Studies On The Early Diagnostic Antigens Of Schistosomula Of Schistosoma Japonicum

Objective To screen the early diagnostic antigen of Schistosomajaponicum(S.J) by SDS poly-acrylamide gel electrophoresis (SDS-PAGE) and enzyme immuno-transfer blotting (EITB). Meanwhile, to screen the simulating antigen epitopes related with early diagnositic antigens of S.j by 12-mers random peptide library , and then to evaluate their early diagnostic value by detecting mice infected with S.j and to identify protective immunity against S.j infection in mice.Methods Schistosomulum and Adult antigens were prepared and separated by SDS-PAGE, then transferred to nitrocellulose membrane. Western blot was performed with infected rabbit sera at 10th, 21th, 42th day and healthy rabbit sera. The positive bands probed with different sera were compared. The specific antigen recognized by early infected sera was screened. After performing three rounds of panning by using the affinity selection with infected rabbit sera at 21th day, target specific phages were effectively enriched from a random 12-peptides phage library. 11 positive clones selected randomly from them were compared and their antigenic ability was identified by ELISA. By using ELISA, phage clones with high affinity to infected rabbit sera were obtained. The diagnositic effect of positive phage clones for the early stage of infected mice was compared with SEA. Simultaneously, mice were immunized by subcutaneously injecting 1×l0~15 pfu positive phages at 0-2-4th week. After 4 weeks of the last immunity, each mouse was challenged by 40 + 1 S.j cercaria. All mice were killed after 42 days post-infection and the reduction rates of adult worms and the liver eggs were observed.Results SDS-PAGE showed that the protein bands of soluble schistosomulum antigen (SSA) were more than soluble adult worm antigen (SAWA), maily within the area of more 97kDa. The other protein patterns of SSA and SAWA were similar which mean stage common antigens. By EITB, SSA andSAWA were screened and analyzed with infected rabbit sera at 10th, 21th, 42th day and normal rabbit sera respectively. Result showed that rabbit sera of before infection did not react with SSA and SAWA. SSA showed 12 reactive bands which were more stronger reaction with early infected sera than SAWA. 97 kDa and 47kDa of SSA were recognized only by infected sera at 10th day, not by infected sera at 21th and 42th day. All other molecules of SSA, including 140 kDa, 107 kDa, 87 kDa, 74 kDa, 67 kDa, 62 kDa, 44 kDa, 32 kDa, 26 kDa, 16 kDa, were recognized by infected sera at 10th, 21th, 42th day, with a stronger reaction after a longer time post-infection, except a weak reaction of 32 kDa and 16 kDa to infected sera at 21th day. SAWA only showed 7 reactive bands, without positive reaction at the positions of 140 kDa, 107 kDa, 97 kDa, 47 kDa, 62 kDa molecules, and without any stage specific antigen molecule recognized merely by infected sera at 10th day. 87 kDa, 74 kDa, 67 kDa of SAWA appeared stronger reaction only with infected sera at 42th day. 11 positive monoclonal phages were obtained. The mixture of 3 positive phage clones were applied to diagnose infected mice of 10 days, 21 days and 42 days post-infetion. Their sensitivity were respectively 48.8 % , 64.4%, 68.9%, without any difference of sensitivity between the mixture of 3 positive phage clones and SEA for the detection of infected mice sera at 10th and 21th day. SEA was superior to positive phage clones in detecting infected mice sera at 42th day. 45 healthy mice were all negative results by the detection of positive phage simulating epitopes, but 11 false positive results by the diagnose of SEA. The reduction rate of adult worms and liver eggs induced by the mixture of positive phage clones and the original peptide library were respectively 29.3 %and 29.7%, 14.1 % and 9.81 %.Conclusion SDS-PAGE and EITB showed that there were many stage common antigen molecules between SSA and SAWA. 97 kDa and 47 kDa of SSA were the potential early diagnostic molecules. The positive phage simulating epitopes of shistosomula japonicum from a random 12-peptides phage library has acertain diagnostic value for the early stage of S.j infection, and can stimulated a protective immunity against S.j infection in mice.