Preparation And Identification Of Monoclonal Antibody Against Matsa

Marek's disease tumor-associated surface antigen(MATSA) is a unique antigen which is associated with MD tumor cells and cells of MD lymphoblastoid tumor cell lines.It plays a major role in stimulating immune effect against tumor and is used for MD differential diagnosis.Balb/c mice were immunized with purified MATSA.And then the spleen cells of the mice were isolated and fused with SP2/0 cells.The positive hybridoma cells were screened by indirect ELISA and its Karyotype was analyzed.At last,Balb/c mice were injected with positive Hybridoma cells and the monoclonal antibody in the ascites was purified by ammonium sulfate salting-out and identified for its subtypes.McAb against MATSA was obtained,which could be applied to the study of Marek's Diease.The results are as follows:1.5 Balb/c mice were immunized with MATSA for 6 times.The titre of MATSA antibody in serum was detected by indirect ELISA.The results showed that,the titer were 2⁶, 2⁶,2⁴,2⁶,2⁶ respectively after the third immunizations,and 2⁶,2⁶,2⁶,2⁶,2⁷ respectively after the fourth immunizations and 2⁸,2⁸,2⁸,2⁸,2⁸ respectively after the fifth immunizations.After the sixth immunizations,the titer 2¹³,2¹³,2¹³,2¹³,2¹³ respectively,were significantly increased then before.Finally,immunized mice with higher level antibody titer were obtained which can be used to produce McAb against MATSA.2.The spleen cells of two immunized mice with higher level antibody were isolated.Cell fusion was made twice.At the first time,the spleen cells of the third mouse was fused with SP2/0 cells.But positive hybridoma cells were not abtained.At the second time,the spleen cells of the first mouse was fused with SP2/0 cells.The fused cells were put in 384 holes of four plates,among which 116 positive hybridoma cells were obtained.Fusion rate of the second time was 30.2%.Detected by indirect ELISA,22 positive cells were obtained.The positive rate was 19%.Cells with higher OD value and perfect growth state were cloned 3 times by limiting dilution.Three strains hybridoma cell lines were established and could secrete MATSA antibodies persistently in the culture medium.The hybridoma cell lines could also secrete the specific antibodies persistently after passage culture,frozen in liquid nitrogen and resuscitated.The antibody titer of cell culture supernatant was 2⁶by ELISA detection.3.Karyotype analysis was carried out by colchicines with three strains;the number of chromosomes of hybridoma cell was 52±2 pairs,similar with the total number of chromosomes of the parent cells,which indicated that the parents' cells fused as hybrids.4.Hybridoma cell line was injected into 100 Balb/c mice by intraperitoneal injection then harvest infected ascites of mice,the antibody titer in ascitic fluid was 2¹⁰.336mL ascitic fluid was isolated by ammonium sulfate salting-out then 16.80g MATSA antibody was obtained after vacuum freeze-drying.The results indicated that the monoclonal antibody has positive reaction with MSB1 cells detected by indirect immunofluorescence staining.