Development and validation of magnetic bead pentaplex immunoassay for simultaneous quantification of murine serum IgG antibodies to acellular pertussis, diphtheria and tetanus antigens used in combination vaccines

Kadam, Laxmikant; Patel, Krunal; Gautam, Manish; Thorat, Shrikant; Kale, Prathamesh; Ghule, Arvind Kumar; Gairola, Akansha; Rao, Harish; Shinde, Yashwant Dattatraya; Shaligram, Umesh; Gairola, Sunil

doi:10.1016/j.ymeth.2019.01.015

Cited by 11 publications

(8 citation statements)

References 15 publications

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“…Two commercially available procedures were evaluated: the first was based on the Luminex cookbook published previously by Kadam L et al. ( 13 , 28 ) and the second was based on the commercially available kit from AnteoTech (Australia) ( 29 ). For the coupling of antigens, microspheres were activated with a carbodiimide derivative, EDAC hydrochloride-containing buffered solution.…”

Section: Methodsmentioning

confidence: 99%

“…However, few studies are available wherein aP antigens are multiplexed with diphtheria and tetanus antigens. A previous study has reported on a pentaplex Luminex assay covering aP, diphtheria, and tetanus antigens to evaluate the immunogenicity of combination vaccines in mouse models ( 13 ). The multiplex assay offers the advantage of lesser turnaround time in simultaneously detecting several antigens utilizing lesser sample volumes.…”

Section: Introductionmentioning

confidence: 99%

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Multiplexed bead-based assay for the simultaneous quantification of human serum IgG antibodies to tetanus, diphtheria, pertussis toxin, filamentous hemagglutinin, and pertactin

et al. 2023

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BackgroundLuminex bead-based assays offer multiplexing to test antibodies against multiple antigens simultaneously; however, this requires validation using internationally certified reference standards. Therefore, there is an urgent need to characterize existing reference standards for the standardization of multiplex immunoassays (MIAs). Here, we report the development and validation of an MIA for the simultaneous estimation of levels of human serum immunoglobulin G (IgG) antibodies for pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), diphtheria toxoid (DT), and tetanus toxoid (TT).MethodsThe MIA was assessed using a panel of human serum samples and WHO reference standards. The WHO reference standards were also studied for suitability in the MIA. Purified antigens (PT, FHA, PRN, DT, and TT) were coupled to the spectrally unique magnetic carboxylated microspheres. The method was validated in accordance with the United States Food and Drug Administration (US FDA), European Medicines Agency (EMA), and the International Committee of Harmonization Multidisciplinary (ICH M10) guidelines, and parameters such as precision, accuracy, dilutional linearity, assay range, robustness, and stability were assessed. Method agreements with commercially available IgG enzyme-linked immunosorbent assay (ELISA) assays were also evaluated. In addition, the study assessed the level of correlation between the IgG levels estimated by the MIA and the cell-based neutralizing antibody assays for PT and DT.ResultsWe identified that an equimix of WHO international standards (i.e., 06/142, 10/262, and TE-3) afforded the best dynamic range for all the antigens in the MIA. For all five antigens, we observed that the back-fitted recoveries using the four-parameter logistic (4-PL) regression fits ranged between 80% and 120% for all calibration levels, and the percentage coefficient of variation (% CV) was < 20%. In addition, the difference in mean fluorescence intensity (MFI) between the monoplex and multiplex format was < 10% for each antigen, indicating no crosstalk among the beads. The MIA also showed good agreement with conventional and commercially available assays, and a positive correlation (> 0.75) with toxin neutralization assays for PT and DT was observed.ConclusionThe MIA that was calibrated in accordance with WHO reference standards demonstrated increased sensitivity, reproducibility, and high throughput capabilities, allowing for the design of robust studies that evaluate both natural and vaccine-induced immunity.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Multiplexed bead-based assay for the simultaneous quantification of human serum IgG antibodies to tetanus, diphtheria, pertussis toxin, filamentous hemagglutinin, and pertactin

et al. 2023

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“…Some bead-based immunoassays have been developed for IgG antibodies and bacteria. A multiplexed magnetic microsphere immunoassay was developed to measure mouse serum antibodies against B. Pertussis antigens , pertussis toxin, filamentous hemagglutinin, pertactin, tetanus, and diphtheria for testing of combination animal vaccines . Antigens were linked to luminescent-labeled magnetic microspheres, and fluorescent output was monitored.…”

Section: Magnetic Bead- and Microsphere-assisted Assaysmentioning

confidence: 99%

“…A multiplexed magnetic microsphere immunoassay was developed to measure mouse serum antibodies against B. Pertussis antigens, pertussis toxin, filamentous hemagglutinin, pertactin, tetanus, and diphtheria for testing of combination animal vaccines. 66 Antigens were linked to luminescent-labeled magnetic microspheres, and fluorescent output was monitored. Appropriate limits of quantitation (LOQs) for the practical measurement of antibodies in vaccines were obtained.…”

Section: Analytical Chemistrymentioning

confidence: 99%

Multiplexed Immunosensors and Immunoarrays

et al. 2019

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Immunoassays make use of highly specific antigen-antibody binding and provide sensitive ways to detect a wide range of biomacromolecules, bacteria, viruses, and small molecules. There are a range of types of immunoassay systems including single analyte sensors, 96well plate formats, arrays, microfluidic sensors, microfluidic arrays, etc. A big target is encompassed by medical diagnostic biomarkers, which are "molecules that can be measured objectively as indicators of normal or disease processes and responses to therapeutic intervention". 1 Accurate, low-cost measurements of multiple proteins are major applications of immunoarrays that are critical for future early detection and monitoring of cancer and other diseases. Multiplexing is very important, since panels of biomarker proteins, as opposed to single biomarkers, are required to provide sufficient information content for reliable disease diagnostics.

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“…For many decades, enzyme-linked immunosorbent assay (ELISA) represented the standard readout method to quantify disease-or antigen-specific serum antibodies. Meanwhile, various multiplex testing platforms, such as bead-based flow cytometric assay and electrochemiluminescence (ECL) immunoassay (ECLIA) have been developed which have led not only to decreased sample and reagent volume needed but also to an increased testing throughput (Bandiera et al 2019;Bolton et al 2020;Bykonia et al 2023;Kadam et al 2019;McAlister et al 2021;Rajam et al 2019;Stenger et al 2011;Thomas et al 2022). Furthermore, multiplex assays were shown to perform at least similarly or even better with regards to sensitivity and reproducibility of antibody quantitation (Bolton et al 2020;Caboré et al 2016;Pickering et al 2002;Reder et al 2008;van Gageldonk et al 2008).…”

Section: Introductionmentioning

confidence: 99%

Determination of DTaP vaccine potency by multiplex immunogenicity testing using electrochemiluminescence

Bekeredjian-Ding,

Friedrichs,

Rehg

et al. 2024

Preprint

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Potency testing of diphtheria and tetanus vaccines traditionally relied on in vivo protection models involving challenge of laboratory animals with toxins while lot release of many other vaccine products is based on serological assays. Here we describe the results of simultaneous serological potency determination of diphtheria (D), tetanus (T) and acellular pertussis (aP) antigens obtained following immunization of guinea pigs with multicomponent pediatric and booster vaccines from different manufacturers. The 4th World Health Organization (WHO) International Standard (IS) for diphtheria toxoid (No. 07/216) and the 4th WHO IS for tetanus toxoid (No. 08/218) were used as reference preparations. For aP, a pediatric vaccine batch containing the antigens pertussis toxoid, filamentous hemagglutinin, pertactin and fimbriae proteins type 2/3 was established as internal control. Quantification of IgG against D, T and aP antigens in guinea pig sera was performed using a hexaplex electrochemi-luminescence immunoassay. We further provide proof-of-concept using experimental vaccine samples lacking or containing reduced amounts of diphtheria toxoid in the presence of full amounts of tetanus and pertussis antigens and alum adjuvant. Importantly, the assay confirmed dose-response relationships for all antigens tested and was able to detect subpotent batches. The results confirmed the suitability of the protocol for combined serology batch release testing of DTaP combination vaccines. This report summarizes the data and the protocol used for validation prior to implementation of this method in routine batch release testing of DTaP vaccines, which led to replacement of in vivo challenge experiments in our laboratory following the 3R (replace, reduce, refine) principle.

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Development and validation of magnetic bead pentaplex immunoassay for simultaneous quantification of murine serum IgG antibodies to acellular pertussis, diphtheria and tetanus antigens used in combination vaccines

Cited by 11 publications

References 15 publications

Multiplexed bead-based assay for the simultaneous quantification of human serum IgG antibodies to tetanus, diphtheria, pertussis toxin, filamentous hemagglutinin, and pertactin

Multiplexed bead-based assay for the simultaneous quantification of human serum IgG antibodies to tetanus, diphtheria, pertussis toxin, filamentous hemagglutinin, and pertactin

Multiplexed Immunosensors and Immunoarrays

Determination of DTaP vaccine potency by multiplex immunogenicity testing using electrochemiluminescence

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