| Cryptosporidium is an important zoonotic opportunistic pathogenic protozoa transmitted mostly through waterborne and foodborne, which can cause severe or chronic diarrhoea in immunocompromised persons and susceptible animals. So it may poss a significant public healthy risk. Immunologic detection methods has been widely applied owing to its advantanges of high specificity and strong sensitivity. Immunofluorescence assay(IFA), enzyme immunoassay (EIA),immunochromatograp-hic method (IC), enzyme-linked immunoelectro-transfer blot (EITB),immunomagne-tic beads Separation (IMS)and flow cytometry test (FCM) in the immunologic detec tion of Cryptosporidium was reviewed and the advantages and disadvantages of each method were briefly analyzed in this paper.The epidemiologic survey of Cryptosporidium infection in cows was used with both Sheather's floatation and Modified Ziehl-Neelsen staining technique on one dairy farm which was Zhengzhou suburban area. Results showed that infected with oocysts of Cryptosporidium andersoni wered positive samples. The Oocysts of C. andersoni were purified by discontinuous sucrose gradients, then treated by filtration and centrifugation, freeze-thawed and sonicated. Balb/c mice were immunized for six times with purified oocyst antigens of C. andersoni. BALB/C mice were immunized with purified oocyst antigens of C. andersoni which had been freezed and thawed for several times and treated by ultrasonic wave. Spleen cells of the mice were selected to fuse with SP2/0 cells.The positive wells were selected by indirect ELISA,and after three to five generations of cloning four hybridoma cell clones named 4F8-E4-E3,4F8-D10,4G3-F3 and 4G3-F7 were obtained.The characterization of McAbs were studied by ELISA,IFA and EITB. Results:The supernant titers ranged from 1:100 to 1:12800 and ascites titers between 1:6400 to 1:102400.The monoclonal ascites showed all against oocyst wall antigen,examined with IFA, The four McAbs could be recognized respectively by 38～97.2 KDa polypeptide antigen bands of C.andersoni oocysts.Four specific McAbs against C. andersoni hybridoma cell were obtained in this study, which provide the foundation for deep study on Cryptosporidium biological characteristic and establishment of diagnostic techniques.An indirect immunofluorescence assay for the antigen detection of Cryptosporidium oocysts was established using 4F8 monoclonal antibody against C. andersoni oocyst wall and fluorescein isothiocyanate conjugate of goat anti-mouse immunoglobulin G.The most suitable concentration of 4F8 monoclonal antibody was 1:800 and the best incubated time of 4F8 monoclonal antibody and fluorescein isothiocyanate conjugate of goat anti-mouse immunoglobulin G were 30min, respectively. Specificity testing of the IFA revealed no reactivity with Eimieria oocyst and Giardia lamblia cyst and only reactivity with Cryptosporidium oocysts and the detection limit was determined as 2×102 Cryptosporidium oocysts/g of fecal samples. The IFA procedure may be useful in the clinical diagnosis of human and animal cryptosporidiosis and also in the detection of oocysts in environmental samples.Immunogen was prepared using purified C.andersoni oocyst with same methods as above, rabbits had been injected four times with oil-adjuvant-vaccines to produce antibody against Candersoni. The mass concentration of IgG was 2.183mg/mL. The sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for detecting Cryptosporidium oocysts from faeces was successful established.Cryptosporidium oocysts were captured with rabbit anti-oocysts IgG and detected with monoclonal antibody. Following the addition of a horseradish peroxidase conjugate of goat anti mouse immunoglobulin. The assay was specific for Cryptosporidium.The results showed that the most suitable concentration of coating antibody was 5.5mg.L-1 and monoclonal antibody was 1:1600,1% BSA-PBST was used for Block, the best substrate coloration time was 15min, Assay sensitivity allowed detection of 2×104 C. andersoni oocysts per mL of feces. The methed of sandwich ELISA is specific, sensitive and suitable for routine diagnosis and large-scale epidemiological studies of Cryptosporidiosis by the specificity blocking test cross test and reproducibility test. |
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