Serum-equivalency Comparison, Detection, and Quantification of Group B Streptococcus Anti-capsular Polysaccharide Antibodies from Dried Blood Spots

Serum-equivalency Comparison, Detection, and Quantification of Group B Streptococcus Anti-capsular Polysaccharide Antibodies from Dried Blood Spots | 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 Serum-equivalency Comparison, Detection, and Quantification of Group B Streptococcus Anti-capsular Polysaccharide Antibodies from Dried Blood Spots Shanna Bolcen, Palak Y. Patel, Bailey Alston, Yikun Li, Panagiotis Maniatis, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5347662/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 standardized multiplex immunoassay (MIA) to quantify group B Streptococcus (GBS) anti-capsular polysaccharide (CPS) IgG serum concentrations was adopted by the G roup B streptococcal A ssay St andardizati on (GASTON) consortium as a standardized assay with the most immediate applications for facilitating the licensure of GBS vaccines. However, dried blood spot (DBS) samples offer advantages for immunological studies, including cost-effectiveness, ease of transport, and storage. To determine suitability of DBS as an alternative sample matrix to serum in multiplex immunoassays, a contrived GBS seropositive panel, including matched DBS and serum samples, was prepared using established methods. The calculated geometric mean titers of GBS anti-CPS IgG values by individual serotype were compared using a paired t-test to establish serum equivalency. Geometric mean values for the matched panel were assessed via Deming regression for precision, accuracy, and concordance correlation coefficient (CCC). The initial acceptance criterion was set at 0.95 for CCC. Two additional criteria based on confidence intervals of CCC, slope, and intercept were used to determine the necessity of a serotype-specific conversion factor. The paired t-test p-values were > 0.05 for serum equivalency. For sample matrix concordance, CCC values were > 0.95 and met correlation criteria for all serotypes. Conversion factors were applied to four serotypes (II, III, IV, and V) that did not meet the criteria for slope, intercept, or both. This demonstration of equivalency between DBS and serum supports the hypothesis that DBS is a suitable testing matrix from which to elucidate anti-CPS IgG concentrations in seroepidemiological and vaccine evaluation studies. GBS Antibody Dried blood spot sample Serum Multiplex immunoassay Figures Figure 1 Figure 2 Figure 3 Introduction Group B Streptococcus (GBS), an encapsulated gram-positive pathogen, is a leading cause of neonatal and infant sepsis and meningitis in the first 90 days of life, contributing to infant morbidity and mortality globally. It has been associated with stillbirth, preterm delivery, and maternal invasive disease during pregnancy and in the post-partum period [ 1 , 2 ]. Given the limitations of the currently recommended intrapartum antibiotic prophylaxis (IAP), development of standard of care maternal vaccines has been identified as a critical intervention to reduce the disease burden of neonatal GBS [ 2 – 5 ]. High concentrations of serotype-specific maternal anti-capsular polysaccharide (CPS) IgG antibody are associated with protection and reduced infant disease via natural immunity [ 7 – 9 ]. The CPS envelopes the bacterial cell and is a critical component in the ability to evade the host’s immune system defense mechanism of opsonophagocytosis and is therefore a key contributor to GBS’s virulence [ 6 ]. Quantified anti-CPS IgG from case-control natural history studies, may be used to determine antibody levels associated with protection via risk reduction curves and establish a correlate of protection for vaccine licensure [ 2 , 7 – 9 ]. However, the use of variable assay methods and reagents in studies attempting to define anti-CPS IgG protective thresholds has complicated interpretation and limited progress towards globally accepted correlates of protection for neonatal invasive GBS disease [ 2 , 9 ]. A Pfizer-developed 6-plex GBS CPS IgG direct immunoassay was adopted by the G roup B streptococcal A ssay St andardizati on (GASTON) consortium as a standardized assay for quantification of IgG concentrations for the six predominant capsular serotypes of GBS: Ia, Ib, II, III, IV, and V [ 9 , 10 ]. The GASTON assay was originally developed for serum, but alternative sample matrices will allow for greater flexibility in future studies. Dried blood spot (DBS) samples offer advantages over serum including overall reduced sample volume, cost, and need for special transport and storage conditions. In high-resource countries, DBS are routinely collected from neonates soon after birth as part of newborn screening programs to assess other health conditions. Remnant DBS samples from these collections offer an alternative to cord blood samples, which are not routinely stored for retrospective studies [ 11 – 14 ]. DBS have proven to be a simple and powerful tool for large-scale investigation and seroepidemiological studies across multiple pathogens [ 11 – 17 ]. The choice of punch size (3mm versus 6mm) depends on specific test requirements and sample availability, and the average volume for 3- and 6-mm punches is 1.6 ± 0.4 µL and 8.7 ± 1.9 µL, respectively [ 11 , 18 – 20 ]. In this study, IgG concentrations were measured to assess DBS punch size conditions for equivalency in addition to evaluating the correlation between serum and DBS anti-GBS CPS IgG concentrations, thus establishing DBS as a suitable alternative sample matrix for the standardized GASTON multiplex immunoassay (MIA). Materials and methods DBS and Serum Panel Preparation Thirty-one adult and two infant cord blood samples that spanned the dynamic range of the assay were prepared using fresh EDTA whole blood samples received from commercial biorepositories or through the Occupational Health and Wellness program at Pfizer (Pearl River, NY). Upon receipt, blood samples were centrifuged at 950 x g for 20 minutes. A calculated volume of the plasma fraction was replaced with an equivalent volume of sera from GBS6 immunized subjects (NCT03170609, [ 21 ]) to obtain appropriate GBS anti-CPS IgG concentrations. Minimal volumes of the plasma fraction were replaced with GBS6 immunized sera to maintain EDTA concentrations to prevent coagulation. This contrived blood sample was then resuspended and used to prepare the DBS cards (PerkinElmer Health Sciences Inc., Catalog # GR2261002). DBS cards were prepared by adding 50 µl of the contrived blood sample onto the indicated spot on the DBS card, allowed to dry for ≥ 4 hours, and then stored at -80°C with humidity sponges. The remaining contrived blood sample was then centrifuged at 950 x g for 20 minutes and the serum was removed and stored at -80°C until testing in the MIA. Elution of DBS DBS were eluted using standardized practices [ 11 ] with the MIA assay buffer serving as the elution buffer (0.5% BSA in 10mM PBS/0.05% Tween-20/0.02% NaN 3 , pH 7.2). In brief, punches were taken from DBS cards using a manual, hand-held hole punch at the appropriate diameter and punched spots were collected directly into spin columns (0.45 µm Spin-X microcentrifuge filter tubes, Costar Catalog #8163). After adding 150 µL of elution buffer to the column, filter tubes were placed on an orbital shaker for 10 ± 5 minutes at room temperature (15°C to 30°C), after which the punched spots were checked for complete immersion and the tubes incubated overnight (16–20 hours) at 2° to 8°C. After incubation, the orbital shaker procedure was repeated, and tubes were centrifuged at 15,000 x g for 3–5 minutes to collect the eluent. Total volume recovered was ≥ 90% of original assay buffer volume. Multiplex Immunoassay (MIA) The previously published MIA [ 9 , 10 ] was modified for application to DBS eluents. Elution of the DBS into 150 µL of buffer served as the initial dilution and dilution factor was determined by the number and size of DBS punches used [Table 1 ]. Table 1 Initial dilution and dilution series by sample condition Sample Initial Dilution Dilution Series One 3 mm punch 1:100 1:200, 1:2,000, 1:20,000 Two 3 mm punches 1:50 1:100, 1:1,000, 1:10,000 Three 3 mm punches 1:33.3 1:66.7, 1:667, 1:6,670 Four 3 mm punches 1:25 1:50, 1:500, 1:5000 One 6 mm punch 1:25 1:50, 1:500, 1:5000 Punch Size Equivalency Determination Previous data equates a 3 mm punch to approximately 1.5 µL of serum [ 11 , 18 ]. To verify expected serum-equivalent recovery rates between DBS punch sizes (3 mm and 6 mm), a panel of seven individual DBS samples was created. From each sample, one-, two-, three-, and four-3 mm (1/8 in) punches were compared against one-6 mm (1/4 in) punch. Each punch size condition was tested a minimum of three times on three different days by two independent operators resulting in a total ≥ 108 analytic endpoints. DBS to Serum Equivalency To establish the correlation between serum and DBS using a serum reference standard, a panel of 33 matched DBS-serum sample sets as described above were tested for each serotype. Matched sample testing used the optimal number of 3 mm punches, established by the above-described punch size equivalency testing and was performed by two independent operators and repeated four times on four different days. Data and Statistical Analysis Raw MFI values from the MIA were evaluated against a human serum reference standard curve with weight-based anti-CPS IgG assignments (µg/mL) [ 22 ] using a log-log linear regression-based algorithm in a validated SAS application (Pfizer). This approach enables comparison of antibody concentrations across serotypes. All samples with a concentration coefficient of variation (CV) > 30% were excluded from the analyses. Statistical analysis was modeled after previously published serum to DBS bridging experiments [ 11 ]. Equivalence of log 10 -transformed sample anti-CPS IgG concentrations eluted from different punch sizes was assessed based on the coefficient of determination (R²) from regression analysis, with acceptance criteria set at ≥ 0.95. Additionally, the 95% confidence interval (CI) around the intercept needed to contain the value zero (indicating that at the baseline level, the two methods yield similar results with no significant systematic difference), and the 95% CI around the slope needed to contain the value 1 (indicating no significant proportional difference across the range). A paired t-test was used to assess individual serotypes once a target punch condition was selected for comparison against the 6mm punch. Equivalence between log 10 transformed concentrations in paired serum and DBS eluents was assessed using the Concordance Correlation Coefficient (CCC), where the lower 95% of confidence interval (CI) was set at ≥ 0.95 [ 11 , 23 ]. Deming regression [ 11 , 23 ] between the two sample types was assessed to ascertain whether a serum-to-DBS conversion factor was needed. If the 95% CI for the intercept did not contain the value 0 or 95% CI for the slope did not contain the value 1, a conversion factor was applied [ 24, 25]. Results Punch Size Equivalency Determination The anti-CPS IgG geometric mean concentrations of the seven samples tested were compared by sample punch size condition against the one-6 mm punch anti-CPS IgG concentration [Fig. 1 ]. The four-3mm punch condition was found to be equivalent to one-6 mm punch condition and was selected for additional evaluation. Anti-CPS IgG geometric mean concentrations across all six serotypes in the assay between the two punch size conditions were highly correlated (R 2 = 0.9972) by regression analysis [Supplemental Table 1, Fig. 2 ] and met the acceptance criteria set at > 0.95 and 95% CI where the intercept contained the value 0 and slope contained the value 1 [Fig. 2 ]. Paired t-test p-values were > 0.05 [range 0.2031 (GBS Ib) – 0.8317 (GBS IV)] for all serotypes in the four-3mm punches versus one-6 mm punch comparison [Table 2 ]. Table 2 Paired t-test results for one-6 mm punch compared to 3 mm punches by serotype (pr (> |t|)) with final concentrations held at the same dilution Serotype One-6mm/four-3mm One-6mm/three-3mm One-6mm/two-3mm One-6mm/one-3mm GBSIA 0.8304 < .0001 0.0001 < .0001 GBSIB 0.8183 < .0001 < .0001 < .0001 GBSII 0.3409 0.1455 0.0022 < .0001 GBSIII 0.4088 < .0001 < .0001 < .0001 GBSIV 0.1788 0.0286 0.0019 < .0001 GBSV 0.1693 < .0001 < .0001 < .0001 DBS to Serum Equivalency Initial concordance between the DBS and serum tested was established by CCC by individual serotypes and ranged from 0.9899–0.9981 [Fig. 3 ]. Accuracy and precision were ≥ 0.98 for all serotypes [Fig. 3 ]. The slope and intercept of the Deming regression best fit lines varied by serotype [Fig. 3 ; Table 3 ]. Criteria for conversion factor application [Table 3 ; Supplemental Table 2] for increased accuracy acceptance was met for two serotypes (GBS Ia and GBS Ib). For two serotypes (GBS II and GBS V), the 95% CI intercept did not contain the value 0 and, for one serotype (GBS III), the 95% CI for slope did not contain the value 1. Both intercept and slope criteria failed for GBS IV. *Samples excluded from analysis due to sample CV. For detailed reportable values, refer to Supplementary Table 2 Table 3 Summary of conversion formula for each serotype Serotype Observed value with 95% Confidence interval (CI) Conversion factor Applied (Yes/No) Conversion formula Intercept Slope GBSIa -0.0022 [-0.0329, 0.0284] 1.0185 [0.9981, 1.0389] No NA GBSIb 0.0189 [-0.0089, 0.0467] 1.0170 [0.9921, 1.0418] No NA GBSII 0.0945 [0.0189, 0.1701] 0.9889 [0.9312, 1.0467] Yes LOG_VALUE = 0.0945 + log10(DBS_RESULT), CONVERTED_VALUE = 10**LOG_VALUE GBSIII 0.0177 [-0.0098, 0.0453] 1.0251 [1.0044, 1.0459] Yes LOG_VALUE = 1.0251*log10(DBS_RESULT), CONVERTED_VALUE = 10**LOG_VALUE GBSIV 0.0491 [0.0206, 0.0777] 1.0337 [1.0191, 1.0483] Yes LOG_VALUE = 0.0491 + 1.0337*log10(DBS_RESULT), CONVERTED_VALUE = 10**LOG_VALUE GBSV 0.0703 [0.0317, 0.1090] 1.0045 [0.9637, 1.0454] Yes LOG_VALUE = 0.0703 + log10(DBS_RESULT), CONVERTED_VALUE = 10**LOG_VALUE Discussion This manuscript presents a first study of its kind in a controlled setting to systematically demonstrate the suitability of DBS in the standardized multiplex immunoassay adopted by the GASTON consortium to quantify IgG antibodies against the most prevalent GBS CPS serotypes. The use of DBS has expanded to evaluate a plethora of biomarkers [ 26 ] in recent years, given that collection is minimally invasive, requires less skilled labor, and uses fewer materials during sample collection compared to venipuncture. Additionally, DBS are easier and more efficient to transport from collection sites as they can be maintained at room temperature for up to 16 weeks without loss of biomarker functionality once the sample has dried and do not require cold-chain transportation [ 11 , 13 , 14 , 17 , 26 – 28 ]. In addition to the utility of the sample matrix, DBS have been used in newborn screening programs since the 1960s and large sample collections are available for potential use in seroepidemiological studies [ 26 ]. In this study, the use of a matched serum and DBS panel allowed for the direct comparison of quantifiable anti-CPS IgG concentrations from two distinct sample matrices that were created from the same source material. When the matched DBS-serum sample pairs were tested, concordance > 0.95 was demonstrated for all six GBS CPS serotypes captured by the assay. In addition to logarithmically transformed regression analysis, prior studies relied on CCC [ 11 ], Pearson correlation coefficient (PCC) [ 29 ], Spearman's rank correlation coefficient [ 29 ], or Cohen's kappa coefficient [ 13 , 17 ] to assess agreement between the sample matrices. This study used an additional layer of stringency through the examination of the CI around the parameter estimates for the slope and intercept, followed by establishing a DBS-to-serum conversion factor for serotypes that did not meet the added criteria. Of the six GBS CPS serotypes assessed, four (GBS II, III, IV, and V) were found to increase result accuracy with the application of a constant multiplier as the conversion factor. Versatility in sample size collection from the DBS through different punch sizes allows for great flexibility in testing when using remnant dried blood spot samples from historic collections. We successfully demonstrated punch size equivalency that verified previously established DBS-punch-size to serum-volume values [ 18 ]. From the geometric mean anti-CPS IgG concentrations of the seven samples tested, it was determined that the four-3 mm punch size was the best fit for overall sample equivalency to the one-6 mm punch and could be used interchangeably in the assay. This allows the researcher to use 3mm punches when larger punches cannot be accommodated. These results thus add value by allowing the use of different punch sizes; for example, researchers could include specimens that have undergone prior testing such as remnant newborn screening samples, in which punches have already been removed from the filter paper. In this study, bridging between the sample matrixes was performed on primarily adult specimens and may not be fully representative of infant samples that may have higher hematocrit concentrations. Hematocrit levels can affect the diffusion of the blood on the filter paper at the time of collection and influence the distribution of antibodies within the DBS sample [ 30 ]. Conclusion In a controlled setting using contrived panels, the use of DBS as a testing matrix in the quantification of GBS anti-CPS antibodies was successfully demonstrated. These results not only advance GBS-related seroepidemiological studies with DBS but also impact the work of the GASTON consortium and the broader GBS community striving for licensure of a maternal vaccine against invasive GBS disease in infants. Declarations Conflict of interest DGS, DP, MG, RS, and NCSM are employees at Pfizer, Inc, and may be shareholders of Pfizer Inc. No other conflicts of interest for other authors. Competing Interests DGS, DP, MG, RS, and NCSM are employees at Pfizer, Inc, and may be shareholders of Pfizer Inc. No other conflicts of interest for other authors. Funding This work was supported by the Centers for Disease Control and Prevention (CDC) and funded by the Bill & Melinda Gates Foundation, Seattle, WA, INV-008343. The funders had no role in the design, analysis, or interpretation of the data in this research study, in the writing of the report, or the decision to publish. Author Contribution Conceived and designed the experiments: SB, PM, LTJ. Performed the experiments: PYP, BA. Provided critical reagents and technical support: DGS, DP, MG, RS, NCSM, PM, JS, and SAV. Analyzed the data: SB, YL, and LTJ. Wrote the paper: SB, SAV. Consultants: JR and SS. All authors reviewed the manuscript. Acknowledgement We would like to thank Richie Ramdhanie (Pfizer) for their technical assistance in preparing the sample panels. Statement of ethics approval: This activity was reviewed by CDC, deemed research not involving human subjects, and was conducted consistent with applicable federal law and CDC policy.§ §See e.g., 45 C.F.R. part 46; 21 C.F.R. part 56; 42 U.S.C. §241(d), 5 U.S.C. §552a, 44 U.S.C. §3501 et seq. References Le Doare K, Kampmann B, Vekemans J, Heath PT, Goldblatt D, Nahm MH, Baker C, Edwards MS, Kwatra G, Andrews N, Madhi SA, Ter Meulen AS, Anderson AS, Corsaro B, Fischer P, Gorringe A (2019) Serocorrelates of protection against infant group B streptococcus disease. Lancet Infect Dis 19(5):e162–e171. https://doi.org/10.1016/S1473-3099(18)30659-5 Absalon J, Simon R, Radley D, Giardina PC, Koury K, Jansen KU, Anderson AS (2022) Advances towards licensure of a maternal vaccine for the prevention of invasive group B streptococcus disease in infants: a discussion of different approaches. Hum Vaccin Immunother 18(1):2037350. https://doi.org/10.1080/21645515.2022.2037350 Schrag SJ, Verani JR (2013) Intrapartum antibiotic prophylaxis for the prevention of perinatal group B streptococcal disease: experience in the United States and implications for a potential group B streptococcal vaccine. Vaccine 31 Suppl 4D20–26. https://doi.org/10.1016/j.vaccine.2012.11.056 Jordan HT, Farley MM, Craig A, Mohle-Boetani J, Harrison LH, Petit S, Lynfield R, Thomas A, Zansky S, Gershman K, Albanese BA, Schaffner W, Schrag SJ (2008) Revisiting the need for vaccine prevention of late-onset neonatal group B streptococcal disease: a multistate, population-based analysis. Pediatr Infect Dis J 27(12):1057–1064. https://doi.org/10.1097/INF.0b013e318180b3b9 . Active Bacterial Core Surveillance (ABCs)/Emerging Infections Program Network Group B (2021) streptococcus vaccine: full value of vaccine assessment. Executive summary. World Health Organization, Geneva. License: CC BY-NC-SA 3.0 IGO Marques MB, Kasper DL, Pangburn MK, Wessels MR (1992) Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci. Infect Immun 60(10):3986–3993. https://doi.org/10.1128/iai.60.10.3986-3993.1992 Baker CJ (2023) Group B Streptococcal Vaccine - Sisyphus Reconciled. N Engl J Med Jul 20;389(3):275–277. 10.1056/NEJMe2306234 . PMID: 37467503 Madhi SA, Anderson AS, Absalon J, Radley D, Simon R, Jongihlati B, Strehlau R, van Niekerk AM, Izu A, Naidoo N, Kwatra G, Ramsamy Y, Said M, Jones S, Jose L, Fairlie L, Barnabas SL, Newton R, Munson S, Jefferies Z, Pavliakova D, de Silmon NC, Gomme E, Perez JL, Scott DA, Gruber WC, Jansen KU (2023) Potential for Maternally Administered Vaccine for Infant Group B Streptococcus. N Engl J Med Jul 20;389(3):215–227. 10.1056/NEJMoa2116045 . PMID: 37467497 Le Doare K, Gaylord MA, Anderson AS, Andrews N, Baker CJ, Bolcen S, Felek A, Giardina PC, Grube CD, Hall T, Hallis B, Izu A, Madhi SA, Maniatis P, Matheson M, Mawas F, McKeen A, Rhodes J, Alston B, Patel P, Schrag S, Simon R, Tan CY, Taylor S, Kwatra G, Gorringe A (2024) Interlaboratory comparison of a multiplex immunoassay that measures human serum IgG antibodies against six-group B streptococcus polysaccharides. Hum Vaccin Immunother 20(1):2330138. https://doi.org/10.1080/21645515.2024.2330138 Gaylord MA, Larrier M, Giordano-Schmidt D, Grube CD, Singh S, Nguyen HH, McKeen A, Tan CY, Anderson AS, Kalina WV, Pavliakova D, Giardina PC (2024) Development and validation of a 6-plex Luminex-based assay for measuring human serum antibodies to group B streptococcus capsular polysaccharides. Hum Vaccin Immunother 20(1):2311480. https://doi.org/10.1080/21645515.2024.2311480 Schiffer JM, Maniatis P, Garza I, Steward-Clark E, Korman LT, Pittman PR, Mei JV, Quinn CP (2013) Quantitative assessment of anthrax vaccine immunogenicity using the dried blood spot matrix. Biologicals 41(2):98–103. https://doi.org/10.1016/j.biologicals.2012.11.003 Andersen NJ, Mondal TK, Preissler MT, Freed BM, Stockinger S, Bell E, Druschel C, Louis GM, Lawrence DA (2014) Detection of immunoglobulin isotypes from dried blood spots. J Immunol Methods 404:24–32. https://doi.org/10.1016/j.jim.2013.12.001 Kouamé DR, Aboli RA, Kabran M, Adiko AC, Coulibaly M, Dembele B, Inwoley A (2023) Validation of dried blood spots samples for the screening of hepatitis B virus surface by enzyme immunoassay method. J Immunol Methods 513:113412. https://doi.org/10.1016/j.jim.2022.113412 Villar LM, de Oliveira JC, Cruz HM, Yoshida CF, Lampe E, Lewis-Ximenez LL (2011) Assessment of dried blood spot samples as a simple method for detection of hepatitis B virus markers. J Med Virol 83(9):1522–1529. https://doi.org/10.1002/jmv.22138 Rajam G, Carlone G, Kim E, Choi J, Paulos S, Park S, Jeyachandran A, Gorantla Y, Wong E, Sabnis A, Browning P, Desai R, Quinn CP, Schiffer J (2019) Development and validation of a robust multiplex serological assay to quantify antibodies specific to pertussis antigens. Biologicals 57:9–20. https://doi.org/10.1016/j.biologicals.2018.11.001 Zhao H, Wang M, Muthelo P, Lof L, Sterky F, Gallini R, Kumar NV, Monsen T, Nilsson K, Aberg M, Kamali-Moghaddam M, Mei YF, Landegren U (2022) Detection of SARS-CoV-2 antibodies in serum and dried blood spot samples of vaccinated individuals using a sensitive homogeneous proximity extension assay. N Biotechnol 72:139–148. https://doi.org/10.1016/j.nbt.2022.11.004 Omosule CL, Conklin J, Seck S, Howell R, Hock KG, Ballman C, Freeman J, Du Toit L, Dubberke E, Farnsworth CW (2023) Qualitative and quantitative detection of SARS-CoV-2 antibodies from dried blood spots. Clin Biochem 117:16–22. https://doi.org/10.1016/j.clinbiochem.2021.12.012 National Center for Environmental Health (2020) Newborn Screening Quality Assurance Program 2021 Annual Summary Report, Volume 39a. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. https://www.cdc.gov/labstandards/pdf/nsqap/NSQAP_Annual_Summary_2019-508.pdf Mercader S, Featherstone D, Bellini WJ (2006) Comparison of available methods to elute serum from dried blood spot samples for measles serology. J Virol Methods Oct 137(1):140–149. 10.1016/j.jviromet.2006.06.018 Epub 2006 Jul 24. PMID: 16860401 Hewawasam E, Liu G, Jeffery DW, Gibson RA, Muhlhausler BS (2018) Estimation of the Volume of Blood in a Small Disc Punched From a Dried Blood Spot Card. Eur J Lipid Sci Technol Vol.120(3);n pag. Web. 10.1002/ejlt.201700362 Absalon J, Segall N, Block SL, Center KJ, Scully IL, Giardina PC, Peterson J, Watson WJ, Gruber WC, Jansen KU, Peng Y, Munson S, Pavliakova D, Scott DA, Anderson AS (2021) Safety and immunogenicity of a novel hexavalent group B streptococcus conjugate vaccine in healthy, non-pregnant adults: a phase 1/2, randomised, placebo-controlled, observer-blinded, dose-escalation trial. Lancet Infect Dis Feb 21(2):263–274. 10.1016/S1473-3099(20)30478-3 Esadze A, Grube CD, Wellnitz S, Singh S, Nguyen HH, Gaylord MA, Zhu A, Gribenko A, Tan CY, Anderson AS, Simon R (2023) Calibration of a serum reference standard for Group B streptococcal polysaccharide conjugate vaccine development using surface plasmon resonance. NPJ Vaccines 8(1):71. https://doi.org/10.1038/s41541-023-00667-1 Lin L, Torbeck LD (1998) Coefficient of accuracy and concordance correlation coefficient: new statistics for methods comparison. PDA J Pharm Sci Technol 52(2):55–59 Armitage P, Berry G, Matthews JNS (2002) The Scope of Statistics. Blackwell Science Ltd Lahdentausta L, Kivimaki A, Oksanen L, Tallgren M, Oksanen S, Sanmark E, Salminen A, Geneid A, Sairanen M, Paju S, Saksela K, Pussinen P, Pietiainen M (2022) Blood and saliva SARS-CoV-2 antibody levels in self-collected dried spot samples. Med Microbiol Immunol 211(4):173–183. https://doi.org/10.1007/s00430-022-00740-x Gruner N, Stambouli O, Ross RS (2015) Dried blood spots–preparing and processing for use in immunoassays and in molecular techniques. J Vis Exp (97) https://doi.org/10.3791/52619 Behets F, Kashamuka M, Pappaioanou M, Green TA, Ryder RW, Batter V, George JR, Hannon WH, Quinn TC (1992) Stability of human immunodeficiency virus type 1 antibodies in whole blood dried on filter paper and stored under various tropical conditions in Kinshasa, Zaire. J Clin Microbiol 30(5):1179–1182. https://doi.org/10.1128/jcm.30.5.1179-1182.1992 Zhang SL, McGann CM, Duranova T, Strysko J, Steenhoff AP, Gezmu A, Nakstad B, Arscott-Mills T, Bayani O, Moorad B, Tlhako N, Richard-Greenblatt M, Planet PJ, Coffin SE, Silverman MA (2024) Maternal and neonatal IgG against Klebsiella pneumoniae are associated with broad protection from neonatal sepsis: a case-control study of hospitalized neonates in Botswana. medRxiv [Preprint] May 28. 2024.05.28.24308042 Cornbleet PJ, Gochman N (1979) Incorrect least-squares regression coefficients in method-comparison analysis. Clin Chem 25(3):432–438. https://doi.org/10.1093/clinchem/25.3.432 Denniff P, Spooner N (2010) The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs. Bioanalysis Aug 2(8):1385–1395. 10.4155/bio.10.103 Erratum in: Bioanalysis Additional Declarations Competing interest reported. DGS, DP, MG, RS, and NCSM are employees at Pfizer, Inc, and may be shareholders of Pfizer Inc. No other conflicts of interest for other authors. Supplementary Files SerumequivalencyComparisonDetectionandQuantificationofGBSAntibodiesfromDBSSupplementaryInformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5347662","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":377443098,"identity":"5179cdc4-3804-4e0b-8307-d25c21ec7d56","order_by":0,"name":"Shanna Bolcen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoklEQVRIiWNgGAWjYBAC9h4GhgMfGJhJ0MJzhoHx4QxStTAb85CmheeMmbRNjbU8A//iYxLEaeHtMZPOOZZu2CDxLI04Lfb8PGbSuQ2HExgkzhgbEGcLSIslaVp4e4yNGUFa+HsMHxCnhedY4cMeoF/aJNgSidWSvOHAD2CI8fMfPnCAKC0MDBwQH7BJJBCpAZhkoM7hJ9aOUTAKRsEoGHEAAEzhKMmdQ1EtAAAAAElFTkSuQmCC","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":true,"prefix":"","firstName":"Shanna","middleName":"","lastName":"Bolcen","suffix":""},{"id":377443099,"identity":"a9889d72-4108-423b-9626-10e9bf23bbf5","order_by":1,"name":"Palak Y. Patel","email":"","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Palak","middleName":"Y.","lastName":"Patel","suffix":""},{"id":377443100,"identity":"26cad7da-e669-4626-9dad-c935597b046f","order_by":2,"name":"Bailey Alston","email":"","orcid":"","institution":"Eagle Global Scientific","correspondingAuthor":false,"prefix":"","firstName":"Bailey","middleName":"","lastName":"Alston","suffix":""},{"id":377443101,"identity":"8d0e9e9c-3d7d-47c1-8637-d03151b080b2","order_by":3,"name":"Yikun Li","email":"","orcid":"","institution":"IHRC, Inc","correspondingAuthor":false,"prefix":"","firstName":"Yikun","middleName":"","lastName":"Li","suffix":""},{"id":377443102,"identity":"390ed7ad-b808-48e1-957b-4e3efc53a68f","order_by":4,"name":"Panagiotis Maniatis","email":"","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Panagiotis","middleName":"","lastName":"Maniatis","suffix":""},{"id":377443103,"identity":"1ce34d06-0297-4693-a640-b2ec645bfe78","order_by":5,"name":"Donna Giordano Schmidt","email":"","orcid":"","institution":"Pfizer Inc. Pearl River","correspondingAuthor":false,"prefix":"","firstName":"Donna","middleName":"Giordano","lastName":"Schmidt","suffix":""},{"id":377443104,"identity":"3a440807-621b-42f8-864a-4259fa9ba2fc","order_by":6,"name":"Danka Pavliakova","email":"","orcid":"","institution":"Pfizer Inc. Pearl River","correspondingAuthor":false,"prefix":"","firstName":"Danka","middleName":"","lastName":"Pavliakova","suffix":""},{"id":377443105,"identity":"8d6722b3-2518-48ac-8b63-2c115f3a32be","order_by":7,"name":"Jessica E. Southwell","email":"","orcid":"","institution":"Eagle Global Scientific","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"E.","lastName":"Southwell","suffix":""},{"id":377443106,"identity":"03339600-7b99-4cc9-897d-ce8bbab93016","order_by":8,"name":"Lily Tao Jia","email":"","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Lily","middleName":"Tao","lastName":"Jia","suffix":""},{"id":377443107,"identity":"a6bf9373-d488-4f90-8a0e-6b9ba30707b9","order_by":9,"name":"Michelle Gaylord","email":"","orcid":"","institution":"Pfizer Inc. Pearl River","correspondingAuthor":false,"prefix":"","firstName":"Michelle","middleName":"","lastName":"Gaylord","suffix":""},{"id":377443108,"identity":"87faff52-43de-409c-a6f7-cb7bc677da56","order_by":10,"name":"Raphael Simon","email":"","orcid":"","institution":"Pfizer Inc. Pearl River","correspondingAuthor":false,"prefix":"","firstName":"Raphael","middleName":"","lastName":"Simon","suffix":""},{"id":377443109,"identity":"466e2e5c-0f17-466f-94c9-d5f4cd9d83db","order_by":11,"name":"Natalie Clare Silmon Monerri","email":"","orcid":"","institution":"Pfizer Inc. Pearl River","correspondingAuthor":false,"prefix":"","firstName":"Natalie","middleName":"Clare Silmon","lastName":"Monerri","suffix":""},{"id":377443110,"identity":"0ec6107a-8fa1-483f-bab2-ca0f3828ec0d","order_by":12,"name":"Julia Rhodes","email":"","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Rhodes","suffix":""},{"id":377443111,"identity":"8891666a-3386-4924-82de-975ab542518d","order_by":13,"name":"Stephanie Schrag","email":"","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Schrag","suffix":""},{"id":377443112,"identity":"e7569777-5c18-4d55-9806-f9ffccc4f8dd","order_by":14,"name":"Sundaram Ajay Vishwanathan","email":"","orcid":"","institution":"Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Sundaram","middleName":"Ajay","lastName":"Vishwanathan","suffix":""}],"badges":[],"createdAt":"2024-10-28 13:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5347662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5347662/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69793614,"identity":"bab21035-5693-461c-9e36-68636cabffae","added_by":"auto","created_at":"2024-11-25 09:55:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":466283,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Box and scatter plot comparisons for the five sample punching conditions across all six serotypes with final concentrations adjusted for individual sample dilution\u003cstrong\u003e (B)\u003c/strong\u003e Box and scatter plot comparisons for the five sample punching conditions across all six serotypes with final concentrations held at the same dilution to assess the effect of the number of DBS punches\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5347662/v1/7da9dd36e10bb2f5f53adf4d.jpg"},{"id":69793615,"identity":"2af8578f-0082-4e30-bfe6-a68a4045b6ce","added_by":"auto","created_at":"2024-11-25 09:55:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39277,"visible":true,"origin":"","legend":"\u003cp\u003eLinear regression with coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) and 95% CI of slope and intercept between sample IgG concentration eluted from four-3 mm DBS punch size and one-6 mm punch size\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5347662/v1/63373c17016da81a1e8901e2.jpg"},{"id":69793610,"identity":"be3b8f1d-5dde-4dbc-ad8d-add5f96de262","added_by":"auto","created_at":"2024-11-25 09:55:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":887309,"visible":true,"origin":"","legend":"\u003cp\u003eDeming regression of log-transformed serotype-specific anti-GBS IgG geometric mean concentrations determined in serum and DBS, showing CCC and other parameters A - F.\u003c/p\u003e\n\u003cp\u003e*Samples excluded from analysis due to sample CV. For detailed reportable values, refer to Supplementary Table 2\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5347662/v1/95d267023a214969375928ed.jpg"},{"id":71399674,"identity":"826e086b-a646-4f58-8222-f90e9b36d0a0","added_by":"auto","created_at":"2024-12-14 09:02:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1718934,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5347662/v1/f30558be-5b9c-4a4a-83ab-4cf006117c3e.pdf"},{"id":69793611,"identity":"add2a26e-6ddc-4564-a6c3-1ee68df04247","added_by":"auto","created_at":"2024-11-25 09:55:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":244079,"visible":true,"origin":"","legend":"","description":"","filename":"SerumequivalencyComparisonDetectionandQuantificationofGBSAntibodiesfromDBSSupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5347662/v1/b5d34b349e32bdeac2c6ae3d.pdf"}],"financialInterests":"Competing interest reported. DGS, DP, MG, RS, and NCSM are employees at Pfizer, Inc, and may be shareholders of Pfizer Inc. No other conflicts of interest for other authors.","formattedTitle":"Serum-equivalency Comparison, Detection, and Quantification of Group B Streptococcus Anti-capsular Polysaccharide Antibodies from Dried Blood Spots","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGroup B \u003cem\u003eStreptococcus\u003c/em\u003e (GBS), an encapsulated gram-positive pathogen, is a leading cause of neonatal and infant sepsis and meningitis in the first 90 days of life, contributing to infant morbidity and mortality globally. It has been associated with stillbirth, preterm delivery, and maternal invasive disease during pregnancy and in the post-partum period [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Given the limitations of the currently recommended intrapartum antibiotic prophylaxis (IAP), development of standard of care maternal vaccines has been identified as a critical intervention to reduce the disease burden of neonatal GBS [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHigh concentrations of serotype-specific maternal anti-capsular polysaccharide (CPS) IgG antibody are associated with protection and reduced infant disease via natural immunity [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The CPS envelopes the bacterial cell and is a critical component in the ability to evade the host\u0026rsquo;s immune system defense mechanism of opsonophagocytosis and is therefore a key contributor to GBS\u0026rsquo;s virulence [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Quantified anti-CPS IgG from case-control natural history studies, may be used to determine antibody levels associated with protection via risk reduction curves and establish a correlate of protection for vaccine licensure [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the use of variable assay methods and reagents in studies attempting to define anti-CPS IgG protective thresholds has complicated interpretation and limited progress towards globally accepted correlates of protection for neonatal invasive GBS disease [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA Pfizer-developed 6-plex GBS CPS IgG direct immunoassay was adopted by the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eG\u003c/span\u003eroup B streptococcal \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eA\u003c/span\u003essay \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSt\u003c/span\u003eandardizati\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eon\u003c/span\u003e (GASTON) consortium as a standardized assay for quantification of IgG concentrations for the six predominant capsular serotypes of GBS: Ia, Ib, II, III, IV, and V [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The GASTON assay was originally developed for serum, but alternative sample matrices will allow for greater flexibility in future studies. Dried blood spot (DBS) samples offer advantages over serum including overall reduced sample volume, cost, and need for special transport and storage conditions. In high-resource countries, DBS are routinely collected from neonates soon after birth as part of newborn screening programs to assess other health conditions. Remnant DBS samples from these collections offer an alternative to cord blood samples, which are not routinely stored for retrospective studies [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. DBS have proven to be a simple and powerful tool for large-scale investigation and seroepidemiological studies across multiple pathogens [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The choice of punch size (3mm versus 6mm) depends on specific test requirements and sample availability, and the average volume for 3- and 6-mm punches is 1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 \u0026micro;L and 8.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 \u0026micro;L, respectively [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, IgG concentrations were measured to assess DBS punch size conditions for equivalency in addition to evaluating the correlation between serum and DBS anti-GBS CPS IgG concentrations, thus establishing DBS as a suitable alternative sample matrix for the standardized GASTON multiplex immunoassay (MIA).\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eDBS and Serum Panel Preparation\u003c/p\u003e \u003cp\u003eThirty-one adult and two infant cord blood samples that spanned the dynamic range of the assay were prepared using fresh EDTA whole blood samples received from commercial biorepositories or through the Occupational Health and Wellness program at Pfizer (Pearl River, NY). Upon receipt, blood samples were centrifuged at 950 x g for 20 minutes. A calculated volume of the plasma fraction was replaced with an equivalent volume of sera from GBS6 immunized subjects (NCT03170609, [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]) to obtain appropriate GBS anti-CPS IgG concentrations. Minimal volumes of the plasma fraction were replaced with GBS6 immunized sera to maintain EDTA concentrations to prevent coagulation. This contrived blood sample was then resuspended and used to prepare the DBS cards (PerkinElmer Health Sciences Inc., Catalog # GR2261002). DBS cards were prepared by adding 50 \u0026micro;l of the contrived blood sample onto the indicated spot on the DBS card, allowed to dry for \u0026ge;\u0026thinsp;4 hours, and then stored at -80\u0026deg;C with humidity sponges. The remaining contrived blood sample was then centrifuged at 950 x g for 20 minutes and the serum was removed and stored at -80\u0026deg;C until testing in the MIA.\u003c/p\u003e \u003cp\u003eElution of DBS\u003c/p\u003e \u003cp\u003eDBS were eluted using standardized practices [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] with the MIA assay buffer serving as the elution buffer (0.5% BSA in 10mM PBS/0.05% Tween-20/0.02% NaN\u003csub\u003e3\u003c/sub\u003e, pH 7.2). In brief, punches were taken from DBS cards using a manual, hand-held hole punch at the appropriate diameter and punched spots were collected directly into spin columns (0.45 \u0026micro;m Spin-X microcentrifuge filter tubes, Costar Catalog #8163). After adding 150 \u0026micro;L of elution buffer to the column, filter tubes were placed on an orbital shaker for 10\u0026thinsp;\u0026plusmn;\u0026thinsp;5 minutes at room temperature (15\u0026deg;C to 30\u0026deg;C), after which the punched spots were checked for complete immersion and the tubes incubated overnight (16\u0026ndash;20 hours) at 2\u0026deg; to 8\u0026deg;C. After incubation, the orbital shaker procedure was repeated, and tubes were centrifuged at 15,000 x g for 3\u0026ndash;5 minutes to collect the eluent. Total volume recovered was \u0026ge;\u0026thinsp;90% of original assay buffer volume.\u003c/p\u003e \u003cp\u003eMultiplex Immunoassay (MIA)\u003c/p\u003e \u003cp\u003eThe previously published MIA [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] was modified for application to DBS eluents. Elution of the DBS into 150 \u0026micro;L of buffer served as the initial dilution and dilution factor was determined by the number and size of DBS punches used [Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\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\u003eInitial dilution and dilution series by sample condition\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 \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial Dilution\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDilution Series\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOne 3 mm punch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:200, 1:2,000, 1:20,000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTwo 3 mm punches\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:100, 1:1,000, 1:10,000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThree 3 mm punches\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:33.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:66.7, 1:667, 1:6,670\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFour 3 mm punches\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:50, 1:500, 1:5000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOne 6 mm punch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1:25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:50, 1:500, 1:5000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003ePunch Size Equivalency Determination\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePrevious data equates a 3 mm punch to approximately 1.5 \u0026micro;L of serum [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To verify expected serum-equivalent recovery rates between DBS punch sizes (3 mm and 6 mm), a panel of seven individual DBS samples was created. From each sample, one-, two-, three-, and four-3 mm (1/8 in) punches were compared against one-6 mm (1/4 in) punch. Each punch size condition was tested a minimum of three times on three different days by two independent operators resulting in a total\u0026thinsp;\u0026ge;\u0026thinsp;108 analytic endpoints.\u003c/p\u003e \u003cp\u003eDBS to Serum Equivalency\u003c/p\u003e \u003cp\u003eTo establish the correlation between serum and DBS using a serum reference standard, a panel of 33 matched DBS-serum sample sets as described above were tested for each serotype. Matched sample testing used the optimal number of 3 mm punches, established by the above-described punch size equivalency testing and was performed by two independent operators and repeated four times on four different days.\u003c/p\u003e \u003cp\u003eData and Statistical Analysis\u003c/p\u003e \u003cp\u003eRaw MFI values from the MIA were evaluated against a human serum reference standard curve with weight-based anti-CPS IgG assignments (\u0026micro;g/mL) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] using a log-log linear regression-based algorithm in a validated SAS application (Pfizer). This approach enables comparison of antibody concentrations across serotypes. All samples with a concentration coefficient of variation (CV)\u0026thinsp;\u0026gt;\u0026thinsp;30% were excluded from the analyses. Statistical analysis was modeled after previously published serum to DBS bridging experiments [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEquivalence of log\u003csub\u003e10\u003c/sub\u003e-transformed sample anti-CPS IgG concentrations eluted from different punch sizes was assessed based on the coefficient of determination (R\u0026sup2;) from regression analysis, with acceptance criteria set at \u0026ge;\u0026thinsp;0.95. Additionally, the 95% confidence interval (CI) around the intercept needed to contain the value zero (indicating that at the baseline level, the two methods yield similar results with no significant systematic difference), and the 95% CI around the slope needed to contain the value 1 (indicating no significant proportional difference across the range). A paired t-test was used to assess individual serotypes once a target punch condition was selected for comparison against the 6mm punch.\u003c/p\u003e \u003cp\u003eEquivalence between log\u003csub\u003e10\u003c/sub\u003e transformed concentrations in paired serum and DBS eluents was assessed using the Concordance Correlation Coefficient (CCC), where the lower 95% of confidence interval (CI) was set at \u0026ge;\u0026thinsp;0.95 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Deming regression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] between the two sample types was assessed to ascertain whether a serum-to-DBS conversion factor was needed. If the 95% CI for the intercept did not contain the value 0 or 95% CI for the slope did not contain the value 1, a conversion factor was applied [ 24, 25].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003ePunch Size Equivalency Determination\u003c/p\u003e \u003cp\u003eThe anti-CPS IgG geometric mean concentrations of the seven samples tested were compared by sample punch size condition against the one-6 mm punch anti-CPS IgG concentration [Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e]. The four-3mm punch condition was found to be equivalent to one-6 mm punch condition and was selected for additional evaluation. Anti-CPS IgG geometric mean concentrations across all six serotypes in the assay between the two punch size conditions were highly correlated (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9972) by regression analysis [Supplemental Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e] and met the acceptance criteria set at \u0026gt;\u0026thinsp;0.95 and 95% CI where the intercept contained the value 0 and slope contained the value 1 [Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e]. Paired t-test p-values were \u0026gt;\u0026thinsp;0.05 [range 0.2031 (GBS Ib) \u0026ndash; 0.8317 (GBS IV)] for all serotypes in the four-3mm punches versus one-6 mm punch comparison [Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePaired t-test results for one-6 mm punch compared to 3 mm punches by serotype (pr (\u0026gt; |t|)) with final concentrations held at the same dilution\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOne-6mm/four-3mm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOne-6mm/three-3mm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOne-6mm/two-3mm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOne-6mm/one-3mm\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSIA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.8304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSIB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.8183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3409\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4088\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1788\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.0001\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\u003eDBS to Serum Equivalency\u003c/p\u003e \u003cp\u003eInitial concordance between the DBS and serum tested was established by CCC by individual serotypes and ranged from 0.9899\u0026ndash;0.9981 [Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e]. Accuracy and precision were \u0026ge;\u0026thinsp;0.98 for all serotypes [Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e]. The slope and intercept of the Deming regression best fit lines varied by serotype [Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e]. Criteria for conversion factor application [Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Supplemental Table\u0026nbsp;2] for increased accuracy acceptance was met for two serotypes (GBS Ia and GBS Ib). For two serotypes (GBS II and GBS V), the 95% CI intercept did not contain the value 0 and, for one serotype (GBS III), the 95% CI for slope did not contain the value 1. Both intercept and slope criteria failed for GBS IV.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e*Samples excluded from analysis due to sample CV. For detailed reportable values, refer to Supplementary Table\u0026nbsp;2\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of conversion formula for each serotype\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSerotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eObserved value with 95% Confidence interval (CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eConversion factor Applied (Yes/No)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eConversion formula\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntercept\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSlope\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSIa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.0022\u003c/p\u003e \u003cp\u003e[-0.0329, 0.0284]\u003c/p\u003e\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0185\u003c/p\u003e \u003cp\u003e[0.9981, 1.0389]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSIb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0189\u003c/p\u003e \u003cp\u003e[-0.0089, 0.0467]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0170\u003c/p\u003e \u003cp\u003e[0.9921, 1.0418]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0945\u003c/p\u003e \u003cp\u003e[0.0189, 0.1701]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9889\u003c/p\u003e \u003cp\u003e[0.9312, 1.0467]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLOG_VALUE\u0026thinsp;=\u0026thinsp;0.0945\u0026thinsp;+\u0026thinsp;log10(DBS_RESULT),\u003c/p\u003e \u003cp\u003eCONVERTED_VALUE\u0026thinsp;=\u0026thinsp;10**LOG_VALUE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0177\u003c/p\u003e \u003cp\u003e[-0.0098, 0.0453]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0251\u003c/p\u003e \u003cp\u003e[1.0044, 1.0459]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLOG_VALUE\u0026thinsp;=\u0026thinsp;1.0251*log10(DBS_RESULT),\u003c/p\u003e \u003cp\u003eCONVERTED_VALUE\u0026thinsp;=\u0026thinsp;10**LOG_VALUE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0491\u003c/p\u003e \u003cp\u003e[0.0206, 0.0777]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0337\u003c/p\u003e \u003cp\u003e[1.0191, 1.0483]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLOG_VALUE\u0026thinsp;=\u0026thinsp;0.0491\u0026thinsp;+\u0026thinsp;1.0337*log10(DBS_RESULT),\u003c/p\u003e \u003cp\u003eCONVERTED_VALUE\u0026thinsp;=\u0026thinsp;10**LOG_VALUE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGBSV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0703\u003c/p\u003e \u003cp\u003e[0.0317, 0.1090]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0045\u003c/p\u003e \u003cp\u003e[0.9637, 1.0454]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLOG_VALUE\u0026thinsp;=\u0026thinsp;0.0703 +\u0026nbsp; log10(DBS_RESULT),\u003c/p\u003e \u003cp\u003eCONVERTED_VALUE\u0026thinsp;=\u0026thinsp;10**LOG_VALUE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis manuscript presents a first study of its kind in a controlled setting to systematically demonstrate the suitability of DBS in the standardized multiplex immunoassay adopted by the GASTON consortium to quantify IgG antibodies against the most prevalent GBS CPS serotypes. The use of DBS has expanded to evaluate a plethora of biomarkers [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] in recent years, given that collection is minimally invasive, requires less skilled labor, and uses fewer materials during sample collection compared to venipuncture. Additionally, DBS are easier and more efficient to transport from collection sites as they can be maintained at room temperature for up to 16 weeks without loss of biomarker functionality once the sample has dried and do not require cold-chain transportation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition to the utility of the sample matrix, DBS have been used in newborn screening programs since the 1960s and large sample collections are available for potential use in seroepidemiological studies [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, the use of a matched serum and DBS panel allowed for the direct comparison of quantifiable anti-CPS IgG concentrations from two distinct sample matrices that were created from the same source material. When the matched DBS-serum sample pairs were tested, concordance\u0026thinsp;\u0026gt;\u0026thinsp;0.95 was demonstrated for all six GBS CPS serotypes captured by the assay. In addition to logarithmically transformed regression analysis, prior studies relied on CCC [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], Pearson correlation coefficient (PCC) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], Spearman's rank correlation coefficient [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], or Cohen's kappa coefficient [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] to assess agreement between the sample matrices. This study used an additional layer of stringency through the examination of the CI around the parameter estimates for the slope and intercept, followed by establishing a DBS-to-serum conversion factor for serotypes that did not meet the added criteria. Of the six GBS CPS serotypes assessed, four (GBS II, III, IV, and V) were found to increase result accuracy with the application of a constant multiplier as the conversion factor.\u003c/p\u003e \u003cp\u003eVersatility in sample size collection from the DBS through different punch sizes allows for great flexibility in testing when using remnant dried blood spot samples from historic collections. We successfully demonstrated punch size equivalency that verified previously established DBS-punch-size to serum-volume values [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. From the geometric mean anti-CPS IgG concentrations of the seven samples tested, it was determined that the four-3 mm punch size was the best fit for overall sample equivalency to the one-6 mm punch and could be used interchangeably in the assay. This allows the researcher to use 3mm punches when larger punches cannot be accommodated. These results thus add value by allowing the use of different punch sizes; for example, researchers could include specimens that have undergone prior testing such as remnant newborn screening samples, in which punches have already been removed from the filter paper.\u003c/p\u003e \u003cp\u003eIn this study, bridging between the sample matrixes was performed on primarily adult specimens and may not be fully representative of infant samples that may have higher hematocrit concentrations. Hematocrit levels can affect the diffusion of the blood on the filter paper at the time of collection and influence the distribution of antibodies within the DBS sample [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn a controlled setting using contrived panels, the use of DBS as a testing matrix in the quantification of GBS anti-CPS antibodies was successfully demonstrated. These results not only advance GBS-related seroepidemiological studies with DBS but also impact the work of the GASTON consortium and the broader GBS community striving for licensure of a maternal vaccine against invasive GBS disease in infants.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eDGS, DP, MG, RS, and NCSM are employees at Pfizer, Inc, and may be shareholders of Pfizer Inc. No other conflicts of interest for other authors.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eDGS, DP, MG, RS, and NCSM are employees at Pfizer, Inc, and may be shareholders of Pfizer Inc. No other conflicts of interest for other authors.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Centers for Disease Control and Prevention (CDC) and funded by the Bill \u0026amp; Melinda Gates Foundation, Seattle, WA, INV-008343. The funders had no role in the design, analysis, or interpretation of the data in this research study, in the writing of the report, or the decision to publish.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceived and designed the experiments: SB, PM, LTJ. Performed the experiments: PYP, BA. Provided critical reagents and technical support: DGS, DP, MG, RS, NCSM, PM, JS, and SAV. Analyzed the data: SB, YL, and LTJ. Wrote the paper: SB, SAV. Consultants: JR and SS. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank Richie Ramdhanie (Pfizer) for their technical assistance in preparing the sample panels.\u003c/p\u003eStatement of ethics approval: This activity was reviewed by CDC, deemed research not involving human subjects, and was conducted consistent with applicable federal law and CDC policy.§ §See e.g., 45 C.F.R. part 46; 21 C.F.R. part 56; 42 U.S.C. §241(d), 5 U.S.C. §552a, 44 U.S.C. §3501 et seq."},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLe Doare K, Kampmann B, Vekemans J, Heath PT, Goldblatt D, Nahm MH, Baker C, Edwards MS, Kwatra G, Andrews N, Madhi SA, Ter Meulen AS, Anderson AS, Corsaro B, Fischer P, Gorringe A (2019) Serocorrelates of protection against infant group B streptococcus disease. Lancet Infect Dis 19(5):e162\u0026ndash;e171. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1473-3099(18)30659-5\u003c/span\u003e\u003cspan address=\"10.1016/S1473-3099(18)30659-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbsalon J, Simon R, Radley D, Giardina PC, Koury K, Jansen KU, Anderson AS (2022) Advances towards licensure of a maternal vaccine for the prevention of invasive group B streptococcus disease in infants: a discussion of different approaches. Hum Vaccin Immunother 18(1):2037350. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/21645515.2022.2037350\u003c/span\u003e\u003cspan address=\"10.1080/21645515.2022.2037350\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchrag SJ, Verani JR (2013) Intrapartum antibiotic prophylaxis for the prevention of perinatal group B streptococcal disease: experience in the United States and implications for a potential group B streptococcal vaccine. Vaccine 31 Suppl 4D20\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vaccine.2012.11.056\u003c/span\u003e\u003cspan address=\"10.1016/j.vaccine.2012.11.056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJordan HT, Farley MM, Craig A, Mohle-Boetani J, Harrison LH, Petit S, Lynfield R, Thomas A, Zansky S, Gershman K, Albanese BA, Schaffner W, Schrag SJ (2008) Revisiting the need for vaccine prevention of late-onset neonatal group B streptococcal disease: a multistate, population-based analysis. Pediatr Infect Dis J 27(12):1057\u0026ndash;1064. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/INF.0b013e318180b3b9\u003c/span\u003e\u003cspan address=\"10.1097/INF.0b013e318180b3b9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Active Bacterial Core Surveillance (ABCs)/Emerging Infections Program Network\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroup B (2021) streptococcus vaccine: full value of vaccine assessment. Executive summary. World Health Organization, Geneva. License: CC BY-NC-SA 3.0 IGO\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarques MB, Kasper DL, Pangburn MK, Wessels MR (1992) Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci. Infect Immun 60(10):3986\u0026ndash;3993. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/iai.60.10.3986-3993.1992\u003c/span\u003e\u003cspan address=\"10.1128/iai.60.10.3986-3993.1992\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaker CJ (2023) Group B Streptococcal Vaccine - Sisyphus Reconciled. N Engl J Med Jul 20;389(3):275\u0026ndash;277. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMe2306234\u003c/span\u003e\u003cspan address=\"10.1056/NEJMe2306234\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 37467503\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadhi SA, Anderson AS, Absalon J, Radley D, Simon R, Jongihlati B, Strehlau R, van Niekerk AM, Izu A, Naidoo N, Kwatra G, Ramsamy Y, Said M, Jones S, Jose L, Fairlie L, Barnabas SL, Newton R, Munson S, Jefferies Z, Pavliakova D, de Silmon NC, Gomme E, Perez JL, Scott DA, Gruber WC, Jansen KU (2023) Potential for Maternally Administered Vaccine for Infant Group B Streptococcus. N Engl J Med Jul 20;389(3):215\u0026ndash;227. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMoa2116045\u003c/span\u003e\u003cspan address=\"10.1056/NEJMoa2116045\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 37467497\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe Doare K, Gaylord MA, Anderson AS, Andrews N, Baker CJ, Bolcen S, Felek A, Giardina PC, Grube CD, Hall T, Hallis B, Izu A, Madhi SA, Maniatis P, Matheson M, Mawas F, McKeen A, Rhodes J, Alston B, Patel P, Schrag S, Simon R, Tan CY, Taylor S, Kwatra G, Gorringe A (2024) Interlaboratory comparison of a multiplex immunoassay that measures human serum IgG antibodies against six-group B streptococcus polysaccharides. Hum Vaccin Immunother 20(1):2330138. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/21645515.2024.2330138\u003c/span\u003e\u003cspan address=\"10.1080/21645515.2024.2330138\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaylord MA, Larrier M, Giordano-Schmidt D, Grube CD, Singh S, Nguyen HH, McKeen A, Tan CY, Anderson AS, Kalina WV, Pavliakova D, Giardina PC (2024) Development and validation of a 6-plex Luminex-based assay for measuring human serum antibodies to group B streptococcus capsular polysaccharides. Hum Vaccin Immunother 20(1):2311480. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/21645515.2024.2311480\u003c/span\u003e\u003cspan address=\"10.1080/21645515.2024.2311480\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchiffer JM, Maniatis P, Garza I, Steward-Clark E, Korman LT, Pittman PR, Mei JV, Quinn CP (2013) Quantitative assessment of anthrax vaccine immunogenicity using the dried blood spot matrix. Biologicals 41(2):98\u0026ndash;103. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biologicals.2012.11.003\u003c/span\u003e\u003cspan address=\"10.1016/j.biologicals.2012.11.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndersen NJ, Mondal TK, Preissler MT, Freed BM, Stockinger S, Bell E, Druschel C, Louis GM, Lawrence DA (2014) Detection of immunoglobulin isotypes from dried blood spots. J Immunol Methods 404:24\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jim.2013.12.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jim.2013.12.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKouam\u0026eacute; DR, Aboli RA, Kabran M, Adiko AC, Coulibaly M, Dembele B, Inwoley A (2023) Validation of dried blood spots samples for the screening of hepatitis B virus surface by enzyme immunoassay method. J Immunol Methods 513:113412. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jim.2022.113412\u003c/span\u003e\u003cspan address=\"10.1016/j.jim.2022.113412\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillar LM, de Oliveira JC, Cruz HM, Yoshida CF, Lampe E, Lewis-Ximenez LL (2011) Assessment of dried blood spot samples as a simple method for detection of hepatitis B virus markers. J Med Virol 83(9):1522\u0026ndash;1529. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jmv.22138\u003c/span\u003e\u003cspan address=\"10.1002/jmv.22138\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajam G, Carlone G, Kim E, Choi J, Paulos S, Park S, Jeyachandran A, Gorantla Y, Wong E, Sabnis A, Browning P, Desai R, Quinn CP, Schiffer J (2019) Development and validation of a robust multiplex serological assay to quantify antibodies specific to pertussis antigens. Biologicals 57:9\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biologicals.2018.11.001\u003c/span\u003e\u003cspan address=\"10.1016/j.biologicals.2018.11.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao H, Wang M, Muthelo P, Lof L, Sterky F, Gallini R, Kumar NV, Monsen T, Nilsson K, Aberg M, Kamali-Moghaddam M, Mei YF, Landegren U (2022) Detection of SARS-CoV-2 antibodies in serum and dried blood spot samples of vaccinated individuals using a sensitive homogeneous proximity extension assay. N Biotechnol 72:139\u0026ndash;148. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.nbt.2022.11.004\u003c/span\u003e\u003cspan address=\"10.1016/j.nbt.2022.11.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOmosule CL, Conklin J, Seck S, Howell R, Hock KG, Ballman C, Freeman J, Du Toit L, Dubberke E, Farnsworth CW (2023) Qualitative and quantitative detection of SARS-CoV-2 antibodies from dried blood spots. Clin Biochem 117:16\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.clinbiochem.2021.12.012\u003c/span\u003e\u003cspan address=\"10.1016/j.clinbiochem.2021.12.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNational Center for Environmental Health (2020) Newborn Screening Quality Assurance Program 2021 Annual Summary Report, Volume 39a. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cdc.gov/labstandards/pdf/nsqap/NSQAP_Annual_Summary_2019-508.pdf\u003c/span\u003e\u003cspan address=\"https://www.cdc.gov/labstandards/pdf/nsqap/NSQAP_Annual_Summary_2019-508.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMercader S, Featherstone D, Bellini WJ (2006) Comparison of available methods to elute serum from dried blood spot samples for measles serology. J Virol Methods Oct 137(1):140\u0026ndash;149. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jviromet.2006.06.018\u003c/span\u003e\u003cspan address=\"10.1016/j.jviromet.2006.06.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eEpub 2006 Jul 24. PMID: 16860401\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHewawasam E, Liu G, Jeffery DW, Gibson RA, Muhlhausler BS (2018) Estimation of the Volume of Blood in a Small Disc Punched From a Dried Blood Spot Card. Eur J Lipid Sci Technol Vol.120(3);n pag. Web. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ejlt.201700362\u003c/span\u003e\u003cspan address=\"10.1002/ejlt.201700362\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbsalon J, Segall N, Block SL, Center KJ, Scully IL, Giardina PC, Peterson J, Watson WJ, Gruber WC, Jansen KU, Peng Y, Munson S, Pavliakova D, Scott DA, Anderson AS (2021) Safety and immunogenicity of a novel hexavalent group B streptococcus conjugate vaccine in healthy, non-pregnant adults: a phase 1/2, randomised, placebo-controlled, observer-blinded, dose-escalation trial. Lancet Infect Dis Feb 21(2):263\u0026ndash;274. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/S1473-3099(20)30478-3\u003c/span\u003e\u003cspan address=\"10.1016/S1473-3099(20)30478-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEsadze A, Grube CD, Wellnitz S, Singh S, Nguyen HH, Gaylord MA, Zhu A, Gribenko A, Tan CY, Anderson AS, Simon R (2023) Calibration of a serum reference standard for Group B streptococcal polysaccharide conjugate vaccine development using surface plasmon resonance. NPJ Vaccines 8(1):71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41541-023-00667-1\u003c/span\u003e\u003cspan address=\"10.1038/s41541-023-00667-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin L, Torbeck LD (1998) Coefficient of accuracy and concordance correlation coefficient: new statistics for methods comparison. PDA J Pharm Sci Technol 52(2):55\u0026ndash;59\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmitage P, Berry G, Matthews JNS (2002) The Scope of Statistics. Blackwell Science Ltd\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLahdentausta L, Kivimaki A, Oksanen L, Tallgren M, Oksanen S, Sanmark E, Salminen A, Geneid A, Sairanen M, Paju S, Saksela K, Pussinen P, Pietiainen M (2022) Blood and saliva SARS-CoV-2 antibody levels in self-collected dried spot samples. Med Microbiol Immunol 211(4):173\u0026ndash;183. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00430-022-00740-x\u003c/span\u003e\u003cspan address=\"10.1007/s00430-022-00740-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGruner N, Stambouli O, Ross RS (2015) Dried blood spots\u0026ndash;preparing and processing for use in immunoassays and in molecular techniques. J Vis Exp (97) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3791/52619\u003c/span\u003e\u003cspan address=\"10.3791/52619\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBehets F, Kashamuka M, Pappaioanou M, Green TA, Ryder RW, Batter V, George JR, Hannon WH, Quinn TC (1992) Stability of human immunodeficiency virus type 1 antibodies in whole blood dried on filter paper and stored under various tropical conditions in Kinshasa, Zaire. J Clin Microbiol 30(5):1179\u0026ndash;1182. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/jcm.30.5.1179-1182.1992\u003c/span\u003e\u003cspan address=\"10.1128/jcm.30.5.1179-1182.1992\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang SL, McGann CM, Duranova T, Strysko J, Steenhoff AP, Gezmu A, Nakstad B, Arscott-Mills T, Bayani O, Moorad B, Tlhako N, Richard-Greenblatt M, Planet PJ, Coffin SE, Silverman MA (2024) Maternal and neonatal IgG against Klebsiella pneumoniae are associated with broad protection from neonatal sepsis: a case-control study of hospitalized neonates in Botswana. medRxiv [Preprint] May 28. 2024.05.28.24308042\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCornbleet PJ, Gochman N (1979) Incorrect least-squares regression coefficients in method-comparison analysis. Clin Chem 25(3):432\u0026ndash;438. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/clinchem/25.3.432\u003c/span\u003e\u003cspan address=\"10.1093/clinchem/25.3.432\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDenniff P, Spooner N (2010) The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs. Bioanalysis Aug 2(8):1385\u0026ndash;1395. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4155/bio.10.103\u003c/span\u003e\u003cspan address=\"10.4155/bio.10.103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eErratum in: Bioanalysis\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":"GBS, Antibody, Dried blood spot sample, Serum, Multiplex immunoassay","lastPublishedDoi":"10.21203/rs.3.rs-5347662/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5347662/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA standardized multiplex immunoassay (MIA) to quantify group B \u003cem\u003eStreptococcus\u003c/em\u003e (GBS) anti-capsular polysaccharide (CPS) IgG serum concentrations was adopted by the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eG\u003c/span\u003eroup B streptococcal \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eA\u003c/span\u003essay \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSt\u003c/span\u003eandardizati\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eon\u003c/span\u003e (GASTON) consortium as a standardized assay with the most immediate applications for facilitating the licensure of GBS vaccines. However, dried blood spot (DBS) samples offer advantages for immunological studies, including cost-effectiveness, ease of transport, and storage.\u003c/p\u003e \u003cp\u003eTo determine suitability of DBS as an alternative sample matrix to serum in multiplex immunoassays, a contrived GBS seropositive panel, including matched DBS and serum samples, was prepared using established methods. The calculated geometric mean titers of GBS anti-CPS IgG values by individual serotype were compared using a paired t-test to establish serum equivalency. Geometric mean values for the matched panel were assessed via Deming regression for precision, accuracy, and concordance correlation coefficient (CCC). The initial acceptance criterion was set at 0.95 for CCC. Two additional criteria based on confidence intervals of CCC, slope, and intercept were used to determine the necessity of a serotype-specific conversion factor.\u003c/p\u003e \u003cp\u003eThe paired t-test p-values were \u0026gt;\u0026thinsp;0.05 for serum equivalency. For sample matrix concordance, CCC values were \u0026gt;\u0026thinsp;0.95 and met correlation criteria for all serotypes. Conversion factors were applied to four serotypes (II, III, IV, and V) that did not meet the criteria for slope, intercept, or both. This demonstration of equivalency between DBS and serum supports the hypothesis that DBS is a suitable testing matrix from which to elucidate anti-CPS IgG concentrations in seroepidemiological and vaccine evaluation studies.\u003c/p\u003e","manuscriptTitle":"Serum-equivalency Comparison, Detection, and Quantification of Group B Streptococcus Anti-capsular Polysaccharide Antibodies from Dried Blood Spots","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 09:54:40","doi":"10.21203/rs.3.rs-5347662/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":"3658c987-e080-4c99-844f-d4c794ebc875","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-14T08:53:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-25 09:54:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5347662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5347662","identity":"rs-5347662","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}