Preparation Of Polyclonal Antibodies Against Shigella And Establishment Of ELISA Methods

Shigella is an important Enteric pathogens, which cause serious diarrhea in clinical. now the method for the detection of Shigella included general detect assay, molecular biological detect assay, toxin detection, immunological detect assay. General detect assay is veracity, but it is time-consuming; molecular biological assay is speediness, delicacy specificity, and apply for the detection of unfamiliar and new pathogens, but it is expensive and need high technic level, Vitek Automated System (VITEK), vitek immune diagnostic assay system(VIDAS) is celerity, exact, but expensive. ELISA assay of immunological apply abroadly in the detection of pathogens for it is simple, convenient, speediness and delicacy.This article by high titer polyclonal antibodies against Shigella were produced from rabbits immunized with the immunogen. The result show the rising trend of the antibodies to the immunity after 50d. Through the indirect enzyme-linked immunosorbent assay (ELISA). The final titer of antibodies were 1:51200, getting high purity IgG by Caprylic acid-Ammonium sulfate assay purificate antibodies.Conditions were optimized for using these preparations for an inderecte ELISA a. The result was shown that ELISA plates from Costar was suitable for ELISA kit; the confining liquid was 5% defatted milk powder, 37℃, 1h; the dilution of antibody was 1.25μg/mL, serum sample for detecting and HRP-labeled goat anti-rabbit should be incubated at 37℃for 1h respectively, the substrate for inderecte ELISA was incubated at room temperature for 5min before reading the OD492. Using this technique, a minimum detectable level of 10~5-10~6cfu/mL Shigella in buffer was achieved.Conditions were optimized for using these preparations for anDot-ELISA. The confining liquid was 5% defatted milk powder, 37℃, 1h; the dilution of antibody was 2.5μg/mL, serum sample for detecting and HRP-labeled goat anti- rabbit should be incubated at 37℃for 1h respectively, the substrate for Dot-ELISA was incubated at room temperature for 15min before reading the OD492. Using this technique, a minimum detectable level of 10~6cfu/mL Shigella in buffer was achieved.Specific-block test and crossing test indicated that antibody has good specificity for the detection of Shigella. It was confirmed that these was no cross-reaction between Salmonella. spp, Proleus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Vibrio, Bacillus subtilis. this research establish a specific, sensitive, stabile and repeatable method. which offers a new analysis method for the detection Shigella.