Isolation and purification of apolipoprotein H with heparin affinity column and preparation and identification of anti-ApoH
- VernacularTitle:自制肝素亲和柱分离纯化载脂蛋白H及其抗体制备与鉴定
- Author:
Xiangdong WANG
;
Chunni ZHANG
;
Junjun WANG
;
Ying TIAN
- Publication Type:Journal Article
- Keywords:
Affinity chromatography;
Heparin;
Apolipoprotein H;
Antibody;
Purification and identification
- From:
Chinese Journal of Immunology
2001;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To purify apolipoprotein H (apoH) from fresh human serum with heparin affinity column self-prepared and to prepare polyclonal antibody anti-ApoH.Methods:Affinity column was made by reaction of indirect reductive amination and operating conditions were optimized and their effects were investigated.The apolipoprotein H antigen was purified from human serum by methods of perchloric acid precipitation,ion exchange and affinity chromatography.The polyclonal antiserum against the antigen was obtained by immuning rabbits.Results:The amount of heparin immobilized on the synthesized gel was 5 mg/ml (medium).Molecular weight of the purified antigen was about 50 kD by SDS-PAGE and no other bands were seen,the percentages of 16 amino acids were consistent with that reported previously.In ELISA and dot-ELISA,the polyclonal antibody displayed a cross reaction with bovine serum albumin,while no such reaction was observed in result of Western blot under reduced and denatured condition.Conclusion:The self-prepared affinity column costs very low,but has excellent affinity performance and stable heparin linking,and high purified apoH can be obtained by using the reagent.Polyclonal antibody needs to be further purified before detecting serum apolipoprotein H.
