Rajam Gowrisankar, Carlone George, Kim Ellie, Choi Jin, Paulos Simon, Park SoHee, Jeyachandran Amilia, Gorantla Yamini, Wong Emily, Sabnis Amit, Browning Peter, Desai Rita, Quinn Conrad P, Schiffer Jarad
Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, 30329, USA.
Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, 30329, USA.
Biologicals. 2019 Jan;57:9-20. doi: 10.1016/j.biologicals.2018.11.001. Epub 2018 Nov 18.
Despite wide spread vaccination, the public health burden of pertussis remains substantial. Current acellular pertussis vaccines comprise upto five Bordetella pertussis (Bp) antigens. Performing an ELISA to quantify antibody for each antigen is laborious and challenging to apply to pediatric samples where serum volume may be limited. We developed a microsphere based multiplex antibody capture assay (MMACA) to quantify antibodies to five pertussis antigens; pertussis toxin, pertactin, filamentous hemagglutinin and fimbrial antigens 2/3, and adenylate cyclase toxin in a single reaction (5-plex) with a calibrated reference standard, QC reagents and SAS based data analysis program. The goodness of fit (R) of the standard curves for five analytes was ≥0.99, LLOQ 0.04-0.15 IU or AU/mL, accuracy 1.9%-23.8% (%E), dilutional linearity slopes 0.93-1.02 and regression coefficients r = 0.91-0.99. MMACA had acceptable precision within a median CV of 16.0%-22.8%. Critical reagents, antigen conjugated microsphere and reporter antibody exhibited acceptable (<12.3%) lot-lot variation. MMACA can be completed in <3 h, requires low serum volume (5μL/multiplex assay) and has fast data turnaround time (<1 min). MMACA has been successfully developed and validated as a sensitive, specific, robust and rugged method suitable for simultaneous quantification of anti-Bp antibodies in serum, plasma and DBS.
尽管广泛接种了疫苗，但百日咳的公共卫生负担仍然很大。目前的无细胞百日咳疫苗包含多达五种百日咳博德特氏菌（Bp）抗原。通过酶联免疫吸附测定（ELISA）对每种抗原的抗体进行定量既费力，又难以应用于血清量可能有限的儿科样本。我们开发了一种基于微球的多重抗体捕获测定法（MMACA），以在单一反应（5重）中对五种百日咳抗原的抗体进行定量；这五种抗原分别是百日咳毒素、丝状血凝素、百日咳杆菌粘附素、菌毛抗原2/3和腺苷酸环化酶毒素，并配备了校准参考标准品、质量控制试剂和基于SAS的数据分析程序。五种分析物标准曲线的拟合优度（R）≥0.99，最低定量限（LLOQ）为0.04 - 0.15 IU或AU/mL，准确度为1.9% - 23.8%（%E），稀释线性斜率为0.93 - 1.02，回归系数r = 0.91 - 0.99。MMACA在16.0% - 22.8%的中位数变异系数（CV）范围内具有可接受的精密度。关键试剂，即抗原偶联微球和报告抗体，批次间变异可接受（<12.3%）。MMACA可在3小时内完成，所需血清量低（5μL/多重测定），数据周转时间快（<1分钟）。MMACA已成功开发并验证为一种灵敏、特异、稳健且耐用的方法，适用于同时定量血清、血浆和干血斑中的抗Bp抗体。
