Establishment Of A Diagnostic Method For Giardia Colloidal Gold Test Strip Based On Monoclonal Antibody Technology And Its Preliminary Application

Giardia lamblia is a common zoonotic parasitic protozoa which parasitize in the small intestines of humans and other mammals.G.lamblia can lead to serious abdominal cramps,acute or chronic diarrhea and malabsorption.Giardiasis has been listed as one of ten major parasitic diseases threatening human health in world,which has got our attention for the seriousness and perniciousness.Nowdays,the traditional parasitological method of detecting the cysts and trophozoites with microscope is the "gold standard" for diagnosis on the disease.However,it is labor-intensive and time-consuming along with low sensitivity.In the past few years,the immunodiagnostic methods have opened a new route for detection of giardiasis,showing a good prospect of application and development for the higher sensitivity,specificity,easy to operate in the field.With the application of McAb technique,immunodiagnostic method has increased the specificity for the detection of giardiasis.The aim of this study is to prepare the the monoclonal antibodies against G.lamblia antigens and to establish an immunodiagnostic method for detection of Giardiasis.1.Preparation of monoclonal antibodies targeting at Giardia lambliaBALB/c mice were immunized with antigens extracted from G.lamblia trophozoites,McAbs were prepared from anti-Giardia antigens hybridoma cells based on cell fonjugation technology,McAbs isotyping was performed with the immunoglobulin subtype ELISA kit.The McAb cross reactions were examined by enzyme-linked immunosorbent assay(ELISA)with other parasitic species.12 McAb hybridoma cell lines were obtained successfully and the subtypes were all IgG1.The McAb EB2 specifically recognized two bands of antigens extracted from G.lamblia trophozoites with Mr of 175 kDa and 191 kDa by Western blot.It was found that the EB2 had no cross reaction with other species such as the soluble extract from Escherichia coli,Ascari ssuum,Blastocystis hominis,soluble egg antigen and soluble adult worm antigen of Schistosomia japonicam,and Paragonimus westermani.2.Establishment of double-antibody sandwich ELISA method for detecting Giardia fecal antigenWe performed the preparation of monoclonal antibody and polyclonal antibody to establish double-antibody sandwich ELISA method for detecting Giardia fecal antigen.The ELISA thus established was a fast,specific and sensitive detection method with 1:10 of sensitivity for detecting fecal antigen and less than 5%of within-coefficient of variation,which had no cross reaction with other species and would make a foundation for field application.3.Preparation of colloidal gold strip for detecting Giardia fecal antigenThe colloidal gold strips were prepared and they exhibited higher sensitivety,specificity and stability with 1:5 of sensitivity for detecting fecal antigen and 0.25μg of minimum detectable quantity of soluble trophozoites extract,which had no cross reaction with other species.The accuracy of colloidal gold strip was the same as the strip saling in the market,and lower than ELISA kit saling in the market by detecting 10 fecal samples coming from clinical patients with diarrhea.Conclusion1.We have cultured the trophozoites in vitro successfully and obtained the soluble trophozoites extract and excretory-secretory products.2.Rabbit Polyclonal antibody was obtained by immunizing rabbit with antigens extracted from G.lamblia trophozoites.3.BALB/c mice were immunized with antigens extracted from G.lamblia trophozoites,based on which 12 McAb hybridoma cell lines were obtained successfully using traditional hybridoma technology.The McAb EB2 specifically recognized two bands of antigens extracted from G.lamblia trophozoites with Mr of 175 kDa and 191 kDa by Western blot.It was found that the McAb EB2 had no cross reaction with other species such as the soluble extract from E.coli,A.ssuum,B.hominis,soluble egg antigen and soluble adult worm antigen of S.japonicam,and P.westermani.4.We performed the preparation of monoclonal antibody and polyclonal antibody to establish double-antibody sandwich ELISA method for detecting Giardia fecal antigen.5.We performed the preparation of monoclonal antibody and polyclonal antibody to establish colloidal gold strip method with higher sensitivety,specificity and stability for detecting Giardia fecal antigen.