Preparation Of Egg Yolk Antibody Against Laribacter Hongkongensis And Primary Application

Laribacter hongkongensis was firstly isolated from the blood and empyema of a patient with hepatic cirrhosis in HongKong in2001. This isolate was identified that was different from other bacteria species and belonged to a new bacterial genus by biochemical identification and gene sequence analysis of16S rRNA. In2004, a multicenter case-control study research published in journal Lancet showed that L. hongkongensis was associated with community-acquired gastroenteritis and traverller’s diarrhoea. And risk factors for infection included consumption of fish and minced freshwater fish meat. As a newly discovered food-borne pathogen, there are bacteria cultivation, biochemical test, PCR detection and LAMP detection methods for Laribacter hongkongensis. But there was no standard detection method for this bacteria and it is never reported about immunological detection method.Immunoglobulin G in avian blood which could be transferred to egg yolk, can provide protection for chick embryo during development. And this immunoglobulin G called egg yolk immunoglobulin---IgY. IgY belong to polyclonal antibody, play an important role in diagnosis and treatment of disease. Usually polyclonal antibody is quite hard for large-scale preparation and multiple preparation may induce bad homogeneity of antibody. In the egg, chicken IgY is found mainly in the egg yolk, whereas the concentration in egg white is very low. Chicken IgY that had advantages such as high purity and specificity,no cross-reaction with mammalian serum, could be mass produced antibody substituting rat, rabbit, and so on. Recently, the investigation and application of IgY is a hot research spot in the field of immunology. In this study, we would prepare the chicken IgY against L.hongkongensis, then do some researches about its physicochemical properties, detection method, antibacterial activity, etc.Objective:1to prepare the chicken IgY against Laribacter hongkongensis2to establish and evaluate a preliminary direct ELISA for identification of Laribacter hongkongensis3to test the growth inhibition of Laribacter hongkongensis in vitro by the chicken egg yolk antibodies.Methods:1. Anti-LH chicken IgY were obtained from the egg of Hylan white hens immunitied by Laribacter hongkongensis.2. The anti-LH chicken IgY were purified by the methods of saturate ammonium sulfate fractional precipitation and antigen absorption.3. The titer concentration and purity coefficient of the chicken IgY were analyzed by indirect ELISA, Bradford, and SDS-PAGE electrophoresis respectively.4. The chicken IgY which conjugated with horseradish peroxidase were established direct ELISA method for identification of Laribacter hongkongensis. Then, the sensitivity and specificity of this method were analyzed, by detecting bacterial samples from intestine of freshwater product.5. The growth inhibition of Laribacter hongkongensis were cultured by liquid medium, and determined by scattering turbidity. Results:1. Chicken IgY against Laribacter hongkongensis were obtained successfully.The titer of Chicken IgY were1:128000approximately after purified, only1:8000after antigen absorption method. The purity was mainly shown two straps of60KD and30KD by SDS-PAGE electrophoresis. The concentration of the chicken IgY against L.hongkongensis were7-10mg per milliliter egg yolk.2. The chicken IgY were identified to have favourable heat stability and tolerance to acid and base.3. We established direct ELISA method with chicken IgY conjugated with horseradish peroxidase. And the determine conditions were optimized as the following:confining time was60min; the titer of HRP enzyme-labeled antibody was1:160, the cut off value for reporting L.hongkongensis positive status was X+3SD=0.181. The limit of detection for bacteria concentration was106cfu/ml.4. Forty-three bacteria isolates from intestines of freshwater products were measured by this direct ELISA method, with90%sensitivity and91.5%specificity.5. The results from the inhibition in vitro studies demonstrated that at the concentration of106cfu/ml, the growth of L.hongkongensis can be inhibited in liquid culture adding the chicken IgY(15mg/ml). But the activity and mechanism of this action need further research.Conclusion:1. Chick IgY that isolated and purified by methods of water dilution and saturate ammonium sulfate fractional precipitation, had high purity and activity.2. The direct ELISA method for L.hongkongensis identification had its feasibility, and provided new ideas for L.hongkongensis identification methods.3. The results from the inhibition in vitro studies demonstrated that the growth of L.hongkongensis can be inhibited in liquid culture. But activity and mechanism of the action need further research.