| Objective: Ancylostomiasis is the common intestinal parasitic diseases in developing countries all over the world. Since repeated deworming leads to a potential risk of drug resistance as well as the drug toxicity and side-effect limits the application of deworming drugs in pregnant woman and infant, the treament depending on anthelmintic can not satisfy the demands on preventing and curing global ancylostomiasis..The safe and effective substitute drug or functional food needs to be developed. In this study, we prepared the adult worm antigen(AWA) using the ancylostomes obtained from the patients'feces who had taken anthelmintic to expel the parasite. Then the ancylostome specific yolk immunoglobulin (IgY) was prepared and identified from the 25-week old HY-line hens immunized by AWA. We monitored the dynamic a ntibody titer of IgY,and purified the antibody at higher titer. The IgY was culture with the third-stage infective larvae(L3) in vitro and in vivo to investigate the effect of IgY upon ancylostome and to lay the foundation for the production of anti-hookworm drugs or functional foods.Methods: Twenty-three patients from the first affiliated hospital of AnHui medical university were screened by brine flotation and sedimentation method. The dejecta from 4 patients with positive kopr-test was collected for consecutive five days after treated with anthelmintic, and the ancylostomes were sieved to produce AWA. The 25 week-old egg-laying HY-line hens were immunized with 200μg AWA by subcutaneous injection and intramuscular injection.On the day 28th and the day 38th after initial immunization, the hens were immunized repeatedly.The eggs layed before and after immunization were collected to make anti-AWA IgY antibody(Storage Temperature is 4℃).IgY was extracted from the eggs by WD (water-dilution) method and purified by dialysis and salting out. The IgY was analyzed by SDS-PAGE and 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 L3 were 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 SDS-PAGE was 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 IgY was purified heavy by EGG stractTMIgY Purification System and the concentration ranged from 5.9 mg/ml to 10.1mg/ml. A floc or granular attachment formed on the surface of L3 after culture in vitro. The activity of L3 had been restricted by attachments. Statistical analysis showed that the IgY with the concentration of 50% or 70% could decrease the livability of L3. It was found that the IgY antibodies could increase chemotaxis and adhesion of neutrophil through the in vivo experiment.TEM observation showed that L3 exhibited a damage of its surface including swelling, roughness, uneven electron density, and the transverse striations of L3 squeezed each other.A same antigen component of adult worm existed on the superficial membrane of L3 disclosed by indirect immunofluorescence.The purified IgY could react with antigen on the cuticular surface of L3.Conclusion: We have successfully prepared the IgY antibody specific to ancylostome with good security and stability. The yield of IgY is generous and easy to store.The IgY can react with antigen on cuticular surface of L3 and increase chemotaxis and adhesion effect of neutrophil. The IgY bined to L3 may damage its superficial membrane and the antibody with high concentration can decrease the livability of L3.In short, the specific IgY is harmful to larvae, and can be used for immunotherapy against ancylostomiasis. |
