A New Electrochemiluminescence-Based Multiplex Assay for the Assessment of Human Antibody Responses to Bordetella pertussis Vaccines

Abstract

Introduction: Commercially available enzymelinked immunosorbent assay (ELISA) kits designed for pertussis diagnostic purposes are frequently used to assess antibody responses to pertussis vaccines in clinical trials, but have limited accuracy and are not calibrated against international standards. We developed a new electrochemiluminescence (ECL)-based multiplexed assay and compared its performance to two commercial Bordetella pertussis ELISA kits and to historical in-house ELISAs. Methods: The ECL assay quantifies serum concentrations of antibodies against four B. pertussis antigens: pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbrial agglutinogen (FIM). The assay was validated for precision, accuracy, dilutability, lower limit of quantification, and specificity.Sera from a clinical trial (CTRI/2016/11/007434) were used to compare the ECL assay to two commercial ELISA kits available from GenWay BioTech and Demeditec Diagnostics for accuracy, linearity, specificity, and concordance to both internal (WWO-2-043) and international (NIBSC 06/140) references. Sera from four clinical trials (NCT02587520, NCT00255047, NCT00347958, NCT01346293) were used to compare the concordance to clinical ELISAs. Informed consent was ensured prior to using any sera. Results: Precision, accuracy, dilutability, lower limit of quantification, and specificity were demonstrated for the ECL assay. Concordance between the ECL assay and established clinical ELISAs was met for antibody responses to PT, FIM, and PRN, but not for FHA. The ECL assay demonstrated higher accuracy and linearity than the ELISA kits. While concordance between the ECL and commercial kits was low, the ECL assay better distinguished between preand post-vaccination clinical samples. Conclusion:The new ECL assay was validated for the quantitative evaluation of anti-PT, anti-FHA, anti-FIM, and anti-PRN IgG antibodies in samples from clinical trials, and demonstrated equivalent or better performance than two commercially available ELISA kits.

Lot-to-lot consistency of a hexavalent DTwP-IPV-HB-PRP∼T vaccine and non-inferiority to separate DTwP-HB-PRP∼T and IPV antigen-matching vaccines at 6–8, 10–12, and 14–16 weeks of age co-administered with oral rotavirus vaccine in healthy infants in India: A multi-center, randomized, controlled study

“…Furthermore, anti-PT antibodies are considered important for protection against clinical symptoms, and WHO recommendations include the documentation of anti-PT responses [20] , [21] . For these reasons, anti-PT and anti-FIM were selected as the primary endpoints for the evaluation of the pertussis response using a robust qualified assay [8] , and were included in the main statistical analyses. A descriptive comparison of all immune response thresholds, GMCs, and GMTs confirmed the robust response.…”

“…Anti-D (IU/mL), anti-T (IU/mL), anti-PT (EU/mL), anti-FHA (EU/mL), anti-PRN (EU/mL), anti-FIM (EU/mL) antibody concentrations were measured by a multiplexed chemiluminescence assay using the Meso Scale Discovery platform (DTP-ECL) [7] , [8] . For the B. pertussis antigens, results were expressed in EU calibrated against the US reference pertussis antiserum (human) lot 3 and lot 4 for IgG antibodies [9] , [10] .…”

Antibody persistence following administration of a hexavalent DTwP-IPV-HB-PRP∼T vaccine versus separate DTwP-HB-PRP∼T and IPV vaccines at 12–24 months of age and safety and immunogenicity of a booster dose of DTwP-IPV-HB-PRP∼T in healthy infants in India

“…The ECL assay used has been shown to perform better than commercially available assays developed for pertussis diagnostic purposes, which are not suited to the precise, accurate, and reproducible evaluation that is required in clinical trials. The rationale for the use of the ECL assay in assessing the vaccine responses elicited by wP vaccines has recently been described [19] .…”

Safety and immunogenicity of a hexavalent DTwP-IPV-HB-PRP∼T vaccine versus separate DTwP-HB-PRP∼T and IPV vaccines in healthy infants in India

“…The DTP-ECL assay has previously been validated for the analysis of antibody responses to B. pertussis vaccines [17] . Additionally, in our study, anti-D and anti-T antibody titers determined by ELISA were similar to those determined by DTP-ECL at GCI, supporting the use of DTP-ECL in future studies.…”

“…At GCI, a validated MesoScale Discovery Multiplexed Electro Chemoluminescence (DTP-ECL) immunoassay [16] , [17] was used to measure anti-D, anti-T, and anti-pertussis (anti-pertussis toxin [PT], anti-filamentous hemagglutinin [FHA], anti-pertactin [PRN], and anti-fimbriae 2/3 [FIM]) antibodies. Poliovirus neutralizing antibody titers were measured at GCI by micro metabolic inhibition testing (MIT) against wild type strains.…”