Development Of ELISA KIT For Rapid Detection Of Bifidobacterium

In recent years, along with the rapid development of immunology and the application of serological technology, the identification and detection technology of Bifidobacterium has already developed from the traditonal plate count technique to the application of various enzyme-immuno assay technologies based on the reaction between antigen and antibody. Among all the improved methods based on the reaction between antigen and antibody, ELISA method is the fasted developed and fairly consummate way at present time. ELISA is based on antigen or antibody as solid phase and enzyme-antigen conjugates or enzyme-antibodyconjugates, then according to the color of enzyme reaction to qualitation and quantitative analysis. Because of using high efficiency catalytic reaction of enzyme, this method amplifies the results of immunoreactive indirectly, and possesses highly sensitivity. Moreover, ELISA detection is reacting in the microwell plate and can detect many samples at the same time, so time is saved. Doubole-antibody sandwish ELISA is usually used for detection of antigen. As it was reported at present, no research is about founding reaction conditions of Doubole-antibody sandwish ELISA and quantitation detection of Bifidobacterium. ELISA method overcomes traditonal plate count technique's trouble and expensive cost of molecular biology detection technology. If to develop a ELISA Kit for detection of Bifidobacterium, it will have significance sense to practical application. Therefore, the purpose of this research was to study and research about this aspect.This research gained high titer immune serum, established and optimized Doubole-antibody sandwish ELISA method for detection of Bifidobacterium. ELISA kit was developed. Evaluating kit by methodology, other strains had no influence on detection results, good reproducibility and the detection limit was 106cfu/mL(g). the detection time was 4h. The results were as follow:1. Preparation of Immune SerumThe serum titer of the prepared rabbit anti-B.longum was 1:20480, and the serum titer of the prepared the mouse anti-B.longum was 1:10240. Prepared Immune Serum had high titer.2. Optimize reaction conditions for Double-antibody Sandwich ELISA for Rapid Detection of BifidobacteriumTo optimize reaction conditions for Double-antibody Sandwich ELISA for Rapid Detection of Bifidobacterium were investigated. The optimization reaction conditions: Density of anti-rabbit and anti-rat immune serums was 1:160 and 1:5120; coating buffer was 0.05mol/L pH9.6 Na2CO3-NaHCO3; confining liquid was 1%BSA-PBS; confining time was 30min; the density enzyme labelled antibody was 1:2000; enzyme labelled antibody reaction time was 90min; substrate action time was 20min; Standard Curve of Bifidobacterium with Double-antibody Sandwich ELISA was drawed, curvilinear equation was obtained: y=0.4461x-2.1503, and coefficient correlation was R2=0.9881.3. Development of the ELISA KitThe key component of kit including 8×12 microwell plate coated by anti-rabbit immune serums and confined; anti-rat immune serums; enzyme labelled antibody; Bifidobacterium longum standard preparation; substrate; stop buffer; concentration eluant; method of operation and illustration.4. Methodology Evaluation of ELISA kitThe kit was evaluated by methodology, including specificity, reproducibility and sensitivity. Using the Kit to detect the strains of Bifidobacterium and other strains, the results showed that other strains had no influence on detection results, so this kit had quite good specificity; reproducibility was good; the detection limit was 106cfu/mL(g).5. Keeping Time Experiment of the KitThe keeping time of coated microwell plate was evaluated. The experiment certified that this kit could maintain over one year.6. Application of ELISA Kit for Detection of BifidobacteriumUsing this Kit detected yoghurt and milk powder bought in the market and prepared in our expereiment which included Bifidobacterium, and to compare with traditonal plate count technique. The the results of respectively detecting Yoghurt(1), milk powder milk powder bought in the market and powder bought (2) prepared in our expereiment by kit were not significantly different from by the traditonal plate count technique (p>0.05). The the results of milk powder(1) prepared in our expereiment by the kit had significantly difference from the traditonal plate count technique (p<0.05).