Preparation Of Monoclonal Antibodies Against Spring Viremia Of Carp Virus (SVCV) And Development Of Double-Antibody Sandwich Indirect Elisa To Detect SVCV

To prepare specific monoclonal antibodies against Spring Viremia of Carp Virus, mouse myeloma cells (SP2/0) were fused with spleen cells from BALB/c immunized with purified SVCV. After fusion and selection, the specify monoclonal antibodies against SVCV were produced. Analyzed the immune biological characteristics and established the method of double antibody sandwich ELISA method against SVCV. The main esults were as following.1. SVCV inoculated on sensitive cell lines of EPC.Repeated freezing and thawing several times when Cytopathogenic effected and collected cell suspension. SVCV in cell suspension was centrifugated by sucrose density gradient. Three concentrations of sucrose(60%,45%,30%) were used to purify SVCV. After that three protein bands were collected, SDS-PAGE analysis showed that virus was mainly concentrated in the second protein bands. In this research, the mice were immunized 6 times. Immunized BALB/c mice with purified SVCV, 100μg of each. The first immunogen emulsified with Freund complete adjuvant equivalent, the second, third, fourth and fifth antigens with Freund incomplete adjuvant emulsion equivalent.The sixth booster immunization was not adjuvanted. Mouse myeloma cells (SP2/0) were fused with spleen cells from BALB/c immunized with purified SVCV after 3 days of the booster immunization, and the cells were incubated in 37℃5% carbon dioxide incubator. We analysised the number of chromosome, the fusion rate was 85%.2. The microtiter plates was coated with purified SVCV and EPC, unimmuned serum, immuned serum were negative and positive control, respectively. PBS was vacancy control. Supernatant was added to each well of 100μL. After incubated at 37℃for 1h, washing 3 times with PBST; then added 1:4000 IgG-HRP to each well of 100μL, After incubated at 37℃for 1h, washing 5 times with PBST; colored with TMB substrate system for 10min and stopped with 2 mol/L H2SO4.In the end, the absorbance of 450nm was measured using Spectra max Plus 384 microplate Continuous Spectrum Measurement System. Positive colonies were selected by indirect ELISA, and after three times limited dilution cloning, four hybridoma cell lines secreting monocolonal antibodies (Mabs) against SVCV (1F1,3E1,3F5,4F9) were produced.3. The ascites monoclonal antibody were purified with caprylic acid-ammonium sulfate.SDS-PAGE analysis showed that,4 purified monoclonal antibody had two protein bands, one was the IgG heavy chain (about 50 kDa), another was the light chain (about 25 kDa). Before purified there werw more complex protein, while after the purification the complex protein was significantly reduced.It indicated that the purification method could be used for the purification of monoclonal antibodies. Characterization of monoclonal antibody to the Spring Viremia of Carp Virus. In ELISA assays, The titres of ascitic fluids of three Mabs ranged between 1:160000 and 1:640000.The result of isotypes tests showed that the 1F1 and 3E1 were characterized as IgG2a, and 3F5 and 4F9 were IgG1.The light chain of them wasκ.Western-blot analysis revealed that three Mabs (1F1,3F5, and 4F9) recognized specifically to a single protein of 47 kDa (N), while the Mab 3E1 only reacted with proteins of 69 kDa (G). Analysis of epitopes demonstrated that 1F1,3F5,4F9 may recognize the same epitope,while 3E1 maybe different. Indirect immunofluorescence assay showed that all of these Mabs had fluorescence characteristics, and the specific fluorescence signals appeared in the cytoplasm of SVCV-infected (EPC) cells. These antibodies would be useful tools to establish Immunological detection methods of SVCV.4. Double antibody sandwich ELISA method against SVCV was developed. Based on the prepared 4F9 monoclonal antibody (McAb) and goat serum anti-SVCV, a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method for SVCV was developed. The assay was optimized by phalanx titration to use following antibody dilutions:capture antibody was at 2.5μg/mL, the best blocking solution was 2% BSA, and incubated in 37℃for 1 h.detecting antibody at 1:1000 and the optimal conjugate was 1:4000, respectively. This method had high sensitivity and with a detection limit of 40ng/mL. TMB was selected as the substrate and anhydrous ethanol as solvent. The results showed that the best of substrate reaction time was 10min at room temperature.The specificity tests showed that there was no cross-reaction with KHV, HRV, VHSV and IHNV. The assay is sensitive and specific and may be used in the determination of SVCV.