| Clostridium perfringens(Cl. perfringens) that was widespread in the natural world can cause enterotoxaemia of calf even death under certain condition. There is no colloidal gold method in detect the disease caused by Cl. perfringens at the present, so the aim of this research is to establish the immunochromatographic strip method which has high specificity, sensitivity and stability for detection of bovine Cl. perfringens type D.Somatic antigen was isolated from bovine Cl. perfringens type D standard strain C60-2 cultured in vitro, inactivated by formalin and immunized rabbits with Freund's adjuvant to obtain polyclonal antibody. The titer of polyclonal antibody was 1:128 tested by agar gel precipitation, and the polyclonal antibody was purified by caprylic acid-ammonium method coupled with HiTrap Protein G HP affinity chromatography, There were two bands(50 KD heavy chain and 24 KD light chain) in purified antibody analyzed by SDS-PAGE. The polyclonal antibody was labeled with 25 nm colloidal gold particle prepared by citric acid three sodium revert as a detection reagent, and rabbit anti-bovine Cl. perfringens type D antibody was blotted on the test line while a goat anti-rabbit antibody was used on the control line of the nitrocellulose membrane to assemble the strip, and the strip detection effect was evaluated.The results showed that The optimum pH value and combining constration for antibody and colloidal gold particle combining were 8.6 and 30μg/mL. The strip was easy operation, specific for detecting bovine Cl. perfringens type D within 15 min, and no cross reaction with other Serotypes of Cl. perfringens standard strains and other bacteria. Storage of the strips for six months at 4℃had not changed their sensitivity and specificity. In conclusion, Our research revealed that the immunochromatographic strip for detection of bovine Clostridium perfringens type D has high specificity, sensitivity and stability. This, together with the advantages of rapid detection and easy operation without requirement for special skills and equipments, makes it suitable for the on-site detection and differentiation of bovine Cl. perfringens type D from other bacteria in bovine. |
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