A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli.
A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli.
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一种将亲和纯化的 Fab 与大肠杆菌的 β-D-半乳糖苷酶缀合的微量方法。
DOI：
10.1093/oxfordjournals.jbchem.a135406
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发表时间：
1985
影响因子：
2.7
通讯作者：
E. Ishikawa
中科院分区：
文献类型：
--
作者：
S. Inoue;S. Hashida;T. Kohno;K. Tanaka;E. Ishikawa
介绍了一种从大肠杆菌中亲和纯化的Fab‘与β-D-半乳糖苷酶偶联的微型方法。用胃酶消化兔抗人绒毛膜促性腺激素血清(0.2ml)，将其转化为F(ab‘)2，然后用人绒毛膜促性腺激素-Sepharose4B柱，在pH 2.5下洗脱。将亲和纯化的抗人绒毛膜促性腺激素F(ab‘)2与非特异性山羊F(ab’)2(0.5 mg)混合，用2-巯基乙胺还原，将F(ab‘)2裂解成Fab’，用N，N‘-邻苯二亚胺偶联到β-D-半乳糖苷酶上。用4M尿素亲和层析法从非特异性山羊Fab‘-β-D-半乳糖苷酶结合物和未结合的β-D-半乳糖苷酶中分离纯化的兔抗人绒毛膜促性腺激素Fab’-β-D-半乳糖苷酶结合物。亲和纯化的结合物的量为56-69微克。人绒毛膜促性腺激素的夹心酶免疫分析检测下限比亲和纯化前提高了30倍。该方法适用于小牛肠道碱性磷酸酶的偶联反应，也适用于其它在4M尿素中稳定的酶的偶联反应。
A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli is described. Rabbit anti-human chorionic gonadotropin serum (0.2 ml) was digested with pepsin to convert IgG to F(ab')2 and applied to a column of human chorionic gonadotropin-Sepharose 4B, followed by elution at pH 2.5. The affinity-purified anti-human chorionic gonadotropin F(ab')2 was mixed with non-specific goat F(ab')2 (0.5 mg) as a carrier, reduced with 2-mercaptoethylamine to split F(ab')2 to Fab' and conjugated to beta-D-galactosidase using N,N'-o-phenylenedimaleimide. The affinity-purified rabbit anti-human chorionic gonadotropin Fab'-beta-D-galactosidase conjugate was separated from non-specific goat Fab'-beta-D-galactosidase conjugate and unconjugated beta-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B using 4 M urea. The amount of the affinity-purified conjugate obtained was 56-69 micrograms. The detection limit of human chorionic gonadotropin by a sandwich enzyme immunoassay technique was improved 30-fold by using the affinity-purified conjugate as compared with that before affinity-purification. This method is applicable to the conjugation with alkaline phosphatase from calf intestine and probably also other enzymes which are stable in 4 M urea.