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|Title:||Development of monoclonal antibodies against chicken IgM and its application in immunity studies|
Raj, G Dhinakar
|Keywords:||Chicken IgM;ELISA;immunoglobulins;monoclonal antibodies|
|IPC Code:||Int. Cl.⁸ C07K16/08|
|Abstract:||Immunoglobulin M (IgM) from specific pathogen free (SPF) chicken serum was purified and used for production of monoclonal antibodies (MAbs) for subsequent application in the detection of primary immune response to infectious agents. Chicken IgM was fractionated by gel filtration technique using Sephadex G-200. Purity of the fractionated IgM was confirmed by agar gel precipitation test (AGPT) and immunoelectrophoresis (IE) using rabbit anti-chicken serum. In polyacrylamide gel electrophoresis (PAGE), under non-reducing conditions, a single band was noticed and under reduced conditions, a band with molecular weight of 74 kDa was observed. Cell fusion was carried out with spleenocytes from immunized BALB/c mice and Sp2/0 cells, and the high positive clones were selected and characterized for isotype and cross-reactivity with IgY in enzyme linked immunosorbent assay (ELISA). Three MAbs chosen for characterization were of IgG1 isotype and they did not cross-react with standard IgY in ELISA. Concentrated IgM MAbs developed in this study were used in ELISA for the measurement of immune response in sequentially collected serum samples of birds, experimentally infected with egg drop syndrome (EDS-76) virus. The purified virus was coated onto the ELISA plates, followed by the addition of sera samples to be tested, MAbs against chicken IgM, anti-mouse peroxidase conjugate and substrate. The assay revealed an increase in IgM response in individual birds from 5-12 d with a peak on 9th d post-inoculation (PI), followed by an increase in IgG on 12-25 d PI. IgM antibodies against infectious bursal disease (IBD) virus were tested in 34 field serum samples. Comparison with AGPT revealed a marginal increase in sensitivity with IgM ELISA for detection of IBD virus specific IgM.|
|Appears in Collections:|| IJBT Vol.06(2) [April 2007]|
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