Preparation of Monospecific Polyclonal Antibodies Against Glechoma hederacea Agglutinin
- VernacularTitle:抗欧亚活血丹凝集素特异性多克隆抗体的制备
- Author:
Weifang WANG
;
Chaozu HE
- Publication Type:Journal Article
- Keywords:
monospecific polyclonal antibodies;
antibody purification;
Glechoma hederacea agglutinin (Gleheda)
- From:
Progress in Biochemistry and Biophysics
2006;33(11):1113-1119
- CountryChina
- Language:Chinese
-
Abstract:
Glechoma hederocea agglutinin (Gleheda) is a novel glycosylated lectin isolated from the leaves of G. hederacea. Like other glycosylated proteins, the detection of Gleheda by immunological methods is often hampered by the cross-reactivity of the polyclonal antibodies with unrelated glycoproteins. Hence a protocol to purify monospecific polyclonal antibodies from a crude antiserum raised against Gleheda was developed. After selective ammonium sulfate precipitation and successive affinity chromatography on columns of Sepharose 4B with immobilized Gleheda and Robinia pseudoacacia agglutinin (RPA), respectively, ion-exchange chromatography on a column of Q Fast Flow was used for further purification. The specificity of the antibody fractions from each step was tested by double immunodiffusion assay and analyzed by Western blot. Results revealed that affinity chromatography of the immunoglobulin fraction on the immobilized Gleheda antigen yielded an antibody preparation that still cross-reacted with many proteins in leaf extracts. Depletion of nonspecific cross-reacting antibodies directed against the glycan part of the glycoprotein by affinity chromatography on immobilized RPA removed most but not all nonspecifically reacting antibodies. Only upon further purification by ion exchange chromatography an IgG fraction of monospecific antibodies that reacted exclusively with Gleheda could be obtained and accordingly was suitable for immunodetection studies. This antibody purification procedure promises simplicity and efficiency. In addition, this method does not require expensive facilities.
