Kawabata T T, Babcock L S, Gauggel D L, Asquith T N, Fletcher E R, Horn P A, Ratajczak H V, Graziano F M
Human Safety Department, Procter and Gamble Company, Cincinnati, Ohio 45253-8707, USA.
Fundam Appl Toxicol. 1995 Feb;24(2):238-46. doi: 10.1006/faat.1995.1027.
Assessment of the allergenic potency of enzymes involves the use of a guinea pig model in which specific IgG1 antibody titers are used as the endpoint. The in vivo passive cutaneous anaphylaxis (PCA) assay is used to measure specific IgG1 antibody. This report describes the development and validation of an enzyme-linked immunosorbent assay (ELISA) to measure guinea pig specific IgG1 antibody as an in vitro alternative to the PCA assay. Cross reactivity of various rabbit and mouse (monoclonal) anti-guinea pig IgG1 preparations were evaluated using purified IgG1 and IgG2 from serum of guinea pigs immunized with ovalbumin. The two subclasses of guinea pig IgG were purified by first using Protein A affinity chromatography, followed by anion exchange chromatography and fluid phase isoelectric focusing. Affinity-purified rabbit anti-guinea pig IgG1 was shown to have minimal cross reactivity toward IgG2, while providing a strong signal with IgG1. The ELISA was designed as an antigen capture system in which the following are added in sequence: (1) enzyme antigen (passively adsorbed to the plate), (2) diluted serum samples from guinea pigs immunized with enzyme, (3) affinity-purified rabbit anti-guinea pig IgG1, (4) alkaline phosphatase-conjugated donkey anti-rabbit IgG, and (5) p-nitrophenyl phosphate substrate. Three replicate ELISA and PCA analyses were conducted on sera samples of varying titers from guinea pigs immunized with either Alcalase (protease), BPN' (protease), and Termamyl (amylase) enzyme. The correlation coefficients (r2) between the ELISA and PCA assay for Alcalase, BPN', and Termamyl were 0.826, 0.945, and 0.755, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
酶变应原性的评估涉及使用豚鼠模型，其中特定IgG1抗体滴度用作终点指标。体内被动皮肤过敏反应（PCA）试验用于测量特定IgG1抗体。本报告描述了一种酶联免疫吸附测定（ELISA）的开发和验证，该方法用于测量豚鼠特定IgG1抗体，作为PCA试验的体外替代方法。使用从用卵清蛋白免疫的豚鼠血清中纯化的IgG1和IgG2，评估了各种兔和小鼠（单克隆）抗豚鼠IgG1制剂的交叉反应性。豚鼠IgG的两个亚类首先通过蛋白A亲和层析纯化，然后进行阴离子交换层析和液相等电聚焦。亲和纯化的兔抗豚鼠IgG1对IgG2的交叉反应性最小，而与IgG1产生强信号。ELISA设计为抗原捕获系统，按以下顺序添加：（1）酶抗原（被动吸附到板上），（2）用酶免疫的豚鼠稀释血清样本，（3）亲和纯化的兔抗豚鼠IgG1，（4）碱性磷酸酶偶联的驴抗兔IgG，和（5）对硝基苯磷酸底物。对用碱性蛋白酶（蛋白酶）、嗜热菌蛋白酶（蛋白酶）和淀粉酶（淀粉酶）酶免疫的豚鼠不同滴度的血清样本进行了三次重复ELISA和PCA分析。碱性蛋白酶、嗜热菌蛋白酶和淀粉酶的ELISA和PCA试验之间的相关系数（r2）分别为0.826、0.945和0.755。（摘要截断于250字）
