Anti-human E. Coli Immune Egg Yolk Antibodies

Immunoglobulin Y (IgY) is the major antibody found in egg yolk from chicken (Gallus domestics). It is easy to produce a specific IgY at a large-scale since hyperimmunized chicken eggs can be obtained from an industrial chicken farm. It has been shown that IgY could be used in diagnosis and research.To investigate whether or not IgY can be used as a function food preparation for the prevention and treatment of infection in gastrk intestine, anti E. coli IgY was isolated from the egg yolk of chickens immunized with inactivated pathogenic E. coli. The E. coli strain ATCC 23985, a human pathogenic bacterium, was selected The cultures of the bacterium was inactivated and emulsified with adjuvant as the vaccine to immunize hens, and the LPS antigen extracted from ATCC 23985 by heat phenol method was employed to detect the specific antibody in the egg yolk from immunized hens.To detect the titer of the specific IgY against the LPS antigen, the ELISA was established, and the work condition was optimized. The concentration of the antigen coating the microtitre plates was 10 jig/ml. The second antibody, goat anti rabbit IgG, labeled with HRP was diluted to 1:2000. The ELISA titer of the specific IgY against the LPS antigen in the egg yolk was monitored up to 1:10240. ELISA data also revealed that the LPS antigen extracted from Samonella from human and enterohaemorrhagenic E. coli (EHEC) from pigs did not react to the specific IgY against LPS from ATCC 23985 E coli. The result showed that ELISA displayed high specificity.The IgY was extracted by ethanol precipitation and purified by DEAE-Sepharose fast flow followed by Sephacryl S-200 chromatography. The molecular weight of the specific IgY against the LPS antigen was determined as 186.4 kDa by SDS-PAGE and 186.5 kDa gel filtration, respectively. The pI was 5.5 by titration with acid.The stability of the purified specific IgY to heat, pH and proteases was studied. The result showed that the IgY stable at pH 4-8, and below 70℃. The IgY was very resistant to the digestion with trypsin and chemotrypsin, but it was sensitive to pepsin. The titer of the IgY did not decrease stored at 4℃ and room temperature for 12 months.The yolk power containing IgY of high titer prepared by spray drying was orally administrated to dogs of 1-2 month-old. The titer of the IgY in the gastric and the intestine of the animals was detected by ELISA 30 min, 60 min, 90 min, and 120 min post administration. Data revealed that most of the IgY could pass through the gastric and the titer of the specific IgY did not significantly decrease. However, the ELISA titer of the specific IgY dramatically decreased more than 2h postadministration.The addition of lactose, cyclic dextrin, and dextrin to egg yolk powder could improve the stability of the IgY to temperature over 70, pH below 3, and pepsin digestion.The effect of the antigen dose, adjuvant and administration on the antibody titer in immunized egg yolk was detected with plate agglutination test and ELISA. Data showed that the titer of the IgY against LPS antigen extracted from E. coli was not affected with increasing the amount of antigen, but decreased when the concentration of the E. coli lower than l*109 CFU/ ml. The data also revealed that Freund's complete adjuvant (FCD), Freund's incomplete adjuvant (FID) and mineral oil could improve the hen's immune response to LPS antigen of E. coli, and that hens immunized with emulsified antigen with FCD displayed the highest titre of the antibodies. Different administrative ways such as intra-muscular and subcutaneous immunization at wings, legs, necks and chests did not dramatically influence the titre of IgY against the LPS antigen of the E. coli. It was found that the immunization procedure one injection every other week and four consecutive times gave the best response the LPS antigen.Data from the radiation of egg yolk solution with 60Co showed that y-ray could kill the microbial present in the yolk, not affecting the activity of the specific IgY.The finding from this study suggests that the specific IgY...