Serum Institute of India Pvt Ltd., 212/2, Hadapsar, Pune, Maharashtra 411028, India.
Serum Institute of India Pvt Ltd., 212/2, Hadapsar, Pune, Maharashtra 411028, India.
Methods. 2019 Apr 1;158:33-43. doi: 10.1016/j.ymeth.2019.01.015. Epub 2019 Jan 25.
We describe here a magnetic bead-based multiplex (pentaplex) immunoassay (MIA) platform developed as an alternative to enzyme-linked immunosorbent assays (ELISA) used in immunogenicity testing of DTaP/TdaP vaccine in animals. MIA simultaneously measures the concentration of serum (IgG) antibodies against B. Pertussis antigens; pertussis toxin, filamentous hemagglutinin (FHA), pertactin (PRN) and tetanus (T) and diphtheria (D) toxoid in the Tdap vaccine immunized animals. Assay validation experiments were done using a panel of serum samples. The results are expressed in IU/ml using WHO reference mice serum. The standard curve was linear with 4PL logistic fit over an eight 2-fold dilution range with LOQ of 0.003, 0.022, 0.005 IU/ml for PT, FHA and PRN and 0.016 U/ml for T and D antigens indicating sensitivity. No interference was observed in monoplex versus multiplex measurements. Specificity was demonstrated by ≥90% homologous and ≤15% heterologous inhibition for all the antigens. The assay was reproducible, with a mean coefficient of variation (CV) of ≤10% for intra-assay duplicates and ≤25% for interassays using different lots of beads and analyst. Accuracy was demonstrated wherein the ratio of observed vs. assigned unitages were within 80-120%. The study suggests that the Pentaplex (MIA) platform meets all the criteria for the serological assay combination vaccines with additional advantages of high throughput, reduced sample volumes, faster analysis with reduced manpower in contrast to conventional monoplex ELISA.
我们在这里描述了一种基于磁珠的多重（五重）免疫分析（MIA）平台，该平台可作为替代酶联免疫吸附测定（ELISA）用于动物中 DTaP/TdaP 疫苗免疫原性检测。MIA 同时测量针对百日咳杆菌抗原的血清（IgG）抗体浓度；百日咳毒素、丝状血凝素（FHA）、 pertactin（PRN）和破伤风（T）和白喉（D）类毒素在 Tdap 疫苗免疫的动物中。使用一组血清样本进行了测定验证实验。结果以 WHO 参考小鼠血清表示，单位为 IU/ml。标准曲线呈线性，在 8 个 2 倍稀释范围内采用 4PL 逻辑拟合，LOQ 分别为 0.003、0.022、0.005IU/ml 用于 PT、FHA 和 PRN，T 和 D 抗原的 LOQ 为 0.016 U/ml，表明灵敏度。在单重与多重测量中未观察到干扰。所有抗原的同源性≥90%，异源性≤15%，证明了特异性。该测定具有重现性，在使用不同批次珠子和分析人员进行的内、间测定中，平均变异系数（CV）分别≤10%和≤25%。准确性表明，观察到的与分配的单位数之比在 80-120%范围内。该研究表明，Pentaplex（MIA）平台符合组合疫苗血清学测定的所有标准，与传统的单重 ELISA 相比，具有高通量、减少样本量、更快的分析和更少的人力等额外优势。
