Antibody response in immunized rabbits measured with bacterial immunoglobulin-binding proteins.
Protein G, an immunoglobulin (Ig)-binding protein isolated from group C or G streptococci, binds to the Fc portion of IgG. Protein L, from the anaerobic bacterium Peptostreptococcus magnus, specifically binds light chains of Ig. In this study, protein G and L were used to measure the production of antibodies in immunized rabbits. Two rabbits were immunized with a mixture of human urinary proteins from a patient with tubular proteinuria, and blood samples were collected regularly from the animals for 6 weeks after the immunization. The antibody levels of the blood samples against six of the proteins in the antigen mixture were then measured by ELISA. Microtiter plates were coated with each of the antigens, incubated with the rabbit serum samples, and the specific antibodies of the IgG class measured by incubation with biotinylated protein G, and antibodies of all Ig classes with biotinylated protein L. Alternatively, Western blotting was employed, where the antibodies which bound to each antigen after separation by SDS-PAGE and transfer to nitrocellulose membranes, were detected by protein G or L. The results showed that antibody production against five of the antigens, albumin, alpha 1 gamma-acid glycoprotein, alpha 1 gamma-microglobulin, Ig light chains, and retinol-binding protein, showed a similar pattern, although the magnitude of the initial IgM response differed somewhat. After 6 weeks, the levels of the protein G-binding antibodies had reached a plateau, while those of protein L-binding antibodies were still increasing. The response to the sixth antigen, beta 2 microglobulin, was considerably different. A dramatic increase of anti-beta 2 gamma-microglobulin antibodies was seen during the 4th week after immunization when protein L was used.
Elbashir
M I
MI
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
Nilson
B H
BH
Akesson
P
P
Björck
L
L
Akerström
B
B
D000818:Animals; D000917:Antibody Formation; D001426:Bacterial Proteins / Q000276:immunology*; D001710:Biotin; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007114:Immunization; D007141:Immunoglobulin Fc Fragments / Q000032:analysis*; D007158:Immunologic Techniques; D011506:Proteins / Q000008:administration & dosage / Q000276:immunology; D011507:Proteinuria / Q000652:urine; D011817:Rabbits
D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; C034284:IgG Fc-binding protein, Streptococcus; D007141:Immunoglobulin Fc Fragments; D011506:Proteins; D001710:Biotin