Preparation And Identification Of Polyclonal And Monoclonal Antibodies To Brucella

Brucellae are Gram-negative, facultative intracellular bacteria that can infect many species of animals and man.It can cause brucellosis of man and animals. In domestic ruminants, the disease is manifested mostly as abortions and infertility. Humans usually acquire brucellosis from domestic animals and are not themselves a source of contagion. Brucellosis is an extremely important disease around the world. .It was popular in the past and caused serious economic loss.Now this disease has been effectively controlled in some developed countries as America, France, and so on.But for lots of developing countries, the disease is also common. In China, Brucellosis had ever been controlled effectively, but since 1990s, especially recent years, numbers of man and animals infected the brucella are increasing sharply. All of this shows that it is important to study the brucella and exploit good methods to diagnose Brucellosis.Preparation of anti-Brucella polyclonal antibody.Two New Zealand rabbits were vaccinated with Brucella abortus vaccine, live(A19) every two weeks and the antibody titer of rabbit serum was detected through agglutination test after the second weeks of second booster.The results showed that the titer was 1:5120,which have been up to the titer that we needed. Using ammonium sulfate precipitation and Sephadex-G25 chromatography, IgG was extracted from rabbit serum.According to the SDS-PAGE analysis, it was showed that the rabbit-anti-Brucella IgG was successfully produced.The entire bacteria were used as antigen to immunize eight-week-old BALB/c mice in different immunized dosage and methods with interval of three weeks.Three days after booster, SP2/0 myeloma cells and spleen cells of the immunized mouse were fused by PEG-4000.Using brucella cell wall antigen as the screening antigen, hybridomas were screened by Enzyme-linked Immunosorbent Assays (ELISA).Those hybridomas with strong positive supernatants were subcloned by limited dilution methods 3-4 times until the positive rate come up to 100%. Finally, we obtained two McAb-secreting cell lines designated as 3E7 and 1C6. Preliminary characterizations of the two hybridoma cell lines were identificated. Indirect enzyme-linked immunosorbent assays(ELISA)were used to screen those hybridomas producing antibodies.The ELISA titers of the ascites were 104to 105 while the ELISA titers of the 3E7 supernatants is 1:1600 and 1C6 is 1:800.Through antigen specific epitopes analysis of two McAbs, the results show that they are against different epitopes of brucella. The OD450 values of the two McAbs in 4 times cloning culture have the trend of rise and the ELISA titers of the ascites were 1:104 to 1:105, indicating that the two hybridoma cell lines are stable.The results of the SDS-PAGE showed molecular weight of heavy chains of the two McAbs is 50kD roughly and the light chain is 25kD approximately. According to the western-blot analysis of this two McAbs and brucella cell wall antigen, the results illustrated that both two McAbs can bind with brucella cell wall antigen and the band locate at the position of 31kD and 55kD approximately. All of this demostrated that this two McAbs may be generated by the brucella antigens which its molecular weight are 31kD and 55kD approximately.