Preparation And Identification Of The Monoclonal Antibody Against AIB1 And Construction Of The Sandwich ELISA Kit

ObjectiveTo prepare monoclonal antibody against AIBland establish hybridoma celllines secreting immunoglobulin G (IgG) antibodies specific for H extremeand C extreme of human AIB1, subsequently construct the Sandwich ELISAkit for AIB1 detection.Methods and Results1. Synthesized immunogen of GST-AIB1-N and GST-AIB1-C peptides wereused to immune BALB/c mouse。Two hybridoma cell strains were obtainedone can stably secret special antibody against AIB1-N and the other canstably secret special antibody against AIB1-C. Following that, the cells areamplified in vivo of BALB/c mouse. The antibody is purified by repeatedprecipitation using different concentration of saturated amine sulfate.Moreover, the protein A affinity chromatography is used to purify theantibody. The bi-directional agar diffusion, ELISA and Western-blot etc areused to respectively identify the McAb. By western-blot, the McAb againstAIB1 display stronglyspecific and high affinity, and has no cross-reactionwith carrier protein and bovine serum albumin (BSA). Subset of the McAb isIgG. The valence of 1A₂E₁ may be up to 1:409600 and 4B₉F₁₂ is 1:102400. 2.We add the BNHS reagents to the McAb 1A₂E₁, incubate them at indoortemperature for 4 hours and harvest Biotin- 1A₂E₁ with its valence-1: 3200.We apply biotinylated McAb to detect AIB1 antigen of target cells.Immunocytochemistry shows that cells of experimental group are positiveresult while those of the controlling group are negateve, andimmunofluorescence shows that tumor cell of the experimental groupexpresses fluorescence strong,while the fluorescence intensity of the controlgroup is negative.3.The Sandwich ELISA kit is based on two strain McAbs, 1A₂E₁ and4B₉F₁₂.1A₂E₁ is biotinylated and acted as labeling antibody. 4B₉F₁₂ isimmobilized McAb. All experiment data are to set up Sandwich ELISA kitand to find out the most suitable reaction system. The immobilizedmonoclonal antibody is 10μg/mtby 0.01 M pH9.6 CB to dilute, and workingconcentration of Biotin- 1A₂E₁ is 1:500.Conclusion1. The McAb against AIB1-N/AIB1-C is produced successfully.2. The method of preparing biotinylated McAb is Successful, and it can beused to detect target cells which express AIB1 antigen.3. The construction and identification of the Sandwich ELISA kit achievesour desire and paves the way to clinical usage.