Cross-reacting monoclonal anti-alpha 1-microglobulin antibodies produced by multi-species immunization and using protein G for the screening assay.
In order to generate monoclonal antibodies (MAb) directed against the low molecular weight glycoprotein alpha 1-microglobulin, a BALB/c mouse was immunized with a mixture of human, guinea pig, rat and rabbit alpha 1-microglobulin homologues (multi-species immunization) and boosted several times. On day 194, the mouse splenocytes were fused to SP2/0 myeloma cells. The resulting hybridomas were screened for anti-alpha 1-microglobulin activity against the alpha 1-microglobulin mixture or against the individual homologues. For this screening, protein G (the newly described IgG-binding streptococcal protein) was used in a solid-phase radioimmunoassay. The binding of protein G to immobilized antigen-antibody complexes was enhanced by pre-incubation with rabbit anti-mouse immunoglobulin G. The result was a panel of nine established hybridoma lines, all producing unique monoclonal antibodies, of IgG1 or IgG2a class, to alpha 1-microglobulin. The antibodies were not only reactive in solid-phase radioimmunoassay, but they could also immunoprecipitate 125I-labeled soluble alpha 1-microglobulin. Moreover, they reacted specifically with the alpha 1-microglobulin band in Western blots of urinary proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Such monoclonal antibodies are potentially valuable reagents for the further characterization of alpha 1-microglobulin.
D000510:Alpha-Globulins / Q000276:immunology* / Q000652:urine; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D003429:Cross Reactions; D006168:Guinea Pigs; D006801:Humans; D006825:Hybridomas / Q000032:analysis; D007114:Immunization / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D009419:Nerve Tissue Proteins; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats
D000510:Alpha-Globulins; D000911:Antibodies, Monoclonal; C029152:G-substrate; D009419:Nerve Tissue Proteins; C016001:alpha-1-microglobulin