Preparation Of Monoclonal Antibodies Against β2-microglobulin And Initially Established ELISA Detection Method

β2-microglobulin (β2-microglobulin, β2-MG) is an endogenous protein with lowmolecular weight of11.8KD in the serum. It’s the light chain (β-chain) of classⅠinmembrane integrity of major histocompatibility complex (MHC), and plays animportant role in the immune response. With the metabolism and degradation ofMHC, β2-microglobulin is released in free form in the serum, urine, cerebrospinalfluid, saliva and other body fluids, and keeps low and constant levels. In recent years,β2-microglobulin has become a hot topic in basic medical and clinical diagnosis, andthe changes of its levels in serum or urine has become an important indicator forevaluating organ function and some related diseases’ occurrence, development andprognosis. The detection methods of β2-microglobulin mainly includeimmunonephelometry, immune turbidimetric method, chemiluminescence method,ect, in which ELISA is highly popular in clinical practice because of its simpleness,swiftness and high sensitivity. In the light of few ELISA kits are applied to clinicalpractice, the development of fast and effective method of ELISA kit is of greatsignificance.In this study, Balb/c mice were immunized with high purity humanβ2-microglobulin using conventional immunization and spleen immune methods. TheSP2/0cells on logarithmic phase and immunized spleen cells were fused; positiveholes were selected by indirect ELISA method. After3to4times cloning, multiplepassages and recovery after cryopreservation, five strains of McAbs stably secretingMcAbs against β2-microglobulin were obtained, which were named2G7,2H2,3F9,5D10and8E12respectively. Five monoclonal antibody subtypes were all belong toIgG1class, whose ascites titers were between10-7-10-9. The ascites, which werepurified by Protein-A affinity chromatography, were detected by electrophoresis, andthe result showed that the purity of five monoclonal antibodies was good. IndirectELISA and Wersten blot results showed that five monoclonal antibody couldspecifically bind to human β2-microglobulin, but have not cross-reactivity with human serum albumin, hemoglobin, EB virus genetic engineered antigens, ormycoplasma genetic engineering antigens, which proved those five monoclonalantibodies, had high specificity and reactogenicity.The modified sodium iodide method was used to conjugate five monoclonal withHRP. By antibodies matching experiment, a pair of matching antibody suitable fordouble-antibody sandwich ELISA were fond, which was the initial establishment ofthe β2-microglobulin double-antibody sandwich ELISA for the quantitative.Determined by phalanx titration experiments, the best package concentration ofantibody was3ug/mL, enzyme-labeled secondary antibody optimal workingconcentration was1:4000, the linear range of detection was1.25~40ng/mL, thelowest detection limit was1.25ng/mL, intra-assay CV<8%, inter-assay CV<12%, andthe accuracy was good.35copies of serum samples were established the clinicaldetection of β2-microglobulin levels with the detection method in this study, whichhad good correlation compared with Roche’s β2macroglobulin test kits test results,and the correlation coefficient R2=0.9550.This study successfully got anti-human β2-microglobulin monoclonal antibodyhybridism cell lines, and initially established a double-antibody sandwich ELISA forthe detection of quantitative human β2-microglobulin, which laid a good foundationfor the further development of rapid diagnostic kits and other detection methods.