. 2012; 7 (1): e29941. PubMed Abstract | Publisher Full Text | Free Full Text 11. Yao G, Lam KH, Weisemann J, et al. : A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding. Sci. Rep. 2017; 7 (1): 7438. PubMed Abstract | Publisher Full Text | Free Full Text 12. Godakova SA, Noskov AN, Vinogradova ID, et al. : Camelid VHHs fused to human fc fragments provide long term protection against botulinum neurotoxin a in mice. Toxins (Basel). 2019; 11 (8). PubMed Abstract | Publisher Full Text | Free Full Text 13. Pham N: Construction of pET45b-HCA1 plasmid.2025. Accessed January 8, 2025. Publisher Full Text 14. Pham N: Construction of pET45b-HCA2 plasmid.2025. Accessed January 8, 2025. Publisher Full Text 15. Pham N: Construction of pET45b-HCA3 plasmid.2025. Accessed January 8, 2025. Publisher Full Text 16. Pham N: Construction of pET45b-HCA4 plasmid.2025. Accessed January 8, 2025. Publisher Full Text 17. Pham N: Construction of pET45b-HCA5 plasmid.2025. Accessed January 8, 2025. Publisher Full Text 18. Pham N: Construction of pET45b-HCA6 plasmid.2025. Accessed January 8, 2025. Publisher Full Text 19. Pham N: Construction of pET45b-HCA7 plasmid.2025. Accessed January 8, 2025. Publisher Full Text 20. Pham N: Construction of pET45b-HCA8 plasmid.2025. Accessed January 8, 2025. Publisher Full Text 21. Conway JO, Sherwood LJ, Collazo MT, et al. : Llama single domain antibodies specific for the 7 botulinum neurotoxin serotypes as heptaplex immunoreagents. PLoS One. 2010; 5 (1): e8818. PubMed Abstract | Publisher Full Text | Free Full Text 22. Pham N: Construction of pET22b-VHH plasmids.2025. Accessed January 8, 2025. Publisher Full Text 23. Shi DY, Lu JS, Mao YY, et al. : Characterization of a novel tetravalent botulism antitoxin based on receptor-binding domain of BoNTs. Appl. Microbiol. Biotechnol. 2023; 107 (10): 3205–3216. PubMed Abstract | Publisher Full Text | Free Full Text 24. Baldwin MR, Tepp WH, Przedpelski A, et al. : Subunit vaccine against the seven serotypes of botulism. Infect. Immun. 2008; 76 (3): 1314–1318. PubMed Abstract | Publisher Full Text | Free Full Text 25. Miyashita SI, Zhang J, Zhang S, et al. : Delivery of single-domain antibodies into neurons using a chimeric toxin–based platform is therapeutic in mouse models of botulism. Sci. Transl. Med. 2021; 13 (575). PubMed Abstract | Publisher Full Text 26. Pham N: Reactivity of HCA1-HCA8 to the Rabbit Anti-Botulinum Toxin A and B IgG.2025. Accessed January 8, 2025. Publisher Full Text 27. Pham N: Reactivity of HCA1-HCA8 to VHHs.2025. Accessed January 8, 2025. Publisher Full Text 28. Pham N: Determination of EC50 values of ciA-C2 and VHH-A3 against HCA1-HCA8.2025. Accessed January 8, 2025. Publisher Full Text 29. Pham N: The overall amino acid sequence identity among BoNT/A subtypes using A1 as the benchmark.2025. Accessed January 8, 2025. Publisher Full Text 30. Pham N: Raw data for “Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity”. [Dataset]. figshare. 2025 [cited 2025Jan10]. Publisher Full Text Comments on this article Comments (1) Version 1 VERSION 1 PUBLISHED 05 Feb 2025 Reader Comment 07 Mar 2025 Kyle Gregory , Life Sciences, University of Bath Faculty of Science, Bath, UK 07 Mar 2025 Reader Comment An interesting article. The authors state 'SDS-PAGE analysis showed that only one band was observed for all purified samples at the expected molecular mass of 50 kDa', however, based ... Continue reading An interesting article. The authors state 'SDS-PAGE analysis showed that only one band was observed for all purified samples at the expected molecular mass of 50 kDa', however, based on the ladder the protein is between 135 kDa and 100 kDa? Is the correct marker displayed? An interesting article. The authors state 'SDS-PAGE analysis showed that only one band was observed for all purified samples at the expected molecular mass of 50 kDa', however, based on the ladder the protein is between 135 kDa and 100 kDa? Is the correct marker displayed? Competing Interests: No competing interests were disclosed. Close Report a concern Comment ADD YOUR COMMENT Author details Author details 1 School of Chemistry and Life Sciences, Hanoi University of Science and Technology, Hanoi, Vietnam 2 High School for the Gifted in Natural Sciences, Vietnam National University, Hanoi, 120558, Vietnam 3 Hanoi Medical University, Hanoi, 11521, Vietnam 4 Center for Gene and Protein Research, Hanoi Medical University, Hanoi, 11521, Vietnam Nga Quynh Pham Roles: Data Curation, Formal Analysis, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Tam Trang Mai Roles: Investigation, Writing – Original Draft Preparation Tran Bao Anh Dang Roles: Investigation, Writing – Original Draft Preparation Ly Huong Tran Roles: Data Curation, Formal Analysis, Supervision, Writing – Review & Editing Quynh Mai Vu Roles: Data Curation, Formal Analysis, Supervision, Writing – Review & Editing Chien Trong Nguyen Roles: Data Curation, Formal Analysis, Supervision, Writing – Review & Editing Anh Thi Phuong Tran Roles: Data Curation, Formal Analysis, Supervision, Writing – Review & Editing Tran Nhat Minh Dang Roles: Investigation, Writing – Original Draft Preparation Van Anh Tran Roles: Investigation, Writing – Original Draft Preparation Thinh Huy Tran Roles: Investigation Van Khanh Tran Roles: Conceptualization, Investigation, Supervision Hoa Quang Le Roles: Conceptualization, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information Research reported in this publication was supported by Hanoi University of Science and Technology [Grant number: T2022-TĐ-002] The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (1) version 1 Published: 05 Feb 2025, 14:163 https://doi.org/10.12688/f1000research.160607.1 Copyright © 2025 Quynh Pham N et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Quynh Pham N, Mai TT, Dang TBA et al. Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.12688/f1000research.160607.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 05 Feb 2025 Views 0 Cite How to cite this report: Shoemaker CB. Reviewer Report For: Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.5256/f1000research.176528.r366729 ) The direct URL for this report is: https://f1000research.com/articles/14-163/v1#referee-response-366729 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 04 Mar 2025 Charles B Shoemaker , Tufts University, Medford, Massachusetts, USA Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176528.r366729 In this manuscript, the author describe the creation of a set of Botulinum neurotoxin serotype type A (BoNT/A) heavy chain domain (AHc) representing each of the eight reported BoNT/A subtype natural variants. They use this resource to screen the AHc ... Continue reading READ ALL In this manuscript, the author describe the creation of a set of Botulinum neurotoxin serotype type A (BoNT/A) heavy chain domain (AHc) representing each of the eight reported BoNT/A subtype natural variants. They use this resource to screen the AHc subtype specificity of several single-domain antibodies (sdAbs) that are reported to bind to AHc. Comprehensive methods and construction details are provided and the work appears well performed. General comments: The authors suggest the primary accomplishment is their creation of “a resource to comprehensively identify antitoxins conferring broad protection against BoNT/A” which seems a substantial overstatement as the resource is simply a panel of expressed receptor-binding domain subunits of known amino acid sequence. Most capable labs could create a similar set by ordering and cloning a panel of synthetic coding DNA into an expression vector. Furthermore, identifying broad subtype antibody binding to AHc does not demonstrate their antitoxin potential as the VHH binding site must also be at a site that interferes with toxin binding which requires neutralization studies. Authors claim that the ciA-C2 specificity for AHc is unknown though information on subtype specificity was provided in the manuscript cited as the source of this information. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Biotechnology.mmunotherapeutic discovery, engineering and development. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Shoemaker CB. Reviewer Report For: Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.5256/f1000research.176528.r366729 ) The direct URL for this report is: https://f1000research.com/articles/14-163/v1#referee-response-366729 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Lou J. Reviewer Report For: Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.5256/f1000research.176528.r366733 ) The direct URL for this report is: https://f1000research.com/articles/14-163/v1#referee-response-366733 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 03 Mar 2025 Jianlong Lou , University of California San Francisco, San Francisco, California, USA Approved VIEWS 0 https://doi.org/10.5256/f1000research.176528.r366733 "Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity" is a well-written original research report with clear citation of some current literature in the BoNT countermeasure development ... Continue reading READ ALL "Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity" is a well-written original research report with clear citation of some current literature in the BoNT countermeasure development field. The work has some academic merit and potential field application, it is accurately presented with sufficient details, so other researchers should be able to replicate the experiments if desired. The relative simple software generated statistical analysis and its interpretation seems to be appropriate. And the conclusions drawn for in vitro activity were adequately supported by the results, but the author should not expand or imply the potential application for in vivo anti-toxin countermeasure efficiency without solid animal test data. In vitro binding data and in vivo biological function data are somewhat related but of different dimensions. The authors also need to explain the size of HC showing in Figure 1. Why does it look more like 100KD instead of 50KD if the molecular marker on the left side is correct? Although the designed fragment with 426 AA should have the molecular size close to 50KD? Furthermore, Some publications such as the ones added below that discusses VHH specific for BoNT/A-LC or clinical trial using antibody based counter measure for BoNT/A will help reader to grasp the status and current situation of antitoxin development better. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes References 1. Dong J, Thompson AA, Fan Y, Lou J, et al.: A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region. J Mol Biol . 2010; 397 (4): 1106-18 PubMed Abstract | Publisher Full Text 2. Nayak SU, Griffiss JM, McKenzie R, Fuchs EJ, et al.: Safety and pharmacokinetics of XOMA 3AB, a novel mixture of three monoclonal antibodies against botulinum toxin A. Antimicrob Agents Chemother . 2014; 58 (9): 5047-53 PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Human antibody and animal based antibody engineering for BoNT and other antigens. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Lou J. Reviewer Report For: Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.5256/f1000research.176528.r366733 ) The direct URL for this report is: https://f1000research.com/articles/14-163/v1#referee-response-366733 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Esmagambetov IB. Reviewer Report For: Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.5256/f1000research.176528.r366732 ) The direct URL for this report is: https://f1000research.com/articles/14-163/v1#referee-response-366732 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 28 Feb 2025 Ilias B Esmagambetov , Gamaleya Research Center for Epidemiology and Microbiology, Moscow, Russian Federation Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176528.r366732 In general, the article is interesting for the target audience. However, the article contains very little data. In my opinion, it is not entirely correct to draw conclusions based only on ELISA data. Characterization of the interaction between the VHH ... Continue reading READ ALL In general, the article is interesting for the target audience. However, the article contains very little data. In my opinion, it is not entirely correct to draw conclusions based only on ELISA data. Characterization of the interaction between the VHH and HC domain of BoNT/A using SPR or BLI methods would be very useful. I would recommend that the authors add at least one alternative method for characterizing the interaction of VHH with HC domain of BoNT/A. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: mAbs, VHH, recombinant proteins, Adenoviral vectors, rAAV, gene therapy. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Esmagambetov IB. Reviewer Report For: Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.5256/f1000research.176528.r366732 ) The direct URL for this report is: https://f1000research.com/articles/14-163/v1#referee-response-366732 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Comments on this article Comments (1) Version 1 VERSION 1 PUBLISHED 05 Feb 2025 Reader Comment 07 Mar 2025 Kyle Gregory , Life Sciences, University of Bath Faculty of Science, Bath, UK 07 Mar 2025 Reader Comment An interesting article. The authors state 'SDS-PAGE analysis showed that only one band was observed for all purified samples at the expected molecular mass of 50 kDa', however, based ... Continue reading An interesting article. The authors state 'SDS-PAGE analysis showed that only one band was observed for all purified samples at the expected molecular mass of 50 kDa', however, based on the ladder the protein is between 135 kDa and 100 kDa? Is the correct marker displayed? An interesting article. The authors state 'SDS-PAGE analysis showed that only one band was observed for all purified samples at the expected molecular mass of 50 kDa', however, based on the ladder the protein is between 135 kDa and 100 kDa? Is the correct marker displayed? Competing Interests: No competing interests were disclosed. Close Report a concern Comment ADD YOUR COMMENT keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 1 05 Feb 25 read read read Ilias B Esmagambetov , Gamaleya Research Center for Epidemiology and Microbiology, Moscow, Russian Federation Jianlong Lou , University of California San Francisco, San Francisco, USA Charles B Shoemaker , Tufts University, Medford, USA Comments on this article All Comments (1) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Shoemaker C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 04 Mar 2025 | for Version 1 Charles B Shoemaker , Tufts University, Medford, Massachusetts, USA 0 Views copyright © 2025 Shoemaker C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In this manuscript, the author describe the creation of a set of Botulinum neurotoxin serotype type A (BoNT/A) heavy chain domain (AHc) representing each of the eight reported BoNT/A subtype natural variants. They use this resource to screen the AHc subtype specificity of several single-domain antibodies (sdAbs) that are reported to bind to AHc. Comprehensive methods and construction details are provided and the work appears well performed. General comments: The authors suggest the primary accomplishment is their creation of “a resource to comprehensively identify antitoxins conferring broad protection against BoNT/A” which seems a substantial overstatement as the resource is simply a panel of expressed receptor-binding domain subunits of known amino acid sequence. Most capable labs could create a similar set by ordering and cloning a panel of synthetic coding DNA into an expression vector. Furthermore, identifying broad subtype antibody binding to AHc does not demonstrate their antitoxin potential as the VHH binding site must also be at a site that interferes with toxin binding which requires neutralization studies. Authors claim that the ciA-C2 specificity for AHc is unknown though information on subtype specificity was provided in the manuscript cited as the source of this information. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Biotechnology.mmunotherapeutic discovery, engineering and development. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Shoemaker CB. Peer Review Report For: Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.5256/f1000research.176528.r366729) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-163/v1#referee-response-366729 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Lou J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 03 Mar 2025 | for Version 1 Jianlong Lou , University of California San Francisco, San Francisco, California, USA 0 Views copyright © 2025 Lou J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions "Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity" is a well-written original research report with clear citation of some current literature in the BoNT countermeasure development field. The work has some academic merit and potential field application, it is accurately presented with sufficient details, so other researchers should be able to replicate the experiments if desired. The relative simple software generated statistical analysis and its interpretation seems to be appropriate. And the conclusions drawn for in vitro activity were adequately supported by the results, but the author should not expand or imply the potential application for in vivo anti-toxin countermeasure efficiency without solid animal test data. In vitro binding data and in vivo biological function data are somewhat related but of different dimensions. The authors also need to explain the size of HC showing in Figure 1. Why does it look more like 100KD instead of 50KD if the molecular marker on the left side is correct? Although the designed fragment with 426 AA should have the molecular size close to 50KD? Furthermore, Some publications such as the ones added below that discusses VHH specific for BoNT/A-LC or clinical trial using antibody based counter measure for BoNT/A will help reader to grasp the status and current situation of antitoxin development better. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes References 1. Dong J, Thompson AA, Fan Y, Lou J, et al.: A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region. J Mol Biol . 2010; 397 (4): 1106-18 PubMed Abstract | Publisher Full Text 2. Nayak SU, Griffiss JM, McKenzie R, Fuchs EJ, et al.: Safety and pharmacokinetics of XOMA 3AB, a novel mixture of three monoclonal antibodies against botulinum toxin A. Antimicrob Agents Chemother . 2014; 58 (9): 5047-53 PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Human antibody and animal based antibody engineering for BoNT and other antigens. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Lou J. Peer Review Report For: Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.5256/f1000research.176528.r366733) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-163/v1#referee-response-366733 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Esmagambetov I. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 28 Feb 2025 | for Version 1 Ilias B Esmagambetov , Gamaleya Research Center for Epidemiology and Microbiology, Moscow, Russian Federation 0 Views copyright © 2025 Esmagambetov I. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In general, the article is interesting for the target audience. However, the article contains very little data. In my opinion, it is not entirely correct to draw conclusions based only on ELISA data. Characterization of the interaction between the VHH and HC domain of BoNT/A using SPR or BLI methods would be very useful. I would recommend that the authors add at least one alternative method for characterizing the interaction of VHH with HC domain of BoNT/A. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise mAbs, VHH, recombinant proteins, Adenoviral vectors, rAAV, gene therapy. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Esmagambetov IB. Peer Review Report For: Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :163 ( https://doi.org/10.5256/f1000research.176528.r366732) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-163/v1#referee-response-366732 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list: Examples of 'Non-Financial Competing Interests' Within the past 4 years, you have held joint grants, published or collaborated with any of the authors of the selected paper. You have a close personal relationship (e.g. parent, spouse, sibling, or domestic partner) with any of the authors. You are a close professional associate of any of the authors (e.g. scientific mentor, recent student). You work at the same institute as any of the authors. You hope/expect to benefit (e.g. favour or employment) as a result of your submission. You are an Editor for the journal in which the article is published. Examples of 'Financial Competing Interests' You expect to receive, or in the past 4 years have received, any of the following from any commercial organisation that may gain financially from your submission: a salary, fees, funding, reimbursements. You expect to receive, or in the past 4 years have received, shared grant support or other funding with any of the authors. You hold, or are currently applying for, any patents or significant stocks/shares relating to the subject matter of the paper you are commenting on. Stay Updated Sign up for content alerts and receive a weekly or monthly email with all newly published articles Register with F1000Research Already registered? Sign in Not now, thanks close PLEASE NOTE If you are an AUTHOR of this article, please check that you signed in with the account associated with this article otherwise we cannot automatically identify your role as an author and your comment will be labelled as a “User Comment”. If you are a REVIEWER of this article, please check that you have signed in with the account associated with this article and then go to your account to submit your report, please do not post your review here. If you do not have access to your original account, please contact us . All commenters must hold a formal affiliation as per our Policies . The information that you give us will be displayed next to your comment. User comments must be in English, comprehensible and relevant to the article under discussion. We reserve the right to remove any comments that we consider to be inappropriate, offensive or otherwise in breach of the User Comment Terms and Conditions . Commenters must not use a comment for personal attacks. When criticisms of the article are based on unpublished data, the data should be made available. I accept the User Comment Terms and Conditions Please confirm that you accept the User Comment Terms and Conditions. Affiliation ✕ refresh Please enter your institution. Note: To add your institution or organisation, start typing the name and then select the correct name from the list. Where applicable, the name will appear in both the original language and in English. Do not paste in the name. If the name does not appear in the drop-down list, we will display the information you have entered. ✕ refresh Country/Region * USA UK Canada China France Germany Afghanistan Aland Islands Albania Algeria American Samoa Andorra Angola Anguilla Antarctica Antigua and Barbuda Argentina Armenia Aruba Australia Austria Azerbaijan Bahamas Bahrain Bangladesh Barbados Belarus Belgium Belize Benin Bermuda Bhutan Bolivia Bosnia and Herzegovina Botswana Bouvet Island Brazil British Indian Ocean Territory British Virgin Islands Brunei Bulgaria Burkina Faso Burundi Cambodia Cameroon Canada Cape Verde Cayman Islands Central African Republic Chad Chile China Christmas Island Cocos (Keeling) Islands Colombia Comoros Congo Cook Islands Costa Rica Cote d'Ivoire Croatia Cuba Cyprus Czech Republic Democratic Republic of the Congo Denmark Djibouti Dominica Dominican Republic Ecuador Egypt El Salvador Equatorial Guinea Eritrea Estonia Ethiopia Falkland Islands Faroe Islands Federated States of Micronesia Fiji Finland France French Guiana French Polynesia French Southern Territories Gabon Georgia Germany Ghana Gibraltar Greece Greenland Grenada Guadeloupe Guam Guatemala Guernsey Guinea Guinea-Bissau Guyana Haiti Heard Island and Mcdonald Islands Holy See (Vatican City State) Honduras Hong Kong Hungary Iceland India Indonesia Iran Iraq Ireland Israel Italy Jamaica Japan Jersey Jordan Kazakhstan Kenya Kiribati Kosovo (Serbia and Montenegro) Kuwait Kyrgyzstan Lao People's Democratic Republic Latvia Lebanon Lesotho Liberia Libya Liechtenstein Lithuania Luxembourg Macao Madagascar Malawi Malaysia Maldives Mali Malta Marshall Islands Martinique Mauritania Mauritius Mayotte Mexico Minor Outlying Islands of the United States Moldova Monaco Mongolia Montenegro Montserrat Morocco Mozambique Myanmar Namibia Nauru Nepal Netherlands Antilles New Caledonia New Zealand Nicaragua Niger Nigeria Niue Norfolk Island North Korea North Macedonia Northern Mariana Islands Norway Oman Pakistan Palau Palestinian Territory Panama Papua New Guinea Paraguay Peru Philippines Pitcairn Poland Portugal Puerto Rico Qatar Reunion Romania Russian Federation Rwanda Saint Helena Saint Kitts and Nevis Saint Lucia Saint Pierre and Miquelon Saint Vincent and the Grenadines Samoa San Marino Sao Tome and Principe Saudi Arabia Senegal Serbia Seychelles Sierra Leone Singapore Slovakia Slovenia Solomon Islands Somalia South Africa South Georgia and the South Sandwich Is South Korea South Sudan Spain Sri Lanka Sudan Suriname Svalbard and Jan Mayen Swaziland Sweden Switzerland Syria Taiwan Tajikistan Tanzania Thailand The Gambia The Netherlands Timor-Leste Togo Tokelau Tonga Trinidad and Tobago Tunisia Turkey Turkmenistan Turks and Caicos Islands Tuvalu UK USA Uganda Ukraine United Arab Emirates United States Virgin Islands Uruguay Uzbekistan Vanuatu Venezuela Vietnam Wallis and Futuna West Bank and Gaza Strip Western Sahara Yemen Zambia Zimbabwe Please select your country/region. You must enter a comment. Competing Interests Please disclose any competing interests that might be construed to influence your judgment of the article's or peer review report's validity or importance. Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list: Examples of 'Non-Financial Competing Interests' Within the past 4 years, you have held joint grants, published or collaborated with any of the authors of the selected paper. You have a close personal relationship (e.g. parent, spouse, sibling, or domestic partner) with any of the authors. You are a close professional associate of any of the authors (e.g. scientific mentor, recent student). You work at the same institute as any of the authors. You hope/expect to benefit (e.g. favour or employment) as a result of your submission. You are an Editor for the journal in which the article is published. Examples of 'Financial Competing Interests' You expect to receive, or in the past 4 years have received, any of the following from any commercial organisation that may gain financially from your submission: a salary, fees, funding, reimbursements. You expect to receive, or in the past 4 years have received, shared grant support or other funding with any of the authors. You hold, or are currently applying for, any patents or significant stocks/shares relating to the subject matter of the paper you are commenting on. Please state your competing interests The comment has been saved. An error has occurred. Please try again. Cancel Post var lTitle = "Recombinant expression of receptor binding...".replace("'", ''); var linkedInUrl = "http://www.linkedin.com/shareArticle?url=https://f1000research.com/articles/14-163/v1" + "&title=" + encodeURIComponent(lTitle) + "&summary=" + encodeURIComponent('Read the article by '); var deliciousUrl = "https://del.icio.us/post?url=https://f1000research.com/articles/14-163/v1&title=" + encodeURIComponent(lTitle); var redditUrl = "http://reddit.com/submit?url=https://f1000research.com/articles/14-163/v1" + "&title=" + encodeURIComponent(lTitle); linkedInUrl += encodeURIComponent('Quynh Pham N et al.'); var offsetTop = /chrome/i.test( navigator.userAgent ) ? 4 : -10; var addthis_config = { ui_offset_top: offsetTop, services_compact : "facebook,twitter,www.linkedin.com,www.mendeley.com,reddit.com", services_expanded : "facebook,twitter,www.linkedin.com,www.mendeley.com,reddit.com", services_custom : [ { name: "LinkedIn", url: linkedInUrl, icon:"/img/icon/at_linkedin.svg" }, { name: "Mendeley", url: "http://www.mendeley.com/import/?url=https://f1000research.com/articles/14-163/v1/mendeley", icon:"/img/icon/at_mendeley.svg" }, { name: "Reddit", url: redditUrl, icon:"/img/icon/at_reddit.svg" }, ] }; var addthis_share = { url: "https://f1000research.com/articles/14-163", templates : { twitter : "Recombinant expression of receptor binding domains of all eight.... Quynh Pham N et al., published by " + "@F1000Research" + ", https://f1000research.com/articles/14-163/v1" } }; if (typeof(addthis) != "undefined"){ addthis.addEventListener('addthis.ready', checkCount); addthis.addEventListener('addthis.menu.share', checkCount); } $(".f1r-shares-twitter").attr("href", "https://twitter.com/intent/tweet?text=" + addthis_share.templates.twitter); $(".f1r-shares-facebook").attr("href", "https://www.facebook.com/sharer/sharer.php?u=" + addthis_share.url); $(".f1r-shares-linkedin").attr("href", addthis_config.services_custom[0].url); $(".f1r-shares-reddit").attr("href", addthis_config.services_custom[2].url); $(".f1r-shares-mendelay").attr("href", addthis_config.services_custom[1].url); function checkCount(){ setTimeout(function(){ $(".addthis_button_expanded").each(function(){ var count = $(this).text(); if (count !== "" && count != "0") $(this).removeClass("is-hidden"); else $(this).addClass("is-hidden"); }); }, 1000); } close How to cite this report {{reportCitation}} Cancel Copy Citation Details $(function(){R.ui.buttonDropdowns('.dropdown-for-downloads');}); $(function(){R.ui.toolbarDropdowns('.toolbar-dropdown-for-downloads');}); $.get("/articles/acj/160607/176528") new F1000.Clipboard(); new F1000.ThesaurusTermsDisplay("articles", "article", "176528"); $(document).ready(function() { $( "#frame1" ).on('load', function() { var mydiv = $(this).contents().find("div"); var h = mydiv.height(); console.log(h) }); var tooltipLivingFigure = jQuery(".interactive-living-figure-label .icon-more-info"), titleLivingFigure = tooltipLivingFigure.attr("title"); tooltipLivingFigure.simpletip({ fixed: true, position: ["-115", "30"], baseClass: 'small-tooltip', content:titleLivingFigure + " " }); tooltipLivingFigure.removeAttr("title"); $("body").on("click", ".cite-living-figure", function(e) { e.preventDefault(); var ref = $(this).attr("data-ref"); $(this).closest(".living-figure-list-container").find("#" + ref).fadeIn(200); }); $("body").on("click", ".close-cite-living-figure", function(e) { e.preventDefault(); $(this).closest(".popup-window-wrapper").fadeOut(200); }); $(document).on("mouseup", function(e) { var metricsContainer = $(".article-metrics-popover-wrapper"); if (!metricsContainer.is(e.target) && metricsContainer.has(e.target).length === 0) { $(".article-metrics-close-button").click(); } }); var articleId = $('#articleId').val(); if($("#main-article-count-box").attachArticleMetrics) { $("#main-article-count-box").attachArticleMetrics(articleId, { articleMetricsView: true }); } }); var figshareWidget = $(".new_figshare_widget"); if (figshareWidget.length > 0) { window.figshare.load("f1000", function(Widget) { // Select a tag/tags defined in your page. In this tag we will place the widget. _.map(figshareWidget, function(el){ var widget = new Widget({ articleId: $(el).attr("figshare_articleId") //height:300 // this is the height of the viewer part. [Default: 550] }); widget.initialize(); // initialize the widget widget.mount(el); // mount it in a tag that's on your page // this will save the widget on the global scope for later use from // your JS scripts. This line is optional. //window.widget = widget; }); }); } close Error Close Add Reset F1000.MICROSERVICES.AFFILIATION = ''; $(document).ready(function () { $('.js-affiliations-form').each((index, form) => { new AffiliationForm({ formId: form.id, institutionErrorSelector: '.comment-enter-institution', departmentErrorSelector: '.comment-enter-department', placeSelector: '.js-add-comment-place', stateSelector: '.js-add-comment-state', zipCodeSelector: '.js-add-comment-zipcode', countrySelector: '.js-add-comment-country', countryErrorSelector: '.comment-enter-country', }); }); }); $(document).ready(function () { var reportIds = { "366737": 0, "366736": 0, "366738": 0, "366733": 11, "366732": 16, "366735": 0, "366734": 0, "366729": 12, "366731": 0, "366730": 0, }; $(".referee-response-container,.js-referee-report").each(function(index, el) { var reportId = $(el).attr("data-reportid"), reportCount = reportIds[reportId] || 0; $(el).find(".comments-count-container,.js-referee-report-views").html(reportCount); }); var uuidInput = $("#article_uuid"), oldUUId = uuidInput.val(), newUUId = "876ebb08-230e-4fd6-b6bf-5ad92c7b1374"; uuidInput.val(newUUId); $("a[href*='article_uuid=']").each(function(index, el) { var newHref = $(el).attr("href").replace(oldUUId, newUUId); $(el).attr("href", newHref); }); }); An innovative open access publishing platform offering rapid publication and open peer review, whilst supporting data deposition and sharing. Browse Gateways Collections How it Works Contact For Developers Cookie Notice Privacy Notice RSS Submit Your Research Follow us © 2012-2026 F1000 Research Ltd. ISSN 2046-1402 | Legal | Partner of Research4Life • CrossRef • ORCID • FAIRSharing R.templateTests.simpleTemplate = R.template(' $text $text $text $text $text '); R.templateTests.runTests(); var F1000platform = new F1000.Platform({ name: "f1000research", displayName: "F1000Research", hostName: "f1000research.com", id: "1", editorialEmail: "[email protected]", infoEmail: "[email protected]", usePmcStats: true }); $(function(){R.ui.dropdowns('.dropdown-for-authors, .dropdown-for-about, .dropdown-for-myresearch');}); // $(function(){R.ui.dropdowns('.dropdown-for-referees');}); $(document).ready(function () { if ($(".cookie-warning").is(":visible")) { $(".sticky").css("margin-bottom", "35px"); $(".devices").addClass("devices-and-cookie-warning"); } $(".cookie-warning .close-button").click(function (e) { $(".devices").removeClass("devices-and-cookie-warning"); $(".sticky").css("margin-bottom", "0"); }); $("#tweeter-feed .tweet-message").each(function (i, message) { var self = $(message); self.html(linkify(self.html())); }); $(".partner").on("mouseenter mouseleave", function() { $(this).find(".gray-scale, .colour").toggleClass("is-hidden"); }); }); Sign In Remember me Forgotten your password? Sign In Cancel Email or password not correct. Please try again Please wait... $(function(){ // Note: All the setup needs to run against a name attribute and *not* the id due the clonish // nature of facebox... $("a[id=googleSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("GOOGLE"); $("form[id=oAuthForm]").submit(); }); $("a[id=facebookSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("FACEBOOK"); $("form[id=oAuthForm]").submit(); }); $("a[id=orcidSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("ORCID"); $("form[id=oAuthForm]").submit(); }); }); If you've forgotten your password, please enter your email address below and we'll send you instructions on how to reset your password. The email address should be the one you originally registered with F1000. Email address not valid, please try again You registered with F1000 via Google, so we cannot reset your password. To sign in, please click here . If you still need help with your Google account password, please click here . You registered with F1000 via Facebook, so we cannot reset your password. To sign in, please click here . If you still need help with your Facebook account password, please click here . Code not correct, please try again Reset password Cancel Email us for further assistance. Server error, please try again. If your email address is registered with us, we will email you instructions to reset your password. If you think you should have received this email but it has not arrived, please check your spam filters and/or contact for further assistance. Please wait... Register $(document).ready(function () { signIn.createSignInAsRow($("#sign-in-form-gfb-popup")); $(".target-field").each(function () { var uris = $(this).val().split("/"); if (uris.pop() === "login") { $(this).val(uris.toString().replace(",","/")); } }); });