| Ancylostomiasis is still prevalent in the rural areas of China, and results in a serious public health problem causing great harm to people. Effective diagnosis methods are crucial for the control of the disease. Nowadays, the most widely used diagnostic method for Ancylostomiasis is fecal examination. With the advance of prevention and control of the disease and the decrease of the infection rate, this method cannot meet the needs of diagnosis, so it is urgent to develop quick and easy immunological methods for large-scale screening and diagnosis. Therefore, the availability of diagnostic antigens is crucial.In this study four soluble rough antigens (named as A, B, C and D antigen) were prepared, from adult Necator Americanus^ using different methods and were evaluated for their diagnostic efficiency, then one antigen with the highest efficiency was selected for analysing the reactivity of hookworm patients serum antibody to the antigen for determining the prevailing antibody classes or subclasses in sera of hookworm patients. The cDNA entrance and expression library were constructed by Gateway technology, immunoscreening the expression library using the mixed serum of Ancylostomiasis patients and analyzing the selected genes by bioinformatics methods. The positive clones were expressed, purified and finally evaluated by ELISA to determine the diagnostic value to Ancylostomiasis patients’sera.Different antigens were compared by detecting antibody of hookworm patients, other parasitic patients and the healthy serum, the sensitivity of four antigens was 87.75%,86.73%, 87.75%, and 93.87%; the specificity was 76.12%,74.63%,73.13%and 80.60%; the diagnostic efficiency was 83.03%,81.82%,81.82%and 88.48%respectively.Different antibody classes and subclasses were used as secondary antibody to test different serums, the sensitivity of IgM, IgD and IgE, IgG, IgGl, IgG2, IgG3, and IgG4, was 41.84%, 2.04%,1.02%,92.93%,19.39%,25.51%,17.35%and 88.78%; the specificity was 77.61% 97.01%,92.54%,79.10%,95.52%,92.53%,92.53%and 92.53%; the diagnostic efficiency was 56.36%,40.61%,38.18%,87.88%,50.30%,52.7%,47.88%and 90.30%respectively. Results suggest that as a secondary antibody for ELISA, IgG4 antibody subclasses are superior than the other subclasses for detecting Americanus ancylostomiasis.Necator Americanus cDNA entrance library was constructed in this study with an average titer of 3.0×105cfu/ml and a total clones of 3.0×105cfu, also the cDNA expression library with an average titer of 1.9×105cfu/ml and the total clones of 1.9×105cfu. The average length of insert fragment is 1.01kb, and the insert efficiency is 85%. The cDNA expression library was screened with mixed serum of hookworm patients and 2 strong positive clones was selected, Sequence analysis showed that the S1 clone contains an open reading frame of 678bp, encoding a protein that is highly homologous with Necator Americanus beta tubulin protein (82%), and S2 contains 861bp open reading frame encoding protein highly homologous with Necator Americanus hypothetical protein (100%).The structure of S1 and S2 encoding proteins were calculated, result proves that both of them has good antigenicity. The proteins were recombined and purified then, the result of detecting the diagnostic efficiency showed that the sensitivity of the two recombinant protein were 88.42%, and 91.58%; the specificity was 77.27% and 95.45%; diagnostic efficiency was 83.85% and 93.17% respectively. S2 protein shows the diagnostic potential, further study is still required on whether the recombinant proteins can be used for the detection of human Ancylostomiasis. |
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