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Objective To observe the growth situation of Blastocystis hominis in vitro and select the optimal method for culti?vation of B. hominis in different media. Methods Ten positive stools with B. hominis were inoculated in three different media for cultivating,namely 1640,Jone’s medium and vitro medium. And the stools with good growth status and high quantities of B. hominis were chosen to inoculate in the three media with equal amount after subcultivation,and the number of B. hominis was counted every 24 h for ten days,and the morphological changes and growth status were also observed. Results The densities of B. hominis in the 1640 and Jone’s medium were higher than that in the vitro medium 48 h after the inoculation. The same stool sample was inoculated to the three different media and observed for ten days,and the results indicated that the growth of B. homi?nis presented regular changes in the three media,the growth peaks were on the third,sixth and ninth day post inoculation;and the density of B. hominis was the highest in the Jone’s medium. The morphology of B. hominis was the clearest and most dynamic in the vitro medium,while various reproductive forms were observed in the Jone’s medium. Conclusion Jone’s medium is suitable for the growth of B. hominis and can be the first choice for the cultivation of B. hominis in vitro,and vitro medium is the best medium for observing the growth situation of B. hominis.
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Objective To analyze the fractional proteins and immunoreactivity of the soluble antigens from Babesia microti (B.microti),and find the candidate antigens for diagnosis with high sensitivity and specificity.Methods BALB/c mice were inoculated with B.microti-infected red blood cells by intraperitoneal injection.The B.microti were collected from the infected red blood cells when the infection rate reached its peak (infection rate ＞70％),then the soluble antigens were extracted by repeated freezing-thawing and ultrasonic method.The mice sera before and after the infection with B.microti for 7,14,21,28,35,42,49 and 56 days were also collected.The polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze protein components of the soluble antigens of B.microti and the Western blot was used to analyze the immunoreactivity of the soluble antigens with the pooled mice sera before and after the infection.The specific positive protein bands were identified by Liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS),and the amino acid sequences of the proteins were analyzed by bioinformatics tools.Results The results from SDS-PAGE analysis indicated that the soluble antigens of B.microti showed distinct protein bands with the range between 12 and 185 × 103 (kDa,relative molecular mass,Mr),among which 9 main bands and 12 minor bands were obtained.In the Western blot analysis,the protein bands with Mr at 40 and 45 kDa could be recognized by pooled mice sera 7 days after infection;the protein bands with Mr at 40,45,54 and 95 kDa could be recognized by pooled mice sera 14 days after infection;the protein bands with Mr at 27,40,45,54,95 and 110 kDa could be recognized by pooled mice sera 21 days after infection.While,the protein bands with Mr at 27,40,45,54,95,1 10 kDa and other weak-reactive bands were recognized by pooled mice sera 28-56 days after infection,and the reaction became stronger with the infection continued.There were 336 proteins,including surface antigen,heat shock protein 70,seroreactive antigen,Eta subunit of chaperonin containing t-complex polypeptide 1 and unnamed protein products,were identified as the components of soluble antigens after mass spectrometry and sequence analysis.Conclusion The 40,45 and 54 kDa protein components from the soluble antigens of B.microti may be ideal candidate antigens for diagnosis,andtheir potential applications in diagnosing of human babesiosis deserve further study.
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Objective To establish A1E3 and B1C4 monoclonal antibody-based ELISA for detecting circulating antigen of Schistosoma japonicum and explore its application value in the field. Methods The characteristics of A1E3 and B1C4 monoclonal antibodies were analyzed by SDS-PAGE and Western blotting. The SEA-based ELISA was used to evaluate the titers of A1E3 and B1C4. The orthogonal test was used to determine the best concentration of coating antibody B1C4 and optimal working concentra-tion of A1E3-HRP. Under the optimal conditions,the serum samples of 20 acute schistosomiasis cases,46 chronic schistosomiasis cases,and 20 control sera were tested to evaluate its detection sensitivity and specificity. Seventy-two antibody positive serum sam-ples from Jiangling County of Hubei Province were detected and compared to a commercially available ELISA kit,to evaluate the detection effects of this method. Results The results of SDS-PAGE demonstrated that the purified A1E3 and B1C4 contained a clear heavy chain with molecular weight of 88 000 and 52 000 respectively and had the same light chain with molecular weight of 20 000;while Western blotting demonstrated that A1E3 and B1C4 could be recognized by SEA and serum samples of acute schis-tosomiasis cases. The SEA-based ELISA demonstrated the titers of B1C4 and A1E3 were 1∶105 and 1∶30 000,respectively. The serum samples from all the acute cases and 86.9% of the chronic cases showed a positive reaction. All of the control sera from healthy persons gave a negative response. The positive rates of the double monoclonal antibody ELISA and commercial ELISA for detecting the circulating antigen were 45.8%and 43.1%respectively,and there was no significant difference between the results of the two methods. Conclusion A1E3 and B1C4 monoclonal antibody-based ELISA is established successfully. It exhibits a high sensitivity and specificity in detecting circulating antigen of Schistosoma japonicum.