Visual Non-Instrumental On-Site Detection of HSP70 Using a Modified Immune Affinity Gel-column Assay

Visual Non-Instrumental On-Site Detection of HSP70 Using a Modified Immune Affinity Gel-column Assay | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Visual Non-Instrumental On-Site Detection of HSP70 Using a Modified Immune Affinity Gel-column Assay Hongjun Lin, Qiqi Liu, Zheng Zhang, Jingxiang Zhang, Xiaoyan Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4593110/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A modified immune affinity gel-column (IAGC) assay was developed for rapid detection biomarkers of heat shock protein 70 (HSP70). The new method presets antibodies in a gel for rapid detection of the target antigen flowing through it. No special instruments and cumbersome processing steps were required throughout the entire process. The whole detection was completed within 10 min and a high-throughput could be achieved. Because the detection gel is in a transparent cylinder, color changes can be observed more sensitively. The detection limit (LOD) of the newly established method can be as low as 10 µg/L, which is significantly better than the existing lateral-flow strip method. In addition, the method is suitable for outdoor use, especially in laboratories with relatively simple conditions. In addition, the new detection method can also be designed into multiple detection layers in series or multiple detection columns in parallel to further improve the detection efficiency. Therefore, the new method is a fast, simple, high-throughput, qualitative and semi-quantitative detection platform, and a new idea for rapid detection. HSP70 gel-column on-site detection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction In recent years, more and more people have devoted themselves to physical exercise with the gradual deepening of health awareness. With the continuous increasing of training frequency and intensity, various sports injuries have also emerged. 1 , 2 More seriously, some sports injuries are irreversible, which will cause permanent damage and distress to bodies. Especially for athletes and soldiers, their careers may come to an end. Therefore, early prediction and timely treatment will have significant significance. 3 , 4 Accurate and rapid detecting biomarkers of sports injuries is a prerequisite. HSP70 in this study is a heat shock protein, the most important branch of the HSP family, induced by stress and heat shock, and its expression is associated with disease and exercise status. HSP70 plays an important protective role in cells, helping cells cope with various stressful situations, such as high temperature, hypoxia, and injury. For example, under high temperature stress, the synthesis rate of HSP70 is significantly increased and can reach the highest level within minutes. Therefore, HSP70 can be used as an auxiliary diagnostic means to guide sports and avoid the occurrence and further aggravation of sports injuries. 5 – 8 At present, the main detection methods for HSP70 include LC-MS/MS method and immunoaffinity test. The LC-MS/MS method requires expensive instruments, long detection times, and professional operators, which greatly limit its use, especially in resource-poor, wilderness areas, and places without power. Therefore, immunoaffinity tests based on antibody-antigen specific recognition play an increasingly important role in biochemistry and clinical medicine and are well suited for field detection. 9 , 10 However, commonly used immunization methods, such as ELISA and immunoblotting, are cumbersome and time-consuming, and require some electrical equipment. These deficiencies also limit the scope of its application. 11 , 12 The newly developed IAGC assay method is a new detection platform using agarose gel as the carrier with some improvements. The principle is that the enzyme-antigen conjugate and the antigen in the sample compete with the limited antibody coupled on the gel. 13 – 15 Color develops after adding the enzyme substrate, and the results can be visually measured. Negative samples will produce blue, while positive samples will produce light blue or colorless at corresponding positions. And the higher the concentration is, the lighter the color displays. The cutoff value is defined as the concentration that just develops no color. 16 The immunoaffinity reaction with CNBr activated Sepharose 4B gel as the carrier has better sensitivity and versatility, and can be suitable for a wider range of detection conditions. Compared with the test strip, this method has the advantages of higher sensitivity, high-throughput and better compatibility. In addition, the method can also be combined with a purification column for samples with serious pollution or dark colored. 17 2. Methods 2.1. Materials Bovine serum albumin (BSA), horseradish peroxidase (HRP), goat anti rabbit IgG secondary antibody, 3,3,5,5-tetramethylbenzidine (TMB), rabbit anti HRP antibody, dimethyl sulfoxide (DMSO), ProClin 300 purchased from Solarbio, CNBr activated Sepharose 4B purchased from Shenggong Biotechnology (Shanghai) Co., Ltd, phosphate buffered saline (PBS, 0.01 M, pH 7.4), coupling solution (NaHCO 3 buffer, 0.1 M, pH 8.3, containing 0.5 M NaCl), blocking solution (coupling solution, containing 0.2 M glycine, pH 8.0), acetate buffer (pH 4.0, containing 0.5 M NaCl), PBST (PBS buffer, containing 0.05% Tween-20), chromogenic substrate (0.1% TMB, H 2 O 2 in 0.05 M citrate buffer, pH 4.5), HSP70 antibody, GST, PCT, IL-15-HIS, and IL-15-Llag were purchased from Shenbiote Co., Ltd. All other chemicals and solvents were of analytical grade or better. 2.2. Methods 2.2.1. Preparation of anti-HSP70 gel Firstly, CNBr activated Sepharose 4B gel powder (1.0 g) was washed with 100 mL of 1 mM HCl. Anti-HSP70 antibody (diluted by 1/50000) was mixed with 1 mL of coupling agent (0.1 M NaHCO 3 , 0.5 M NaCl, pH 8.3) and gel, and incubated on a shaker at room temperature (RT) for 2 h. Then the gel was washed with 20 mL of coupling agent to remove the excess antibody, and the coupling gel was obtained. Next, the unconnected active groups were blocked with 12 mL of blocking agent (0.2 M glycine, 0.1 M NaHCO 3 , 0.5 M NaCl, pH 8.0) and incubated for 2 h. Finally, after washed with 0.1 M acetate buffer (containing 10 mL of 0.5 M NaCl, pH 4.0) to remove the excessive glycine, the blocking gel was obtained. The prepared gel was suspended in PBS (containing 0.03% Proclin 300) and stored at 4°C for use. 18 – 20 2.2.2. Preparation of anti-HRP gel The method was similar to that above. The coupling gel was mixed with sealing gel (1:4, v/v), and then, 400 µL of goat anti rabbit IgG (2.0 mg/mL) was added to the mixture. Anti-HRP gel was obtained after shaking at 400 rpm for 5 min. 21 2.2.3. Preparation of HSP70-HRP conjugates According to the periodate method: firstly, 2 mg of HRP was activated in 500 µL of sodium periodate solution (1 mol/L) at RT for 20 min. Then, 1 mL of HSP70 solution (1mg of HSP70 dissolved in 1 mL of 0.05 mol/L carbonate buffer, pH 9.5) was added dropwise to above solution, and the mixture was stirred under magnetic force at RT for 2 h. Secondly, 100 µL of sodium borohydride solution (1 mol/L) was added and the mixture was kept at 4 ℃ for 1 hour to terminate the reaction. Finally, the conjugate was dialyzed with PBS (0.01 mol/L, pH 7.4) for 72 h, and stored at 4 ℃ for use. 22 – 23 2.2.4. Assembly of gel-column The gel-column was a syringe shaped hollow tube made of polyethylene material with a capacity of 1 mL (length is 56.5 mm, and inner diameter is 5.8 mm) including control layer, isolation layer and test layer from bottom to top. The gel was fixed by polyethylene frit, and the isolation layer was a 2 ~ 3 mm air gap between the control layer and the test layer to facilitate color observation. 24 , 25 The assembly steps of the control layer were as follows: firstly, a piece of porous polyethylene frit was placed at the bottom of the gel-column. The pore size was appropriate to allow the sample to pass and effectively support the gel. The pore size of frit was 20 µm used in this study. Secondly, 150 µL of anti-HRP gel was added to the gel-column, and the excess liquid was pushed away with a syringe. Finally, the second frit was putted on the upper surface of gel, and the assembly of the control layer was complete. The coupling gel was diluted 5 times with the blocked gel and other assembly steps were the same as the control layer. At this stage, the gel column was assembled. 26 , 27 2.2.5. Procedure of the modified gel-column detection method CNBr activated Sepharose 4B was used as the carrier to couple the antibody through cyanogen bromide in test layer. HSP70-HRP conjugate and HSP70 in the sample competed for the limited anti-HSP70 antibody on the gel. After adding enzyme substrate and color development, the results could be observed with naked eyes. The test layer displayed blue if the sample was negative, and a weaker color or no visible color would be generated if the sample was positive. In the control layer, HSP70-HRP conjugate bound to anti-HRP antibody and appeared color when encountering enzyme substrate. The LOD was the concentration when the color just disappeared completely. The color development in the control layer indicated that the gel-column measurement was effective. If there was no color development in the control layer, the measurement was deemed invalid. The detailed steps were shown as follows: firstly, sample addition. Sample (mixed with an enzyme tracer GST-HRP conjugate, 1 mL) was added to the test column at a rate of 1 drop per second from the inlet. The analyte and GST-HRP conjugate competitively bound to the anti-GST antibody on the test layer. The remaining GST-HRP conjugate leaked into the control layer and bound to anti-HRP antibodies. Secondly, washing. PBS (2 mL) and PBST (3 mL) were added to remove unbound analytes and enzyme tracers. Thirdly, color development. The control layer and test layer were immersed by 300 µL of chromogenic substrate drawn into the column from the outlet. Visual inspection was performed after incubation for 10 seconds. Considering the balance point between non-specific adsorption effect and color intensity, 5 min were chosen as the optimal incubation time. 28 , 29 3. Results and discussion 3.1. Comparison with lateral-flow strip test The identical sample was tested under the same condition using IAGC assay and lateral-flow strip method, and the LODs were compared. The detailed process of IAGC assay was as follows: 100µL of GST solution was dropped onto the test strip. Color with a certain intensity appeared and could be visually observed within 6 min. A booster can be used to accelerate the sample flowing through the detection column if needed. By comparing the two methods, both could be used for on-site test. The differences between them were that IAGC assay had a higher sample loading ability, better sensitivity, and less time used than lateral-flow strip test. In summary, IAGC assay had greater advantages in high sensitivity measurements. 30 , 31 The sensitivity improvement of the IAGC method is mainly attributed to its high throughput. Gel with a certain thickness can enrich and effectively detect samples with lower concentration. 3.2. Validation of specificity To investigate the specificity of IAGC assay, the following experiment was designed. The specific steps were as follows: antibodies of HSP70, Pro Calcitonin (PCT), IL-15-His, and IL-15-Flag were selected as models (concentrations were 10 µg/L,100 µg/L, 100 µg/L, 100 µg/L respectively), and the 4 gel columns were all coupled to anti-HSP70 antibody. The 4 antibodies were added to 4 gel columns respectively, and color development results were observed. c: 100 µg/L of IL-15-His, and d: 100 µg/L of IL-15-Flag. The results showed that only the sample containing anti-HSP70 antigen could make the color of the test layer disappear, while the other 3 columns showed bright blue. This indicated that the IAGC detection method had good specificity. 32 ,33 3.3. Validation of multi test layers in one column In order to investigate the Validation of multi test layers in one column, the following series of experiments were carried out with HSP70 protein used as a model. In order to improve the test efficiency, a gel-column filled with 3 test layers was designed. The purpose was to investigate whether multiple test layers interfered with each other. The detailed steps were as follows: firstly, the sample containing HSP70, PCT and IL-15-His antigens respectively passed through the gel-column with single test layer coupled with anti-HSP70, PCT and IL-15-His antibodies respectively. Secondly, gel-column with 3 test layers coupled with anti- A, B, and C antibody separately was designed. The same sample was used to all the gel-columns (Fig. 4 . a, b, c, and d). Finally, color results were obtained and compared to examine the interference. Results show that the detection layers in gel columns of a, b and c can effectively detect the mixture samples. The same samples can also be effectively detected using gel column d with three test layers, and there is no obvious interference between each detection layers. Therefore, the design of multiple inspection layers in one gel column can effectively detect mixture samples, which can greatly improve detection efficiency. 3.4. Multiple columns detection To eliminate the interference of multiple detection layers in one column completely, a multi-column is designed. That is, the same multiple detection columns are interconnected, and samples can flow freely between the three columns. The advantage of this design is that it cannot only realize multi-component detection, but also eliminate the interference between each detection layer. Since the sample is evenly divided among the three columns, the detection is more accurate. In addition, the detection flux is larger, especially suitable for high throughput, low concentration detection. As shown in the Fig. 5 , Anti-HSP70, PCT and IL-15 His antibodies were chelated respectively in the gel columns of a, b and c. The results showed that the color of columns a and c completely disappeared, while column b did not change, indicating that the sample contained HSP70 and IL-15. 3.5. Validation of min sample volume According to the principle of immunity affinity, the larger the sample volume was, the darker the color produced. But at the same time, the adverse effect was that it produced poorer sensitivity. Therefore, the minimum solution volume was another important factor to be examined. 38 A series of volume gradient 1 µL, 5 µL, 10 µL, 15 µL, 30 µL respectively from left to right was designed. The results shown that the test layer has no color at all when the volume was greater than or equal to 10 µL. It was indicated that the minimum volume of the method was 10 µL. The results indicate that when the volume is equal to or greater than 10 µL, the test layer is no color at all. The reason is that all HSP70 antibody sites in the detection layer are occupied by antigens in the sample. As the increasing sample volume (such as d and e), there are no excess sites available for HSP70-HRP binding, so they all appear colorless. 3.6. Curve fitting Visual inspection could only provide rough qualitative analysis results and low sensitivity. To further improve sensitivity, the app Image Recognition 1.0 is developed. It can directly identify, fit curves, statistics, and save experimental results (Fig. 7 ). The detailed steps as follows: the entire gel-column was placed on a white background, and photos were taken using a smartphone and saved as JPEG format. The average of 3 points from each photo was taken as the color grayscale value. Quantitative analysis could be achieved according to the curve fitted on this numerical value. 39,40 Results showed that the sensitivity was significantly improved benefitting from high pixel of smartphone combined with the app Image Recognition 1.0 . It was approximately 10-fold better with a quantitative LOD of 1 ng/mL than visual detection. 3.7. Comparisons with ELISA The principle of IAGC assay and traditional ELISA was similar, and the main differences between them lied in carriers and the ways of immobilizing antibodies. Compared with ELISA, IAGC assay had unique advantages. Firstly, the sample loading capacity was much larger than ELISA, which could effectively improve the detection sensitivity especially for samples with low concentration. Secondly, the operation was very simple and fast, and there was almost no need for instruments. Finally, repeated washing was avoided, which not only saved time but also reduced the loss of analytes. 41 Table 1 Comparison between IAGC assay with traditional ELISA. IAGC assay ELISA Time 8 min 90 min LOD (ng/mL) 10 ng/mL 10 ng/mL cost low high washing 1 time many times carrier Sepharose gel polystyrene well suitable for the wild Yes No need for instruments No Yes connecting the purification device Yes No In summary, IAGC assay had significant advantages over ELISA in many aspects as shown in Table 1 above. 3.8. Validation of the test using actual sample The experimental process was similar as above. The internal standard antigen was added to actual samples to test the practicality of this method. B: grayscale value of the images, and dashed lines was the cut-off level. 4. Conclusion In this work, we have successfully developed an IAGC assay method able to carry out ultra-sensitive immunoassay with the advantages of sensitivity, velocity, simplicity, high-throughput and suitable for outdoor use. Since the IAGC assay method itself is a widely used technology basing on the immunoaffinity principle, our improvements should make it has more potential application scenarios than other traditional immunoassays. The new assay method can detect actual samples without special instruments and make the sensitivity higher than lateral-flow strip, simpler than ELISA detection kit. Experiments show that detection limit is 10 µg/L, which is 10 times higher than lateral-flow strip. The new method can effectively detect multiple samples in one gel-column by changing the layout of the detection layers in the gel column. Results also showed that the new method has good specificity and broad application prospects. We believe that IAGC assay can be exploited to wider applicability for the analysis of trace biomarkers in clinical analysis, biological research and military training. Abbreviations IAGC immune affinity gel-column HSP70 heat shock protein 70 LOD the detection limit HRP horseradish peroxidase BSA bovine serum albumin TMB 3,3,5,5-tetramethylbenzidine DMSO dimethyl sulfoxide RT room temperature PCT Pro Calcitonin. Declarations Acknowledgements Not applicable Funding This work was financially supported by the Fund of Research on High Throughput Rapid Detection Technology for Pathogens. Availability of data and materials Data supporting our findings is contained within the manuscript; any additional data will be shared upon request to the corresponding author. Authors’ contributions HJL and QQL designed the study, performed the experiments and wrote the manuscript. ZZ, JXZ, XYL and ZZ analysed the data. Competing interests The authors declare that they have no competing interests. Authors' information Prof. Dr. Zhe Zhou is the professor of Bioinformatics Center of AMMS, Beijing, China. He has over 30 years of experience in the field of immunoassay. Dr. Hongjun Linis the associate professor of Bioinformatics Center of AMMS, Beijing, China. He has over 20 years of experience in the field of immunoassay. References Ahmed S, Ashraf M, Sahanand S, Rajan DV. Indian J Orthop. 2021;55:402–8. Andryskova A, Lee JH. Healthcare. 2020;8:144–7. Palmi J, Alcubierre N, Gil M, de Mora G, Reig F, Planas-Anzano A. Int J Environ Res Public Health. 2021;18:5487–94. Arumugam S, Prakash A, Janani G, Vignesh M, Anjanavannan MM, Perumal S, Alwar T. Indian J Orthop. 2021;55:484–91. Kaushik R, Goel A, Rout PK. Scientifc Rep. 2022;12:18310–9. Tabusam J, Shi QL, Feng DL, Zulfiqar S, Shen SX, Ma W, Zhao. J J Cells. 2022;11:2316–39. Cumming KT, Paulsen G, Wernbom M, Ugelstad I, Raastad T. Acta Physiol. 2014;4:634–46. Zhang N, Deng WC, Li Y, Ma YD, Liu Y, Li XJ, Wang H. Anal Chem. 2020;92:1197–204. Gao Y, Li X, Zhao HL, Ling-Hu T, Zhou YZ, Tian JS, Qin XM. J Proteome Res. 2021;20:2477–86. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4593110","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322877029,"identity":"3e787945-5b34-41ca-9eb7-24d1fa10d5d5","order_by":0,"name":"Hongjun Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3RMWrDMBSA4RcEyqJWqwzGuYJCwVMPI2PQ5EBHD4YYGpShKV4TeomMGSUEnpTdo3sDe+vYem6JnK2Dvlk/T08CCIJ/CNNm0ANn22ZlTS/Kyp88Mr02x/J5cTrinPeu9ScJiCdLnFycO5JGnzs042KghTkpi6IPJcusxkD3b+J2gmqtR2UxjW3bZZcYmLuePVNMPU0hUS1llzkMnG18SQ72QVnGdZG+ZArNSSRM63PeFSnMS5iD6ZFFdGhzJlxLvLusmgMafr5S0OWrGb/KKqH799vJL+S+40EQBMGfvgETEFJpWwhMUAAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Hongjun","middleName":"","lastName":"Lin","suffix":""},{"id":322877030,"identity":"394c2509-3d1e-46ad-88cc-7a7cf1395ec5","order_by":1,"name":"Qiqi Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qiqi","middleName":"","lastName":"Liu","suffix":""},{"id":322877031,"identity":"39b4cffe-82f2-4ee9-b65d-93d2a3224a83","order_by":2,"name":"Zheng Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Zhang","suffix":""},{"id":322877032,"identity":"7365f297-99c2-4844-8970-3991b8a0d607","order_by":3,"name":"Jingxiang Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jingxiang","middleName":"","lastName":"Zhang","suffix":""},{"id":322877033,"identity":"f1b882b7-a9e1-42c4-8ae2-36a4c87e6f3c","order_by":4,"name":"Xiaoyan Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Li","suffix":""},{"id":322877034,"identity":"3ab1e104-f752-48fe-8f7d-36038f5d3e55","order_by":5,"name":"Zhe Zhou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2024-06-17 09:21:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4593110/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4593110/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59953431,"identity":"aa597d82-4273-4b8a-919b-81c65036a05d","added_by":"auto","created_at":"2024-07-09 18:26:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58705,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of IAGC assay standard procedure.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-4593110/v1/acfa3b547322709a8f6edf91.png"},{"id":59952497,"identity":"38a09623-f534-46c3-a34e-af3917a45044","added_by":"auto","created_at":"2024-07-09 18:10:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":163108,"visible":true,"origin":"","legend":"\u003cp\u003eLateral-flow strip test of GST in standard solution (A). The concentration of GST is 0 μg/L (A-a), 1 μg/L (A-b), 10 μg/L (A-c) and 100 μg/L (A-d) from left to right, and the LOD is 100 μg/L。IAGC assay of GST in standard solution (B). The concentration of GST is 0 μg/L (B-a), 1 μg/L (B-b), 10 μg/L (B-c) and 100 μg/L (B-d) from left to right, and the LOD is 10 μg/L。\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-4593110/v1/d9542f059eb80035a59d5a2b.png"},{"id":59952498,"identity":"0190fd6f-3394-44d9-b7a6-cf0b548f71c0","added_by":"auto","created_at":"2024-07-09 18:10:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":119066,"visible":true,"origin":"","legend":"\u003cp\u003eResults of specificity. a: 10 μg/L of HSP70, b: 100 μg/L of PCT,\u003c/p\u003e\n\u003cp\u003ec: 100 μg/L of IL-15-His, and d: 100 μg/L of IL-15-Flag.\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-4593110/v1/37472ea1ce9697b1bfc1af26.png"},{"id":59952964,"identity":"8d552153-b592-4594-87ac-9472f6c63af2","added_by":"auto","created_at":"2024-07-09 18:18:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135457,"visible":true,"origin":"","legend":"\u003cp\u003eAnti- HSP70, PCT and IL-15 His antibodies were chelated respectively in the detection layers of a, b and c gel columns, and they were chelated successively from top to bottom in the detection layers of d columns. The sample is a mixed solution containing HSP70, PCT and IL-15 His, and is added to 4 gel columns in turn. The lowest layer was the control layer.\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-4593110/v1/c4b9b6442b663c421d099ae5.png"},{"id":59953718,"identity":"119cbf1d-3851-4991-ae71-c047c5a8a3b8","added_by":"auto","created_at":"2024-07-09 18:34:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":145574,"visible":true,"origin":"","legend":"\u003cp\u003eTriple column detection.\u003c/p\u003e\n\u003cp\u003eAs shown in the figure 5, Anti-HSP70, PCT and IL-15 His antibodies were chelated respectively in the gel columns of a, b and c. The results showed that the color of columns a and c completely disappeared, while column b did not change, indicating that the sample contained HSP70 and IL-15.\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-4593110/v1/a7019573d7402a3a0f38edac.png"},{"id":59952503,"identity":"1d7f8aab-65d0-4eb5-a2e1-bf420c547a14","added_by":"auto","created_at":"2024-07-09 18:10:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":194362,"visible":true,"origin":"","legend":"\u003cp\u003eThe solution volumes are 1 μL, 5 μL, 10 μL, 15 μL, and 30 μL in sequence from column a to e.\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-4593110/v1/ac7eea324b78a965432a2471.png"},{"id":59952965,"identity":"b7f7e6da-4a34-4d73-a0b9-7d29963fcc0a","added_by":"auto","created_at":"2024-07-09 18:18:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":98541,"visible":true,"origin":"","legend":"\u003cp\u003eTest flow chart of app Image Recognition 1.0.\u003c/p\u003e","description":"","filename":"F7.png","url":"https://assets-eu.researchsquare.com/files/rs-4593110/v1/cc23b6ef6b1231fa33c1cf4f.png"},{"id":59952968,"identity":"f6703ab8-2827-496a-9382-eff9cc07bf37","added_by":"auto","created_at":"2024-07-09 18:18:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":170666,"visible":true,"origin":"","legend":"\u003cp\u003e(A): visual outcome; (B): calibration curve, and the data were means of 3 tests and bars gave standard deviations.\u003c/p\u003e","description":"","filename":"F8.png","url":"https://assets-eu.researchsquare.com/files/rs-4593110/v1/86eafa7ccb6fef502777d48c.png"},{"id":59952501,"identity":"d03ca28e-bf27-4a82-9564-dfb431a3869f","added_by":"auto","created_at":"2024-07-09 18:10:02","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":124278,"visible":true,"origin":"","legend":"\u003cp\u003eHSP70 determination with IAGC assay method. A: photos taken by a smartphone;\u003c/p\u003e\n\u003cp\u003eB: grayscale value of the images, and dashed lines was the cut-off level.\u003c/p\u003e","description":"","filename":"F9.png","url":"https://assets-eu.researchsquare.com/files/rs-4593110/v1/7bb744a13b5ef4a20c8aefb8.png"},{"id":72500257,"identity":"3763a18f-bea7-4c48-9646-1d33a812c63d","added_by":"auto","created_at":"2024-12-28 04:31:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1622935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4593110/v1/1a8b84d1-2061-4b55-ad1a-7930fd36e15a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Visual Non-Instrumental On-Site Detection of HSP70 Using a Modified Immune Affinity Gel-column Assay","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn recent years, more and more people have devoted themselves to physical exercise with the gradual deepening of health awareness. With the continuous increasing of training frequency and intensity, various sports injuries have also emerged.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e More seriously, some sports injuries are irreversible, which will cause permanent damage and distress to bodies. Especially for athletes and soldiers, their careers may come to an end. Therefore, early prediction and timely treatment will have significant significance.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Accurate and rapid detecting biomarkers of sports injuries is a prerequisite.\u003c/p\u003e \u003cp\u003eHSP70 in this study is a heat shock protein, the most important branch of the HSP family, induced by stress and heat shock, and its expression is associated with disease and exercise status. HSP70 plays an important protective role in cells, helping cells cope with various stressful situations, such as high temperature, hypoxia, and injury. For example, under high temperature stress, the synthesis rate of HSP70 is significantly increased and can reach the highest level within minutes. Therefore, HSP70 can be used as an auxiliary diagnostic means to guide sports and avoid the occurrence and further aggravation of sports injuries. \u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e At present, the main detection methods for HSP70 include LC-MS/MS method and immunoaffinity test. The LC-MS/MS method requires expensive instruments, long detection times, and professional operators, which greatly limit its use, especially in resource-poor, wilderness areas, and places without power. Therefore, immunoaffinity tests based on antibody-antigen specific recognition play an increasingly important role in biochemistry and clinical medicine and are well suited for field detection. \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e However, commonly used immunization methods, such as ELISA and immunoblotting, are cumbersome and time-consuming, and require some electrical equipment. These deficiencies also limit the scope of its application. \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe newly developed IAGC assay method is a new detection platform using agarose gel as the carrier with some improvements. The principle is that the enzyme-antigen conjugate and the antigen in the sample compete with the limited antibody coupled on the gel.\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Color develops after adding the enzyme substrate, and the results can be visually measured. Negative samples will produce blue, while positive samples will produce light blue or colorless at corresponding positions. And the higher the concentration is, the lighter the color displays. The cutoff value is defined as the concentration that just develops no color.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e The immunoaffinity reaction with CNBr activated Sepharose 4B gel as the carrier has better sensitivity and versatility, and can be suitable for a wider range of detection conditions. Compared with the test strip, this method has the advantages of higher sensitivity, high-throughput and better compatibility. In addition, the method can also be combined with a purification column for samples with serious pollution or dark colored.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eBovine serum albumin (BSA), horseradish peroxidase (HRP), goat anti rabbit IgG secondary antibody, 3,3,5,5-tetramethylbenzidine (TMB), rabbit anti HRP antibody, dimethyl sulfoxide (DMSO), ProClin 300 purchased from Solarbio, CNBr activated Sepharose 4B purchased from Shenggong Biotechnology (Shanghai) Co., Ltd, phosphate buffered saline (PBS, 0.01 M, pH 7.4), coupling solution (NaHCO\u003csub\u003e3\u003c/sub\u003e buffer, 0.1 M, pH 8.3, containing 0.5 M NaCl), blocking solution (coupling solution, containing 0.2 M glycine, pH 8.0), acetate buffer (pH 4.0, containing 0.5 M NaCl), PBST (PBS buffer, containing 0.05% Tween-20), chromogenic substrate (0.1% TMB, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 0.05 M citrate buffer, pH 4.5), HSP70 antibody, GST, PCT, IL-15-HIS, and IL-15-Llag were purchased from Shenbiote Co., Ltd. All other chemicals and solvents were of analytical grade or better.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Methods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Preparation of anti-HSP70 gel\u003c/h2\u003e \u003cp\u003eFirstly, CNBr activated Sepharose 4B gel powder (1.0 g) was washed with 100 mL of 1 mM HCl. Anti-HSP70 antibody (diluted by 1/50000) was mixed with 1 mL of coupling agent (0.1 M NaHCO\u003csub\u003e3\u003c/sub\u003e, 0.5 M NaCl, pH 8.3) and gel, and incubated on a shaker at room temperature (RT) for 2 h. Then the gel was washed with 20 mL of coupling agent to remove the excess antibody, and the coupling gel was obtained. Next, the unconnected active groups were blocked with 12 mL of blocking agent (0.2 M glycine, 0.1 M NaHCO\u003csub\u003e3\u003c/sub\u003e, 0.5 M NaCl, pH 8.0) and incubated for 2 h. Finally, after washed with 0.1 M acetate buffer (containing 10 mL of 0.5 M NaCl, pH 4.0) to remove the excessive glycine, the blocking gel was obtained. The prepared gel was suspended in PBS (containing 0.03% Proclin 300) and stored at 4\u0026deg;C for use.\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Preparation of anti-HRP gel\u003c/h2\u003e \u003cp\u003eThe method was similar to that above. The coupling gel was mixed with sealing gel (1:4, v/v), and then, 400 \u0026micro;L of goat anti rabbit IgG (2.0 mg/mL) was added to the mixture. Anti-HRP gel was obtained after shaking at 400 rpm for 5 min.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Preparation of HSP70-HRP conjugates\u003c/h2\u003e \u003cp\u003eAccording to the periodate method: firstly, 2 mg of HRP was activated in 500 \u0026micro;L of sodium periodate solution (1 mol/L) at RT for 20 min. Then, 1 mL of HSP70 solution (1mg of HSP70 dissolved in 1 mL of 0.05 mol/L carbonate buffer, pH 9.5) was added dropwise to above solution, and the mixture was stirred under magnetic force at RT for 2 h. Secondly, 100 \u0026micro;L of sodium borohydride solution (1 mol/L) was added and the mixture was kept at 4 ℃ for 1 hour to terminate the reaction. Finally, the conjugate was dialyzed with PBS (0.01 mol/L, pH 7.4) for 72 h, and stored at 4 ℃ for use.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. Assembly of gel-column\u003c/h2\u003e \u003cp\u003eThe gel-column was a syringe shaped hollow tube made of polyethylene material with a capacity of 1 mL (length is 56.5 mm, and inner diameter is 5.8 mm) including control layer, isolation layer and test layer from bottom to top. The gel was fixed by polyethylene frit, and the isolation layer was a 2\u0026thinsp;~\u0026thinsp;3 mm air gap between the control layer and the\u003c/p\u003e \u003cp\u003etest layer to facilitate color observation.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe assembly steps of the control layer were as follows: firstly, a piece of porous polyethylene frit was placed at the bottom of the gel-column. The pore size was appropriate to allow the sample to pass and effectively support the gel. The pore size of frit was 20 \u0026micro;m used in this study. Secondly, 150 \u0026micro;L of anti-HRP gel was added to the gel-column, and the excess liquid was pushed away with a syringe. Finally, the second frit was putted on the upper surface of gel, and the assembly of the control layer was complete. The coupling gel was diluted 5 times with the blocked gel and other assembly steps were the same as the control layer. At this stage, the gel column was assembled.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5. Procedure of the modified gel-column detection method\u003c/h2\u003e \u003cp\u003eCNBr activated Sepharose 4B was used as the carrier to couple the antibody through cyanogen bromide in test layer. HSP70-HRP conjugate and HSP70 in the sample competed for the limited anti-HSP70 antibody on the gel. After adding enzyme substrate and color development, the results could be observed with naked eyes. The test layer displayed blue if the sample was negative, and a weaker color or no visible color would be generated if the sample was positive. In the control layer, HSP70-HRP conjugate bound to anti-HRP antibody and appeared color when encountering enzyme substrate. The LOD was the concentration when the color just disappeared completely. The color development in the control layer indicated that the gel-column measurement was effective. If there was no color development in the control layer, the measurement was deemed invalid.\u003c/p\u003e \u003cp\u003eThe detailed steps were shown as follows: firstly, sample addition. Sample (mixed with an enzyme tracer GST-HRP conjugate, 1 mL) was added to the test column at a rate of 1 drop per second from the inlet. The analyte and GST-HRP conjugate competitively bound to the anti-GST antibody on the test layer. The remaining GST-HRP conjugate leaked into the control layer and bound to anti-HRP antibodies. Secondly, washing. PBS (2 mL) and PBST (3 mL) were added to remove unbound analytes and enzyme tracers. Thirdly, color development. The control layer and test layer were immersed by 300 \u0026micro;L of chromogenic substrate drawn into the column from the outlet. Visual inspection was performed after incubation for 10 seconds. Considering the balance point between non-specific adsorption effect and color intensity, 5 min were chosen as the optimal incubation time.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Comparison with lateral-flow strip test\u003c/h2\u003e \u003cp\u003eThe identical sample was tested under the same condition using IAGC assay and lateral-flow strip method, and the LODs were compared. The detailed process of IAGC assay was as follows: 100\u0026micro;L of GST solution was dropped onto the test strip. Color with a certain intensity appeared and could be visually observed within 6 min. A booster can be used to accelerate the sample flowing through the detection column if needed. By comparing the two methods, both could be used for on-site test. The differences between them were that IAGC assay had a higher sample loading ability, better sensitivity, and less time used than lateral-flow strip test. In summary, IAGC assay had greater advantages in high sensitivity measurements.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe sensitivity improvement of the IAGC method is mainly attributed to its high throughput. Gel with a certain thickness can enrich and effectively detect samples with lower concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Validation of specificity\u003c/h2\u003e \u003cp\u003eTo investigate the specificity of IAGC assay, the following experiment was designed. The specific steps were as follows: antibodies of HSP70, Pro Calcitonin (PCT), IL-15-His, and IL-15-Flag were selected as models (concentrations were 10 \u0026micro;g/L,100 \u0026micro;g/L, 100 \u0026micro;g/L, 100 \u0026micro;g/L respectively), and the 4 gel columns were all coupled to anti-HSP70 antibody. The 4 antibodies were added to 4 gel columns respectively, and color development results were observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ec: 100 \u0026micro;g/L of IL-15-His, and d: 100 \u0026micro;g/L of IL-15-Flag.\u003c/p\u003e \u003cp\u003eThe results showed that only the sample containing anti-HSP70 antigen could make the color of the test layer disappear, while the other 3 columns showed bright blue. This indicated that the IAGC detection method had good specificity.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,33\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Validation of multi test layers in one column\u003c/h2\u003e \u003cp\u003eIn order to investigate the Validation of multi test layers in one column, the following series of experiments were carried out with HSP70 protein used as a model. In order to improve the test efficiency, a gel-column filled with 3 test layers was designed. The purpose was to investigate whether multiple test layers interfered with each other. The detailed steps were as follows: firstly, the sample containing HSP70, PCT and IL-15-His antigens respectively passed through the gel-column with single test layer coupled with anti-HSP70, PCT and IL-15-His antibodies respectively. Secondly, gel-column with 3 test layers coupled with anti- A, B, and C antibody separately was designed. The same sample was used to all the gel-columns (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. a, b, c, and d). Finally, color results were obtained and compared to examine the interference.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResults show that the detection layers in gel columns of a, b and c can effectively detect the mixture samples. The same samples can also be effectively detected using gel column d with three test layers, and there is no obvious interference between each detection layers. Therefore, the design of multiple inspection layers in one gel column can effectively detect mixture samples, which can greatly improve detection efficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Multiple columns detection\u003c/h2\u003e \u003cp\u003eTo eliminate the interference of multiple detection layers in one column completely, a multi-column is designed. That is, the same multiple detection columns are interconnected, and samples can flow freely between the three columns. The advantage of this design is that it cannot only realize multi-component detection, but also eliminate the interference between each detection layer. Since the sample is evenly divided among the three columns, the detection is more accurate. In addition, the detection flux is larger, especially suitable for high throughput, low concentration detection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Anti-HSP70, PCT and IL-15 His antibodies were chelated respectively in the gel columns of a, b and c. The results showed that the color of columns a and c completely disappeared, while column b did not change, indicating that the sample contained HSP70 and IL-15.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Validation of min sample volume\u003c/h2\u003e \u003cp\u003eAccording to the principle of immunity affinity, the larger the sample volume was, the darker the color produced. But at the same time, the adverse effect was that it produced poorer sensitivity. Therefore, the minimum solution volume was another important factor to be examined.\u003csup\u003e38\u003c/sup\u003e A series of volume gradient 1 \u0026micro;L, 5 \u0026micro;L, 10 \u0026micro;L, 15 \u0026micro;L, 30 \u0026micro;L respectively from left to right was designed. The results shown that the test layer has no color at all when the volume was greater than or equal to 10 \u0026micro;L. It was indicated that the minimum volume of the method was 10 \u0026micro;L.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results indicate that when the volume is equal to or greater than 10 \u0026micro;L, the test layer is no color at all. The reason is that all HSP70 antibody sites in the detection layer are occupied by antigens in the sample. As the increasing sample volume (such as d and e), there are no excess sites available for HSP70-HRP binding, so they all appear colorless.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Curve fitting\u003c/h2\u003e \u003cp\u003eVisual inspection could only provide rough qualitative analysis results and low sensitivity. To further improve sensitivity, the app \u003cem\u003eImage Recognition 1.0\u003c/em\u003e is developed. It can directly identify, fit curves, statistics, and save experimental results (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The detailed steps as follows: the entire gel-column was placed on a white background, and photos were taken using a smartphone and saved as JPEG format. The average of 3 points from each photo was taken as the color grayscale value. Quantitative analysis could be achieved according to the curve fitted on this numerical value.\u003csup\u003e39,40\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResults showed that the sensitivity was significantly improved benefitting from high pixel of smartphone combined with the app \u003cem\u003eImage Recognition 1.0\u003c/em\u003e. It was approximately 10-fold better with a quantitative LOD of 1 ng/mL than visual detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Comparisons with ELISA\u003c/h2\u003e \u003cp\u003eThe principle of IAGC assay and traditional ELISA was similar, and the main differences between them lied in carriers and the ways of immobilizing antibodies. Compared with ELISA, IAGC assay had unique advantages. Firstly, the sample loading capacity was much larger than ELISA, which could effectively improve the detection sensitivity especially for samples with low concentration. Secondly, the operation was very simple and fast, and there was almost no need for instruments. Finally, repeated washing was avoided, which not only saved time but also reduced the loss of analytes.\u003csup\u003e41\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison between IAGC assay with traditional ELISA.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIAGC assay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOD (ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 ng/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 ng/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecost\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehigh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ewashing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 time\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emany times\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ecarrier\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSepharose gel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epolystyrene well\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esuitable for the wild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eneed for instruments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003econnecting the purification device\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn summary, IAGC assay had significant advantages over ELISA in many aspects as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.8. Validation of the test using actual sample\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe experimental process was similar as above. The internal standard antigen was added to actual samples to test the practicality of this method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eB: grayscale value of the images, and dashed lines was the cut-off level.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this work, we have successfully developed an IAGC assay method able to carry out ultra-sensitive immunoassay with the advantages of sensitivity, velocity, simplicity, high-throughput and suitable for outdoor use. Since the IAGC assay method itself is a widely used technology basing on the immunoaffinity principle, our improvements should make it has more potential application scenarios than other traditional immunoassays. The new assay method can detect actual samples without special instruments and make the sensitivity higher than lateral-flow strip, simpler than ELISA detection kit. Experiments show that detection limit is 10 \u0026micro;g/L, which is 10 times higher than lateral-flow strip. The new method can effectively detect multiple samples in one gel-column by changing the layout of the detection layers in the gel column. Results also showed that the new method has good specificity and broad application prospects. We believe that IAGC assay can be exploited to wider applicability for the analysis of trace biomarkers in clinical analysis, biological research and military training.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIAGC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmune affinity gel-column\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSP70\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eheat shock protein 70\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethe detection limit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehorseradish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebovine serum albumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3,3,5,5-tetramethylbenzidine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edimethyl sulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eroom temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePro Calcitonin.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Fund of Research on High Throughput Rapid Detection Technology for Pathogens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting our findings is contained within the manuscript; any additional data will be shared upon request to the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHJL and QQL designed the study, performed the experiments and wrote the manuscript. ZZ, JXZ, XYL and ZZ analysed the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einterests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProf. Dr. Zhe Zhou is the professor of Bioinformatics Center of AMMS, Beijing, China.\u0026nbsp;He has over 30 years of experience in the field of immunoassay.\u003c/p\u003e\n\u003cp\u003eDr.\u0026nbsp;Hongjun Linis the associate professor of Bioinformatics Center of AMMS, Beijing, China. He has over 20 years of experience in the field of immunoassay.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmed S, Ashraf M, Sahanand S, Rajan DV. Indian J Orthop. 2021;55:402\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndryskova A, Lee JH. 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Anal Methods. 2009;1:170\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai J, Zhang H, Huang C, Chen ZY, Han A. Anal Chem. 2020;92:16122\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim YI, Cho JY. Biochim Biophys Acta Proteins Proteom. 2019;1867:9\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang XM, Luo PJ, Chen JL, Huang YT, Jiang WX. Int Dairy J. 2016;55:59\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe GZ, Dong T, Yang ZC, Branstad A, Huang L, Jiang ZD. Analyst. 2022;147:1273\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUno N, Li Z, Liu CC. Analyst. 2023;148:926\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HSP70, gel-column, on-site detection","lastPublishedDoi":"10.21203/rs.3.rs-4593110/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4593110/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA modified immune affinity gel-column (IAGC) assay was developed for rapid detection biomarkers of heat shock protein 70 (HSP70). The new method presets antibodies in a gel for rapid detection of the target antigen flowing through it. No special instruments and cumbersome processing steps were required throughout the entire process. The whole detection was completed within 10 min and a high-throughput could be achieved. Because the detection gel is in a transparent cylinder, color changes can be observed more sensitively. The detection limit (LOD) of the newly established method can be as low as 10 \u0026micro;g/L, which is significantly better than the existing lateral-flow strip method. In addition, the method is suitable for outdoor use, especially in laboratories with relatively simple conditions. In addition, the new detection method can also be designed into multiple detection layers in series or multiple detection columns in parallel to further improve the detection efficiency. Therefore, the new method is a fast, simple, high-throughput, qualitative and semi-quantitative detection platform, and a new idea for rapid detection.\u003c/p\u003e","manuscriptTitle":"Visual Non-Instrumental On-Site Detection of HSP70 Using a Modified Immune Affinity Gel-column Assay","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-09 18:09:57","doi":"10.21203/rs.3.rs-4593110/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6470d346-f55b-4d09-bf9e-3959c29a49ad","owner":[],"postedDate":"July 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-28T04:23:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-09 18:09:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4593110","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4593110","identity":"rs-4593110","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}