Preparation And Identification Of Monoclonal Antibody Against Fluorescein Isothiocyanate

Fluorescein Isothiocyanate(FITC)is currently the most widely used fluorescein.Because of its special structure?physical characteristic and chemical characteristic,it is usually used as a fluorescent marker to label various biological macromolecules,such as proteins,nucleic acids and polysaccharides.Especially,the FITC-anti-FITC system based on FITC can effectively amplify the sensitivity of the antigen-antibody reaction.In the present study artificial complete antigens FITC-BSA and FITC-OVA were prepared using the direct marking method,hybridomas producing anti-FITC monoclonal antibody with high affinity and specificity were perpared through hybridoma technology,anti-FITC monoclonal antibody was generated by in vivo induced ascites technique,and the monoclonal antibody was identificated.The above work has laid foundation for the efficient and rapid detection of biological macromolecules labelled with FITC,such as proteins,nucleic acids and polysaccharides.1.Preparation and identification of artificial complete antigenFITC was coupled with carrier protein BSA and OVA respectively using the direct marking method.UV scanning method,SDS-PAGE method and animal immunization method were choosed to identify the artificial complete antigens.As a result the coupling rates of FITC-BSA and FITC-OVA were respectively calculated to be 14:1 and 13:1.SDS-PAGE results demonstrated that,compared to the band of BSA or OVA,the band of FITC-BSA or FITC-OVA migrated slower,suggesting that the conjugations were successful.Animal immunization results demonstrated that artificial complete antigen FITC-BSA could induce an immune response in mice,the antiserum titers of immunized mice could be achieved 1:12800,and Mouse 3 had the highest sensitivity(IC50=4.25 ng/mL),suggesting that the two complete antigens obtained has good antigenicity.2.Preparation and identification of monoclonal antibody against Fluorescein IsothiocyanateWith the highest serum titer and sensitivity,Mouse 3 was chosen for cell fusion.Two strong positive clones,named 1F11 and 2A7,displayed highest titer were subjected to three times limiting dilution for single hybridoma selection.After subcloning and indirect ELISA screening,clone 2A7 with higher titer(1:3200)was used to generate ascites fluid,which was purified by using the caprylic/ammonium sulfate precipitation method.SDS-PAGE results showed two distinct bands of purified 2A7 mAb,with the heavy chain at 53 KD and the light chain at 25 KD,which demonstrated that 2A7 mAb had higher purity.Biological analysis of 2A7 mAb showed that the titer was 1:2.048�106,the affinity was 1.72�109 L/mol,and the IC50 against FITC was 1.37 ?g/mL;Most of the 2A7 mAb belonged to the immunoglobulin G1,?-light chain isotype according to the detection result by using Mouse Monoclonal Antibody Isotyping Reagents;2A7 mAb was highly specific to FITC;After three months of subculture and repeated cryopreservation,the 2A7 hybridoma was able to stably secrete anti-FITC mAb after resuscitation.In this study,we prepared the 2A7 mAb against FITC with high titer,high affinity and high specificity,which can lay foundation for efficiently and rapidly detecting various substance labelled with FITC.