基于酶促反应动力学的无标准品ELISA抗体滴度定量方法

Antibody titre quantitation method by ELISA without standard substance based on kinetics of enzyme-catalyzed reactions

目的建立无标准品的酶联免疫吸附测定(ELISA)抗体滴度定量方法。方法对血清抗体ELISA检测设置非特异、阴性对照、特异和总抗体检测组。显色早期的时间-吸光度数据经线性拟合的斜率即吸光度变化速度,分别记为ν0、νC、νS、νT。基于底物过量时ν值与待测抗体浓度(C)呈直线关系,设计ν值函数计算待测样本与阴性对照样本的抗体浓度倍比。以鱼胶原蛋白免疫昆明小鼠的血清特异IgG抗体检测进行实例分析。结果函数C/CC=(ν-ν0)/(νC-ν0)可计算待测样本与阴性对照样本的特异抗体浓度倍比(滴度)。结论该动力学ELISA抗体滴度检测方法适用于无标准品半定量分析。

Objective To establish a antibody titre quantitation method by ELISA without standard substance. Methods The test groups of non-specific,negative control,specific and total antibodies were set for detecting sera antibodies by ELISA. After linear fitting of the time-absorbance data of early developing, the fitted slopes were used as the velocity of absorbance changing, which were denoted by v0 ,vc ,vs ,vr. Based on that the v-value and the concentration （C） of determinand were linear relationship while the substrate was excessive,the function with parameter of v-values for reflecting the multiple proportions of antibodies con- centrations between specific and negative control groups could be deduced. The assessment of specific IgG antibodies in sera of KM mice immunized with fish collagen was used as an instance. Results The function C/Cc = （v--v0 ） / （vc - v0 ） could calculate the mul- tiple proportions （titre） of antibodies concentrations between specific and negative control groups. Conclusion The above method of antibody titre quantitation is suitable for semi-quantitative analysis without standard substance.