Synthesis Of Artificial Digoxin Antigen And Preparation Of Monoclonal Anti-Digoxin Antibody

OBJECTIVE: To investigate the preparation and identification of artificial antigen for digoxin; to screen the optimal condition of preparation of hybridomas; and to study the monoclonal antibody preparation of digoxin.METHODS: Digoxin was conjugated with bovine serum albumin(BSA) and ovalbumin (OVA) using sodium periodate oxidation method to obtain the Dig-BSA and Dig-OVA conjugate, the conjugate and the hapten number in the conjugate was determined by UV-Vis spectrophotometry(UV), Infrared Spectroscopy(IR) and Matrix-Assisted Laser Absorption Ionization Time-Of-Flight Mass Spectrometry(MALDI-TOF-MS); the specificity of the antigen of Dig-BSA was identified by enzyme-linked immunosorbent assay (ELISA); the female BALB/C mice, seven weeks old, were immunized with Dig-BSA to prepare the antiserum against digoxin, the serum titer of the digoxin antibodies were detected by ELISA which based upon Dig-OVA.To study the effects of different PEG concentration, PEG dosage, ratio of SP2/0 cells and spleen cells, mixed cell density and feeder cell density, in which the SP2/0 cells and immunizing mice's spleen cells were induced to fusion with the help of fusogenic agent polyethylene glycol(PEG, MW=4000).The fused cells were cultured in the 96 wells cell culture plates, then the selected solution HAT was used to culture the mixed cell so as to select hybridoma cells. The hybridoma cells was selected and identified by indirect enzyme-linked immunosorbent assay, and re-cloned by limiting dilution assay.RESULTS: The UV spectra and IR spectra of artificial antigen proved that the Dig-BSA conjugate was synthesized successfully; the conjugation ratio of digoxin and the carrier protein BSA was 5:1 confirmed by MALDI-TOF-MS; the antiserum against digoxin with a titer of 1∶62500 was obtained after immunizing the mice with the Dig-BSA conjugate.When the PEG concentration, ratio of SP2/0 cells and spleen cells, mixed cell density and feeder cell density was 45%PEG(5%DMSO), 1︰5, 105/well and 0 respectively, the cell fusion rate reached to 84.7%.The selected hybridoma cells were able to secrete monoclonal antibodies of digoxin stably. CONCLUSIONS: The artificial antigen of digoxin was able to synthetized by this reaction; the antigen possesses good immunogenicity, suggesting the feasibility of preparing the monoclonal antibody against digoxin using this conjugate.Screening the optimum condition for cell fusion can improve the efficiency of preparation of monoclonal antibodies.The hybridoma technique was able to prepare monoclonal antibody of digoxin successfully.