Immunoaffinity purification of recombinant hepatitis B surface antigen from yeast using a monoclonal antibody.
A murine monoclonal antibody developed for the purification of recombinant hepatitis B surface antigen was immobilized on a chromatographic support and used to adsorb and purify the recombinant antigen from yeast. The adsorption-elution behaviour was first investigated using monoclonal antibody-coated enzyme-linked immunosorbent assay plates and performing adsorption, washing and elution procedures with different elution agents. It was found that 3 M KSCN and 8 M urea at neutral pH disrupted antigen-antibody interactions in both systems. The procedure for washing the immunoaffinity column was optimized, using different salts and detergents. The best results were obtained by applying the starting material in 1 M NaCl and washing with the same buffer. The use of 0.1% sodium deoxycholate in the washing buffer reduced about 20-fold lipopolysaccharide contamination in the eluates as compared with washing without detergent. The relationship between bed height and the adsorption capacity of the column was studied, and it was found that the dynamic capacity decreased twice on reducing its length/diameter ratio tenfold. The recovery of antigen was not affected by increasing the flow-rate up to 25 cm/h but decreased at higher values. Using the optimum conditions, the affinity column was able to purify the recombinant hepatitis B surface antigen to more than 90% purity and a 65% antigen recovery was obtained.
Agraz
A
A
Biopharmaceutics Development Department, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Duarte
C A
CA
Costa
L
L
Pérez
L
L
Páez
R
R
Pujol
V
V
Fontirrochi
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006514:Hepatitis B Surface Antigens / Q000276:immunology / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D051379:Mice; D010843:Pichia / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins