Purification of human anti-TF (Thomsen-Friedenreich) and anti-Tn antibodies by affinity chromatography on glycophorin A derivatives and characterization of the antibodies by microtiter plate ELISA.
The TF and Tn antigens were obtained from glycophorin A (GPA) by desialylation under mild acidic conditions and by desialylation followed by Smith degradation, respectively. A method of purification of anti-TF and anti-Tn antibodies from human sera by affinity chromatography on the immobilized asialoGPA (TF antigen) and on asialo-agalactoGPA (Tn antigen), respectively, is described. Purity of the antibodies was demonstrated by SDS-polyacrylamide gel electrophoresis and their specific reactivity with TF or Tn antigens was shown using hemagglutination and the microtiter plate ELISA. A high unspecific binding of human immunoglobulins to the ELISA plates was encountered, therefore optimal conditions for the most specific binding of the antibodies to the target antigens were selected. Problems of the unspecific binding of immunoglobulins were more difficult to overcome when the antibodies were determined in whole sera by their binding to antigen-coated ELISA plates.
Duk
M
M
Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław.
Lisowska
E
E
D000906:Antibodies / Q000097:blood / Q000302:isolation & purification*; D000918:Antibody Specificity; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006021:Glycophorins / Q000031:analogs & derivatives / Q000737:chemistry; D006384:Hemagglutination; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data
D000906:Antibodies; D015295:Antigens, Tumor-Associated, Carbohydrate; D006021:Glycophorins; C040286:Tn antigen; C016467:Thomsen-Friedenreich antigen