| Poliovirus(PV)is the causative agent of polio.It mainly causes flaccid muscle paralysis by invading the central nervous system.Since the World Health Organization(WHO)'s 1988 decision to “eliminate polio globally by the year 2000”,polio cases have fallen by more than 99%,until now,with only very few cases of polio occurred in a few countries,the world is on the verge of eradicating polio.The poliovirus monoclonal antibody is of great significance for epidemiological surveillance of poliovirus,evaluation of vaccine immunization level and derivative case study.This study intends to use monoclonal antibody of poliovirus Sabin strain type I prepare test strip for antibody against type I poliovirus in human serum.Anti-poliovirus Sabin stain type ? monoclonal cell line 62-F5,which was prepared by our laboratory before,was cultivated and the titer of monoclonal antibody was determined by indirect ELISA to be 1:8,indicating that the cells were good condition for this study.The Balb/c female mice were sensitized with Incomplete Freund's adjuvant.One week later,the above hybridoma cells were inoculated intraperitoneally,and the mice was carefully observed after one week of inoculation.When the abdomen of the mouse is swollen,the ascite is taken from the living body;if the vigor of the mouse decreased and the temperature of the body lowered than usaual,the mice should be sacrificed in time to collect the ascites.After ascites was collected,the antibody was purified by caprylic acid-ammonium sulfate precipitation method,and the concentration of the monoclonal antibody was 15.364 mg/mL afterultrafiltration.Reduced SDS-PAGE and Non-reduced SDS-PAGE results showed that the purity of the purified monoclonal antibody was high,a heavy chain of about 70 KDa,and a light chain of about 23 KDa.According to the Non-reduced SDS-PAGE electrophoresis pattern,the molecular weight of the monoclonal antibody is about 800-900 KDa,and the monoclonal antibody type may be IgM.The titer of the concentrated monoclonal antibody was determined by indirect Elisa to be 1:51200.40 nm colloidal gold particles were prepared by trisodium citrate reduction method,and the monoclonal antibody was labeled with the colloidal gold,and then combined with the polio PVI antigen to form a colloidal gold-antibody-antigen complex,which was fixed on the gold standard pad.Adhere the NC membrane to the middle of the PVC plate,the mouse anti-human IgG with a concentration of 1mg/mL was coated at the T line with a concentration of 2 ?g/cm;goat anti-mouse IgG of 1mg/mL was coated at the C line with a concentration 2 ?g/ cm;Adhere the gold standard pad to one end of the NC film and overlap by about 2 mm.The sample pad is glued to the gold standard pad and overlap by about 2 mm.The absorbent pad is bonded to the other end of the NC film and overlaps by about 2 mm.Finally,the test strip was assembled in the plastic case.The result showed that the sensitivity of the colloidal gold test strip for detecting poliovirus antibody standard positive serum was 1:80;the specific result showed positive results for polio positive,positive for hepatitis A virus antibody positive serum,measles virus antibody-positive serum,hepatitis B virus antibody Serum and poliovirus antibody-negative serum showed negative results,indicating that the test strips were specific.The coincidence rate of the test strip with the PV-IgG ELISA was 86.7%.After storage at 4? and room temperature for 3 months,there was no significant change in sensitivity and specificity.This study preliminarily established the detection of poliovirus type I antibody colloidal gold test strips,which is of great significance for the epdemic study PVI and massive inverstigaton of the immune effects of Type ? PV vaccine. |
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