Comparative Research Of Development Of Monoclonal Antibodies Against Enrofloxacin With Rat Lymph Node Cells And Mouse Spleen Cells

Scientists had developed a lot of monoclonal antibodies after Kohler and Milsteind reported the method of producing monoclonal antibody in 1975 at the first time. They had been used widely, for example, they were been used in ELISA kit, colloidal gold, affinity column, immunohistochemistry and so on. But, most of the monoclonal antibodies are produced by hybridomas which were made by B lymphocytes and myeloma cells from mouse at the past. It is so called conventional methods. It has great advantages, for instance, their genetic stability is good, and their titer is high. They had some disadvantages, too. It could not produce monoclonal antibodies when mouse antigens were used as immunogens, at least 4 times of immunes are needed, and it need a lot of antigens. Scientists are looking for another effective method to producing monoclonal antibodies all the time.As the improving of the people's living standard, the demands of the animal derived foods are increasing, and the problem of the veterinary drug residues in them became a big concern. On one hand, these residues can do great harm to people's healthy, on the other hand, a lot of animal derived foods could not be exported to the developed countries because of the quantity of veterinary drug residues were higher than the Maximum Residue Limits. The problem is serious, and it has bad effect to increasing our export. Our country's economy development is also influenced.Enrofloxacin is an antibacterial agent. It is belong to the fluoquinolones (FQs) group with effective, widespread antibacterial role in the clinical cure. In recent years, the more use of enrofloxacin, the more adverse effect of its residues were reported. People in many country paid more attention to the bad effects of its residues than ever before.The high performance liquid chromatography is the standard method for determination of the residues of enrofloxacin in animal derived food at present in our china. It has limited use for its complicated sample pre-treatment and money-costing. Therefore, it is necessary to development a fast, effective kit not only for market monitor but also for elementary determination of large-scale of samples for import and export in our country.The complete antigens (ENRO-BSA) were made of enrofloxacin (ENRO) and bovine serum albumin (BSA) by EDC method according to the principle of immunology. BALB/c mouse were immune, and the monoclonal antibodies against enrofloxacin were obtained by conventional method. The Wister rat is also immune by the same antigens, and the monoclonal antibodies against enrofloxacin were obtained by the novel method reported by Japanese scientists. Two methods as well as the characteristics of two hybridomas and monoclonal antibodies were compared. The reseach confirm the feasibility of novel method for producing monoclonal antibodies reported by Japanese scientists, and provide parts of materials for developing a kit for determination of enrofloxacin residues in animal derived food effectively.The enrofloxacin was conjugated directly to the BSA and ovalbumin (OVA) by EDC method. The conjugates were analyzed by SDS-PAGE and UV absorbance method, the molecular conjugate ratio of enrofloxacin to BSA and OVA were 2:1 and 5:1 respectively. The ENRO-BSA was injected as immnogens to BALB/C mouse and Wister rat respectively. Their antibodies against immnogens in blood serum were determined by indirect ELISA; and their antibodies against enrofloxacin were further determined by competitive indirect ELISA. The mouse and rat with higher titer were used for cell fusion. The hybridomas were selected, screened, and cloned for 3 times after cell fusion. Two hybridomas were obtained by conventional method, and another 2 hybridomas were obtained by novel method. They were called M1B3, M4E6, R2B1 and R4D3. The 4 hybridomas were magnified and conserved in liquid nitrogen.The sterilized wax and M1B3 were injected into abdominal in mouse as the same as R2B1. They were magnified in their abdominal for 7 days, and a lot of ascites including monoclonal antibodies were obtained. They monoclonal antibodies produced by two different methods were collected, purified before they were identified by SDS-PAGE. It is obvious that a lot of monoclonal antibodies against enrofloxacin were obtained by these methods.The hybridomas that secrete monoclonal antibodies against enrofloxacin which were obtained by tratitional method and novel method were compared. It is found that there were 93 chromosomes in M1B3 and there were 96 chromosomes in R2B1 by comparing the quantities of two hybridomas, that is to say the cell fusion were done well. The two hybridomas have high genetic stability after they were cultured and conserved for 16 times. And the comparative researches about the monoclonal antibodies were also done. At the first, the titer of monoclonal antibodies were determined by indirect ELISA, goat anti-mouse IgG(H+L) and goat anti-rat IgG(H+L) were used as secondary antibodies respectively, it is obvious that the titer of R2B1 was higher than M1B3. At the second, the ascites and monoclonal antibodies were indentified by SDS-PAGE. The photography was scanned. A curve was determined by the molecular of standard protein marker. Other protein's weight in the ascites and monoclonal antibodies were calculated by comparing with the standard curve. The molecular weight of M1B3 is 151.242KDa, and the molecular weight of R2B1 is 156.157KDa. At the third, the subtype of both monoclonal antibodies were identified as IgG₁ using the reagent from Sigma and Binding site company. At the fourth, the actual contents of monoclonal antibodies in both purified ascites were determined by the method of standard curve with standard mouse IgG-F (ab')₂ from the Sigma company. There were 0.68mg/ml monoclonal antibodies in the purified ascites made from M1B3, and there were 1.133mg/ml monoclonal antibodies in the purified ascites made from R2B1. At the fifth, the proportions of monoclonal antibodies in the purified ascites were 54.028% and 65.309% according to the result of lamellar scanning. At the sixth, the quantities of protein in the purified ascites were 6.53mg/ml and 14.83mg/ml respectively after the determination by Beckman Du-640 from the USA. At the seventh, the affinity constants of both monoclonal antibodies in the purified ascties were 2.95×10⁷M^-1 and 3.69×10⁷ M^-1. Both of them were high affinity antibodies according to the immunology principle. At the eighth, according to the results of determination of specificity, the monoclonal antibodies can identify the antibiotic except the FQs. They were able to be used for determination of enrofloxacin residues in the animal derived food.The research reported producing rat-monoclonal antibodies using medial lymph node cells for cell fusion firstly in our country. It is also the first time to conformation the feasibility of novel method for producing rat-monoclonal antibodies by comparing it to the conventional method in the world. Our research would be able to be used in the development of ELISA kit for quick determination of enrofloxacin residues in animal derived food. It is also provide good materials for further application for quick producing rat-monoclonal antibodies.