Study On The Purification Of Specific Immunoglubolin Y By Affinity Chromatography With O Specific Chain

Compared with mammalian IgG, immunoglobulin Y (IgY)has advantages in source, avidity, quantity, isolation, stability and productivity. It also has less non-specific binding with complements, rheumatoid factor, IgM or Fc Receptor and Protein A or G. But the amount of specific IgY in total IgY is very low. Therefore, we established a kind of immunoglobulin chromatography method with LPS as the ligand to purify specific IgY, which was produced by chicken after immunization.The chickens were immunized with O157 inactivated vaccines to produce IgY. Immunization proceeded three times by injection on five sites. After immunization, the titer of IgY could reach a high level from 7th to 11th week. The titer showed downtrend from 11th week. Using indirect ELISA to detect the titer of IgY, it was 1:128000. The egg yolk from 7th to 11th week was collected and processed by water solution followed by ammonium sulfate fractionation for total IgY isolation. We used Lab-Image software to analysis the SDS-PAGE, the result showed that the purity of IgY was 48.3%.O chain is one of the components of LPS on bacterial cell walls. It is the basis of bacterial immunology classification. It diversities in different generas, even in different strains. According to these, we purified specific IgY by using LPS as ligand. First, we optimized the extraction method of LPS. And the procedures were bacterial disintegration by ultraphonic, followed by hot phenol water method to isolate LPS, then purification by ultracentrifugation. The results showed that the purity of LPS was almost 99% and the yield was 0.3%.We coupled LPS to the epoxy activated sepharose FF. The efficiency of the column was measured after packing. According to the activation density and references,7 g sepharose FF coupled with 10 mg LPS in NaOH-KCl solution for 24 h. The coupling efficiency for the ligand was 0.66 mg LPS/mL supporter. The capacity was 0.75 mg/mL medium. We chose Tris-HCl(0.1 M, pH 8.9) solution to elute the bound specific IgY. Then the bioactivity of the sample was detected by indirect ELISA and competitive ELISA. The results showed that the activity of the purified specific IgY could be increased at least 3 times. And the purity could rise to 50 folds.