| Schistosomiasis is a serious parasitic disease with public health importance。Diagnosis is the key link for schistosomiasis control. Parasitological and immunological tests are two main techniques used in diagnosis. Parasitological detection was the main method to diagnose the infection before 1970's, but immunodiagnosis is widely used nowadays due to the high efficiency and sensitivity, especially when the detection of circulating antigens opens the possibility to differentiate between acute and chronic infection. Immunological techniques used to detect antibody or antigen(s) include indirect haemagglutination assay (IHA) and enzyme-linked immunosorbent assay (ELISA). However, antibody levels in general show no correlation with the worm burden and the presence of specific antibodies doesn't indicate an active infection since antibodies are demonstrable in serum even a long time after successful chemotherapy. Detection of circulation antigens is preferable since it is a more direct measure of the worm burden, giving an indication of the activity and intensity of infection. Unfortunately, the sensitivity and specificity of current techniques to explore circulation antigens are unsatisfied, how to improve the sensitivity and specificity is the research focus.IgY antibody is the unique immunoglobulin in birds which is transferred in female from serum to egg yolk. Compared with mammalian IgG, birds IgY has attractive advantages in immunological diagnosis: (1) Distinct phylogenetic distance between birds and mammalian antigens facilitates the high immunoreaction; (2) High and long-lasting IgY titers in the yolk can be induced with low amount of mammalian antigens ; (3) IgY can be easily obtained by collecting eggs from immunized hens without invasion; (4) Large yield and scalable production. A chicken usually lays ~280 eggs in a year and an egg yolk contains 100–150 mg of IgY antibodies. This can result in 28-42 g of IgY per year from each chicken; (5) No binding to rheumatoid factor (RF). It can avoid false-positive reaction to use IgY in the test; (6) IgY doesn't react with mammalian complements, thereby, the assay interference could be reduced.Because of increasing sensitivity and low background in the immunoassays, IgY has been increasedly used in the diagnosis of many diseases, however, there is no report about IgY application in diagnosis of schistosomiasis. In this study, we firstly produced and identified anti-SEA IgY, and further developed a novel sandwich ELISA based on specific IgY to detect CAg in serum of schistosomiasis patients.Three 25-week old hens were intravenously immunized with 60μg SEA, and another 3 times injection of 30μg SEA was administered at 10 days interval. IgY was extracted with WD (water-dilution) method and PEG method from eggs laid by hens 35d after primarily immunization. The protein concentration of IgY was measured by BCA method, and analyzed by SDS-PAGE. WD method and PEG method were comparatively evaluated based on the yield and total protein content. Average 61mg IgY could be extracted from one egg with WD method, and average 57 mg IgY with PEG method. The analysis of IgY by non-reduced SDS-PAGE demonstrated that the IgY purified with WD method contained one major protein band with molecular weight of 130 000 while two protein bands with molecular weights of 66 000 and 35 000 indicated by reduced SDS-PAGE. IgY that extracted with PEG contained only one major protein band with molecular weights of 66 000.Specificity of IgY was analyzed by Western blotting. Sensitivity was detected by sandwich ELISA with IgY as captured antibody and IgG as detective antibody. The titer of IgY was measured by indirect ELISA. Western blotting showed IgY could recognize three bands with molecular weights of 140 000, 100 000 and 69 000 of SEA. These three protiens were considered as potential candidates for early diagnosis. IgY based-sandwich ELISA demonstrated the high sensitivity of purified IgY with trace amount as 2.4ng/ml SEA was detected. SEA specific IgY could be explored in immunized hens as early as 10 days after first immunization. On day 31 after primary immunization, the antibody titers reached to 1:1600 as a peak.To investigate the possibility of IgY in application of patients diagnosis, we developed a sandwich ELISA with anti-SEA IgY as captured antibody and IgG as detecting antibody. A 96-well plate was coated with anti-SEA IgY at different concentrations and incubated at 4℃for 12h. After washed three times with PBS-T, the plate was blocked with 1% BSA at 37℃for 1h.The plate was washed as described above, then 200-fold sera from the schistosomiasis patients were added and incubated at 37℃for 1h. After wash, anti-SEA IgG was added and incubated at 37℃for 1h.The plate was washed three times at the same method, and HRP-IgG (dilution 1:100 000) was added to wells and incubated at 37℃for 1h. The enzymatic reaction was terminated by incubation with 40μl of 2 M sulfuric acid at room temperature for 15 min. The optical density was measured by automatic ELISA reader, using 462 nm for the emission wavelength. The positive result of sandwich-ELISA was indicated with S/N value 2.7043 when the concentration of IgY was 2μg/ml.Our results demonstrated IgY based-sandwich ELISA could detect CAg of serum.It was firstly reported that anti-SEA IgY was produced with average amount of 61mg by 30μg SEA immunization for each hen. Anti-SEA IgY based indirect ELISA can explore SEA as low amount as 2.4ng/ml. The analysis of specificity showed IgY could recognize three bands of SEA which are considered as potential candidates for early diagnosis, and would be used for diagnosis of schistosomiasis. IgY based-sandwich ELISA could detect CAg from sera of schistosomiasis patients. It can be presumed that IgY has high potential in schistosomiasis diagnosis. |
PDF Full Text Request