ABSTRACT
<p><b>OBJECTIVE</b>To produce specific monoclonal antibody (mAb) against recombinant human erythropoietin (rHuEPO) for development of highly efficient methods for erythropoietin detection in biological fluids.</p><p><b>METHODS</b>rHuEPO was covalently coupled with bovine serum albumin (BSA) and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western blot.</p><p><b>RESULTS</b>The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1 x 10(8) L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work.</p><p><b>CONCLUSIONS</b>The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.</p>
Subject(s)
Animals , Female , Humans , Mice , Antibodies, Monoclonal , Allergy and Immunology , Antibody Affinity , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Erythropoietin , Allergy and Immunology , Immunoglobulin G , Allergy and Immunology , Immunoglobulin M , Allergy and Immunology , Mice, Inbred BALB C , Molecular Weight , Recombinant ProteinsABSTRACT
<p><b>OBJECTIVE</b>Determination of estrone (E1) levels has a significant meaning in evaluating physiological effect and diagnosing some diseases. In order to detect free E1 in biological fluids, a monoclonal antibody specific for E1 was prepared after the complete antigen of E1 was synthesized. The purified monoclonal antibody was fully characterized for later immunoassay.</p><p><b>METHODS</b>3-O-carboxymethyl ether derivative of E1 was synthesized and in turn coupled to bovine serum albumin (BSA) to form complete antigen E1-BSA. A monoclonal antibody (McAb) specific for E1 was produced both in vitro and in vivo by a hybridoma anti-E1. Anti-E1 was prepared by fusion of SP2/0 murine myeloma cells with spleen cells isolated from immunized BALB/c mouse. The McAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western-blotting. The specificity of the immunoassay was investigated by determining the cross-reactions of E1 analogs when free E1 was detected by competitive indirect enzyme-linked immunosorbent assay (CI-ELISA).</p><p><b>RESULTS</b>Analysis revealed that anti-E1 McAb (E1-McAb) was of the IgG1 type, the molecular weight of E1-McAb was 164,000 daltons. The affinity constant of E1-McAb with coated complete antigen was 8.2 x 10(8) L/mol. The linear range for free E1 determined by CI-ELISA was 10 pg/mL-10 ng/mL. The detection limit was 21.4 pg/mL (defined as twice the standard deviation of the blank).</p><p><b>CONCLUSION</b>The CI-ELISA developed with E1-McAb was both sensitive and specific. The prepared E1-McAb can be used in some immunoassays.</p>