Preparation Of Monoclonal Antibody Against Cryptosporidium Oocysts Wall

Cryptosporidiosis has been a globally recognized zoonotic parasitic disease that is caused by Cryptosporidium.Parasite transmission occurs through ingestion of oocysts,which is a form of Cryptosporidium in the environment,through either direct contact or consumption of contaminated water or food,and there is no effective treatments or vaccines available.At present,the immunological method EPA 1623 is the international standard for detection of Cryptosporidium.The key point of this method is the need for a sensitive and specific monoclonal antibody against the oocyst wall,and no relevant reagent has been developed in China.Therefore,the purpose of this study is to prepare antibodies against Cryptosporidium parvum oocyst wall proteins and and fill the domestic gaps.In this study,two immune strategies were used to prepare monoclonal antibodies.The first was to select four proteins(Gp15,COWP6,POWP6,and MPA2)that may be located in the oocyst wall by selecting the reported and biological analysis.By designing peptides with highly specific amino acid sequences,they were conjugated to carrier proteins KLH and BSA for serum titer detection in immunized mice and ELISA,respectively.The other is to use the traditional method of oocysts for immunization.We used 1×10~6 Cryptosporidium parvum oocysts to be inactivated in a80°C water bath for 30 min,and then emulsified with Freund’s adjuvant to immunize BALB/c mice to prepare monoclonal antibodies.A total of four immunizations,each interval of 14 days.Indirect immunofluorescence was used to screen monoclonal antibodies localized to the oocyst wall.Through bioinformatics analysis,we designed 5 peptides from these 4 proteins,which are c-GGKRIEVAVP(Gp15)、c-FYTGANSGTTNG(Gp15)、CESKIHEKHHGKNTRT(COWP6)、c-DENNKKNDNKKSNQN(POWP6)、和c-EEQPDTSKDEENIAN(MPA2)respectively.After obtaining polyclonal antibodies,IFA test showed that all four protein polyclonal antibodies recognized proteins in the oocyst wall.Through traditional immunization,we prepared two McAb 2-C11 and1-B11 located on the oocyst wall.2-C11 was located on the anterior part of sporozoite and oocyst wall,while 1-B11 was localized on both sides of the sporozoite nucleus and the oocyst wall,and its antibody subtype was IgM.In the sensitivity experiment,we found that the IFA titer of the ascites of the two antibodies could be diluted to more than 1:10,000.The results of specific experiments showed that the antibodies did not cross-react with Eimeria oocysts and E.coli.Western blot results showed that2-C11 antibody recognized a protein larger than 180 KDa,while 1-B11 recognized a protein of approximately 180 KDa.In summary,antibodies against five polypeptides,Gp15,COWP6,POWP6,and MPA2,all recognize the oocyst wall and successfully prepared 2 monoclonal antibodies with good sensitivity and specificity by traditional means.These antibodies lay the foundation for the development of cryptosporidium detection reagents with proprietary intellectual property rights.