Enzyme-Linked Immuno-Sorbent Assay for Bovine Caseins

우 Casein의 면역효소분석법

A rapid, sensitive, and specific enzyme-linked immuno-sorbent assay (ELISA) for bovine casein was developed. Biotinylated casein and peroxidase-conjugated avidin were used in the assay with antibody separated from yolks of immunized hens. Caseins were biotinylated with sulfo-N-hydroxy succinimido biotin and peroxi-dase-conjugated avidin bound the biotinylated casein which became bound to immobilized anti-body on a microplate. The antibodies were specific for bovine $\alpha$- and $\beta$-caseins, and their cross-reactivities with whey proteins, IgG, and serum albumin from bovine were not detectable by ELISA and Western blot. Various sensitivities ranging from 2ng/ml to 20${\mu}\textrm{g}$/ml of casein were achieved, and were controlled by adding vanous concentrations of the biotinylated casein. Parallelism was observed between standard and sample curves. The coefficients of variation of intra-assays and inter-assays from the most sensitive assay were 5.5 and 5.7%, respectively, at the 50% displacement. Casein contents of peripaturient milk samples showed that casein secretion rapidly increased 3d prepartum.

본 연구는 우 casein을 신속, 정확하게 분석할 수 있는 특수 면역효소분석법을 개발하였다. Biotin이 연결된 casein과 peroxidase-conjugated avidin을 사용하였으며 면역화시킨 닭의 난황으로부터 추출한 항체를 이용하여 분석하였다. Sulfo-N-hydroxy succinimido biotin을 사용하여 casein에 biotin을 연결시키고 microplate에 고정한 뒤 peroxidase-conjugated avidin을 결합시켰다. 생산된 항체는 $\alpha$-와 $\beta$-casein에 특이적이었으며, 유청단백질, IgG, 우혈청 알부민과 교차반응은 면역효소분석법과 Western blot에서 나타나지 않았다. 본 분석법의 민감도는 2ng에서 20$\mu\textrm{g}$이었으며 Standard와 시료의 분석시 뚜렷한 평행곡선이 형성되었다. Intra-assay와 Inter-assay의 변이계수는 각각 5.5와 5.7%이었다. 그리고 비유 초기의 casein량을 조사한 결과 분만전 3일경부터 급격히 상승하는 것을 알 수 있었다.