Use of high-performance liquid chromatography for the purification of antibodies and antibody conjugates and the study of antibody-antigen interactions.
The purification and characterization of polyclonal and monoclonal antibodies and antibody-enzyme conjugates by high-performance liquid chromatography (HPLC) using a Protein Pak 300 SW column and a Protein Pak DEAE anion-exchange column is described. The following polyclonal antibodies were examined: (i) donkey anti-mouse immunoglobulin G (IgG), (ii) mouse IgG, (iii) sheep anti-human IgG and (IV) goat anti-human factor VIII. HPLC was used to analyse the purity of horseradish peroxidase conjugates of rabbit anti-mouse IgG and donkey anti-mouse IgG. In the case of donkey anti-mouse IgG, each stage of the production of the purified antibody and antibody-enzyme conjugate was monitored by HPLC. HPLC was used to examine monoclonal mouse anti-human T cell and mouse anti-human apoliproprotein B antibodies. The presence of antibody in ascites fluid from mice bearing Landschütz ascites tumour cells was also detected. The antigen-antibody reaction between human serum albumin and anti-human serum albumin was demonstrated using HPLC and this procedure should offer a novel method for studying antigen-antibody interaction.
Carty
P
P
School of Biological Sciences, National Institute for Higher Education, Dublin, Ireland.
O'Kennedy
R
R
D000645:Ammonium Sulfate; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000937:Antigen-Antibody Reactions; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000032:analysis; D008970:Molecular Weight; D010527:Perissodactyla / Q000276:immunology
D000906:Antibodies; D007074:Immunoglobulin G; D000645:Ammonium Sulfate