Romero-Steiner Sandra, Holder Patricia F, Gomez de Leon Patricia, Spear Willie, Hennessy Thomas W, Carlone George M
MS A-36, Respiratory Diseases Immunology Section, Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Clin Diagn Lab Immunol. 2005 Sep;12(9):1029-35. doi: 10.1128/CDLI.12.9.1029-1035.2005.
Determination of antibody avidity measurements can be difficult in human serum depending on the population evaluated. We evaluated three approaches for the determination of antibody avidity for immunoglobulin G (IgG). These approaches were (i) elution of bound antibody with increasing concentrations of a chaotropic agent using a single serum dilution, (ii) binding interference of multiple serum dilutions by a single concentration of a chaotrope, and (iii) elution of multiple serum dilutions by a single concentration of a chaotrope. Parameters that affect the determination of avidity measurements and their limitations were evaluated with pre- and post-Haemophilus influenzae type b conjugate vaccination sera (n=89). We determined that elution of low-avidity antibodies present in multiple dilutions of the serum sample by a single concentration of a chaotrope (0.15 M sodium thiocyanate [NaSCN]) was optimal for the determination of avidity measurements throughout a wide range of IgG concentrations (0.94 to 304.6 microg/ml). The percent reduction in concentration as determined by the elution assay with 0.15 M NaSCN correlated highly (r=0.84) with weighted averages obtained by an elution assay with multiple solutions of NaSCN. The correlation (r=0.57) between elution and binding interference, when a single concentration of a chaotrope was used, was lower than the correlation between the two elution methods (r=0.84). We found that the serum dilution, the heterogeneity of the antibody population, and the concentration of the chaotrope were the primary variables affecting avidity determinations. In this study, we present multiple analysis methods depending on the methodology used. We also present the factors that affect the analysis of avidity determinations given the polyclonal nature of human sera. This experimental approach should benefit the evaluation of similar antibodies induced by other bacterial polysaccharide vaccines.
根据所评估的人群不同，在人血清中测定抗体亲和力可能会有困难。我们评估了三种测定免疫球蛋白G（IgG）抗体亲和力的方法。这些方法分别是：（i）使用单一血清稀释度，用浓度递增的离液剂洗脱结合的抗体；（ii）用单一浓度的离液剂对多个血清稀释度进行结合干扰；（iii）用单一浓度的离液剂洗脱多个血清稀释度。使用b型流感嗜血杆菌结合疫苗接种前后的血清（n = 89）评估了影响亲和力测定的参数及其局限性。我们确定，用单一浓度的离液剂（0.15 M硫氰酸钠[NaSCN]）洗脱血清样品多个稀释度中存在的低亲和力抗体，对于在广泛的IgG浓度范围（0.94至304.6微克/毫升）内测定亲和力最为合适。用0.15 M NaSCN洗脱试验测定的浓度降低百分比与用多种NaSCN溶液进行的洗脱试验获得的加权平均值高度相关（r = 0.84）。当使用单一浓度的离液剂时，洗脱与结合干扰之间的相关性（r = 0.57）低于两种洗脱方法之间的相关性（r = 0.84）。我们发现血清稀释度、抗体群体的异质性和离液剂的浓度是影响亲和力测定的主要变量。在本研究中，我们根据所使用的方法介绍了多种分析方法。鉴于人血清的多克隆性质，我们还介绍了影响亲和力测定分析的因素。这种实验方法应有助于评估由其他细菌多糖疫苗诱导的类似抗体。
