Development And Characterization Of Monoclonal Antibodies Against Green Fluorescent Protein Based On Genetic Immunization

In the last 10 years green fluorescent protein (GFP) has changed from a nearly unknown protein to a commonly used tool in the field of molecular biology, medicine, and cell biology. GFP is mainly come from jellyfish, hydra and coral that belong to coelenterate. It is particularly useful due to its stability and the fact that its chromophore is formed in an autocatalytic cyclization that does not require any cofactor. This has enabled researchers to use GFP in living systems, and it has led to it's widespread use as a marker in the biomedical field. Furthermore, it appears that fusion of GFP to a protein does not alter the function or location of the protein. However, it was found that there is hindrance in some application fields: GFP's fluorescence is dimness at sometimes and confound with background, moreover, purifying syncretic protein is usually difficult. In order to resolve those problems, the monoclonal antibodies(mAb) against GFP was developed.Production of monoclonal antibodies using genetic immunization is a potential alternative technique when purified antigen is difficult to obtain. To prepare the mAb against GFP, 6-week-old female BALB/c mice were immunized intramuscularly with 100 g of purified recombinant plasmid pVAX1-EGFP at 0, 3, 6, 9 weeks. One week after the last inoculation, splencytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells, and the positive hybridoma clones were screened by indirect ELISA. Four hybridoma cell lines named 1A12, 3C8, 3D6, 4D9 were obtained. The antibodyliters of their culture supernatants were 1:3200, 1:1600, 1:3200, 1:2560 and ascitic fluids were 1:50000, 1:13000, 1:40000, 1:25000 respectively in indirect ELISA. The immunoglobulin subclass of the 4 mAbs were IgG1. In Dot-ELISA, four mAbs reacted with bacteria expressing EGFP, they did not react with other bacteria without EGFP. The immunoblotting confirmed that four mAbs recognized 27kD protein of EGFP. The results suggested that these four mAbs were specific to EGFP.GFP monoclonal antibody can improve the sensitivity of the GFP detection and expand the application of GFP. The GFP mAbs could be very useful in the location of the antigen, purification of fusion protein, interaction between host and pathogen and the development of new detection reagent.