应用异型双功能交联剂制备酶-抗体结合物

酶标免疫测定法(ELISA)中最关键的化合物是酶-抗体结合物,将酶和抗体交联起来需用交联剂。本文作者使用了N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯(简称SPDP)将辣根过氧化物酶(HRP)和兔抗小鼠IgG(兔IgG)交联起来。我们试验了SPDP/HRP,SPDP/IgG和HRP/IgG的不同比例,以期获得活性高的酶-抗体结合物。此外还研究了从结合物中去除自由HRP和自由IgG的方法。用SDS-PAGE及硝酸纤维膜电泳转移法证明本法制备的结合物不含HRP及IgG的自身聚合物。用ELISA法鉴定结合物制品时,一般稀释度可达到1:10,000以上,有的可达到1:20,000(当结合物浓度A_(280nm)=1.0,底物显色A_(492nm)=1.0时)。

PREPARATION OF ENZYME-ANTIBODY CONJUGATE BY HETEROBIFUNCTIONAL REAGENT SPDP

In enzyme linked immunosorbent assay (ELISA) the key reagent is the enzyme-antibody conjugate which determines the specificity and sensitivity of this method. In this article the use of a heterobifunctional crosalinking reagent, N-su ccinimidyl 3- (2-pyridyldithio) propionate (SPDP) , for the conjugation of horseradish peroxidase (HRP) and rabbit anti-mouse IgG (rabbit IgG) was described .Different ratios of SPDP/HRP, SPDP/IgG and HRP/IgG were studied and the removal of free HRP and free IgG was investigated. SDS-PAGE and Western Blot showed that the conjugate products contain no aggregate of either HRP or IgG.In ELISA our HRP-IgG conjugate preparation can be diluted down to 1:20, 000 (or more) when the concentration (A 230nm) of the conjugate is 1.0 and the absorbance of developed color (A 4921nm) produced from substrate and conjugate is 1.0.