Preparation Of Polyclonal Antibodys Against Plasma-coagulase From Staphylococcus Aureus And Establishment Of ELISA Methods

Staphylococcus aureus (S. aureus), belongs to staphylococcus in microbial taxonomy, is a representative of gram positive bacteria and a kind of important human pathogenic microorganism, which can cause lots of serious infections. Therefore, the inspection of S. aureus is an important test item in medicine, health care products, cosmetic and food.The common test methods of S. aureus are national standard, fully automatic biochemical method and real-time fluorescence quantificative PCR (RT-qPCR). Fully automatic biochemical method and RT-qPCR are fast, accurate and can realize high-throughput screen. The high price of instruments and consumable materials is limited for basic level inspection organizations. National standard method is the most widely used due to low cost. Rabbit plasma is uesed to make plasma coagulase test and confirm the result, and the stability and controllability of rabbit plasma is worse, so there are more false-negative and false positive in plasma coagulase test. Therefore, the development of ELISA test method, increased the sensitivity and specificity of plasma coagulase test, could be desired. ELISA test method can also be directly used for rapid screening of S. aureus. The inactivated S. aureus was used as antigen to immunize the New Zealand white rabbits, guinea pig. The rabbit antiserum with 1:4000 titer and guinea pig antiserum with 1:2000 titer were obtained. We used the two antiserums and established double-antibody sandwich ELISA test method, and optimized the experiment condition of double-antibody sandwich ELISA test method. The optimal experimental conditions were as follow:guinea pig antiserum as coated antibody, rabbit antiserum as detecting antibody, goat anti-rabbit immunoglobulin marked by horseradish catalase as enzyme anti-antibody, rabbit antiserum is diluted at the rate of 1:2000, the rate of guinea pig antiserum is 1:1000 and the rate of goat anti-rabbit immunoglobulin marked by horseradish catalase is 1:5000. The results of evaluation for the ELISA method showed that the sensitivity, specificity and repeatability of this method are able to satisfy the requirements of test, and can provide good technical support for the basic level inspection institutions to conduct detection of S. aureus.