IgG3酶联免疫分析方法的建立及初步应用研究

Establishment and Primary Application of a Method of IgG3 ELISA in this Laboratory

目的：建立IgG3酶联免疫分析（ELISA）方法，探讨IgG3在炎症性疾病患者血清中的水平变化。方法：用兔抗人IgG3的多克隆抗体包被成固相酶标板，以识别结合待测标本中的IgG3，再用此多克隆抗体标记生物素（Bio-Ab），用亲和素标记辣根过氧化物酶（HRP-A），向固相酶标板中加入标准品或待测品，反应、洗涤后，加入Bio-Ab，反应、洗涤后，加入HRP-A，反应、洗涤后，酶标板上形成Ab-Ag-Ab-Bio-A-HRP复合物，加酶底物显色，用酶标仪在相应波长下测定光密度（OD值），根据标准曲线，计算出待测标本中的IgG3含量。结果：该法测定范围为（1．875-60）ng／ml，最低检出量1．5ng／ml，批内和批间误差分析〈8％和10％。用该法测得正常青年人血清IgG3含量为（12.91±4．25）ng／ml（n=64），风湿病患者为（11．23±4．64）ng/ml（n=64），肺部感染患者为（9．32±2．00）ng／ml（n=24），后两组血清IgG3水平均显著低于正常青年人（P〈0．05）。结论：我们建立的IgG3 ELISA方法稳定、简便，特异性和灵敏度高，适于检测人血清ISG3水平的变化。

Objective To develop a method of ELISA for detection of IgG3 and its clinical application. Methods Rabbit anti - human IgG3 multiclonic antibody was prepared and used to pack solid enzyme - linked board for recognition and binding of IgG3 in the samples. This multi - clonic antibody was also used to label biotin （ forming Bio - Ab） and avidin was used to label horse radish peroxidase （ forming HRP - A）. These two substances were successively reacted with the samples on the board, i.e. Bio - Ab and HRP - A these were successively reacted with standards or samples on the solid enzyme - labeled board i.e. and HRP - A were added respectively, with a washing between them, and then Ab - Ag - Ab - Bio - A - HRP complex was formed. After color display with enzyme substrate, we used enzymelabeler to check absorbency （OD value） under corresponding wavelength, and calculated IgG3 concentration in the samples according to the standard curve. Results The detective range of this assay was from （ 1. 875 to 60 ＋ 2.5 ） ng/ml with the lowest detection of 1.5ng/ml, the intra - and inter - assay deviation were less than 8% and 10%, respectively. Using this assay, the detected serum IgG3 level of normal youth was 12.91 ± 4. 25 ng/ml （ n = 64）, the level of rheumatic patients was 11.23 ± 4.64ng/ml （ n = 64）, that of pulmonary infection patietns was 9.32 ± 2. 00ng/ml （ n = 24）. And serum IgG3 levels of the latter two groups were significantly lower than normal youth （ both P 〈 0.05）. Conclusion The IgG3 ELISA we established is simple and stable, has pretty good specificity and sensitivity, and is qualified for clinical use.