Separation And Purification Of Marek's Disease Tumor-Associated Surface Antigen And Its Monoclonal Antibody Development

Marek's disease (MD) is a contagious phymatosis of chickens caused by a herpesvirus, Marek's disease virus (MDV). It is characterized by lymphoproliferation, lymphoma formation and monocytes infiltration in peripheral nerve, gonad, iris, various visceral organ, muscles and skin. MD can not only cause tumor in chickens but also damage immune organs of infected chickens and cause immunosuppression state which can depress resistance of infected chickens and cause immune response fall or no immune response to vaccination. This state can initiate coincidence of many other ailments and secondary infection, then results in large economy loss. MD can be precluded by vaccination with attenuated MDV, non-pathogenic MDV or turkey herpes virus (IIVT). MD is a successful paradigm of viral phymatosis that can be precluded by vaccination. Studies of MD not only help to development of poultry industry but also offer perfect models to search pathogenic mechanism of virus and precaution and control neoplastic disease of mankind and animals.The transformation of T cells can lead to lymphomas formation in chickens after three to five weeks of MDV infection. MD lymphoblastoid tumor cell lines can be established from MD lymphomas. 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 is identification marker of cell transformation. MATSA is not expression on lymphomas cells of avian lymphocytic leukemia (ALL) and avian reticuloendotheliosis (ARE).Anti-tumor targeted drugs using monoclonal antibody as carrier not only are study hot spot of medicine biotechnology territory but also are great topic of anti-tumor targeted drugs studies. This study is about separation and purification of MATSA and its monoclonal antibody development.1. MDV stain GA was resuscitated from liquid nitrogen and rejuvenated in nine-day-old SPF chick embryo fibroblast (CEF). One-day-old SPF chicks were infected with rejuvenated MDV stain GA. Morbility and death started from thirty-five days after infection. Livers and spleens intumesced in typical visceral cases when dissect. Tumor nodules of inequable size formed in livers and spleens. These nodules were gray and their texture was hard and compact. Tumors in some cases grew diffusible. Whole organs intumesced obviously. Incanous tumor tissue and original tissue existed together. Cross sections of organs showed appearance of marble figure. Ratio of tumor formation was percent 35(14/40).2. Anadesma was removed from tumor tissue of livers and spleens of MD chickens. Tumor tissue was washed several times with phosphate buffer saline (PBS) and cut tablets about lmm3. Then, those tablets were washed with PBS to almost no redness of the skin. Tumor tissue tablets were homogenated and washed three times with PBS. Tumor cells were suspended in citrate buffer solution as proportion of 1:20 and incubated 25min in 4°C. Mixed liquor was centrifuged and tumor cells were suspended with PBS. Added 2IU papain and lmmol/L L-cystine 0.1 mL to 2*106 tumor cells and incubated lh in 37°C to remove MATSA from tumor cells. Normal tumor cells and tumor cells treated by papain were detected with indirect fluorescent antibody assay (IFA). The results indicated that MATSA had been removed from tumor cells. The results of indirect enzyme linked immunosorbent assay (ELISA) indicated that interest protein MATSA resided in extraction liquid.3. MSB1 cells were cultured with DMEM and fetal bovine serum (FBS). MSB1 cells were harvested and washed three times with PBS to remove serum. Added 2IU papain and lmmol/L L-cystine 0.1 mL to 2x106 MSB1 cells, then incubated lh in 37°C to remove MATSA from MSB1 cells. Normal MSB1 and MSB1 treated by papain were detected with IFA and the results indicated that MATSA had been removed from MSB1 cells. Crude extract of MATSA was concentrated and purified by ultrafiltration and gel filtration of Sephadex G-75. Purified result was detected with sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE). It is proved that purified MATSA had been obtained. Molecular wieght of purified MATSA is about 35kuo4. Antiserum against MATSA was prepared in rabbits by inoculated rabbits with MSB1 cells. The results of microagglutination test indicated that valences agglutinating full cells of four immunized rabbits all reached 61og2. The results of suspension cell ELISA indicated that valences of four immunized rabbits all reached 1:3200.5. Detection conditions of MATSA antibody by indirect ELISA were investigated and many factors influencing test results were screened. The results showed that the optimal reaction conditions as follow. Coating liquid was buffer bicarbonate of O.lmol/L pH9.6. Optimal coating concentration of antigen was 4ug/mL. Confining liquid was PBST containing 1% BSA. Optimal dilution of antiserum was 1:800. Optimal dilution of enzyme labelled antibody was 1:1200. Action time of enzyme labelled antibody was 45min in 37°C. Action time of substrate was 15min in 37°C. Stop buffer was 2mol/L sulfuric acid. Washings way was lmin with four times.6. Balb/c mice were immunized with purified MATSA. The results of indirect ELISA indicated that valences of immunized mice were all high and the best were 11 mouse and 12mouse. Their sera valences were all reached 1:6400. Spleen cells of 11 mouse and 12 mouse were prepared and mixed with SP2/0 cells. One hybridoma cell was obtained after screen, clone culture and monoclone culture. This hybridoma cell grew stable and could secrete bulk monoclonal antibody of MATSA. It was named 1D5. Valences of monoclonal antibody of supernatant and ascites of hybridoma cell 1D5 were detected. That of supernatant was 1:800 and that of ascites was 1:106. Relative affinity of monoclonal antibody in supernatant of hybridoma cell 1D5 was detected by indirect ELISA. It was 1.3ng/100uL.This study was about culturing MSBl cell, then separating and purifying MATSA from MSBl cells. Antiserum and monoclonal antibody of MATSA were prepared successfully by immunized rabbits and Balb/c mice. Those results established fundament to correlated studies of MATSA and prevention and cure MD by anti-tumor targeted drugs using MATSA monoclonal antibody conjugated with "bullet" drugs which could anti-tumor. It also could explore new way to precaution and therapy neoplastic disease of mankind and animal.