抗钩虫特异性卵黄抗体的制备与鉴定及其抗钩蚴作用分析-preparation and identification of yolk antibody against hookworm and analysis of its anti - hookworm effect.docx

hens were immunized repeatedly.The eggs layed before and after immunization were collected to make anti-AWA IgY antibody(Storage Temperature is 4℃).IgY wasextracted from the eggs by WD (water-dilution) method and purified by dialysis and salting out. The IgY was analyzed by SDSand Western-blotting. Indirect ELISA was used to determine the titer of the antiserum. Large amount IgY was purified while the antibody had a high titer. The patients with severe ancylostomiasis infection in Mengcheng County, Anhui Province diagnosed by Kato-Katz were included in the following experiment. Their dejecta were cultured by solid medium-filter paper cultivation of ancylostoma caninum larvae and tube-filter paper cultivation and the L3were isolated. The L3 was cultured with IgY at the concentration of 30％, 50% and 70%respectively in vitro or in the abdominal cavity of mice.The larvae cultured in vitro were observed by inverted microscope and their survival rate was calculated. The larvae obtained from the mice were observed by transmission electron microscopy(TEM) and graded according to the neutrophil adhesion by inverted microscope. The distribution of ancylostome antigen on L3 was observated by indirect immunofluorescence staining using anti-IgY and FITC conjugated chicken IgY antibody.Result: The concentration of AWA was 1.21mg/ml.The concentration of IgY obtained by Lowry method was 1.8mg/ml. The molecular size of IgY detected by SDSwas equal to its predictive value. Western blotting results showed that the AWA could be recognized by IgY collected from immunizsed hens while not by IgY collected before immunization. Using indirect ELISA, the facts were observed that the IgY antibody began to produce by hens on the tenth day after the initial immunization, and the antibody titer increased gradually over time while reached the peak with a titer overtoped 1:10000 on the 55th day after immunization. There is a discrepancy on IgY antibody titer between different hens. The Ig