Preparation Of Egg Yolk Antibody Against Norovirus And Primary Application

Backgroud:Norovirus and Sapporo-like virus collectively referred to as human calicivirus. Norovirus was detected from the faeces of patients with acute gastroenteritis outbreaks which happened in Norwalk, America in1968with immune electron microscopy method by Kapikian in1972with the diameter was27-32nm. Now, Nov has become an important non-bacterial gastroenteritis pathogens. It can cause an outbreak in kindergarten, summer camps, schools, hospitals, nursing homes, restaurants, troops and maritime fleet units and all ages can be infected. GⅡ type Norovirus had become the most common strain of virus which was found from the analysis of several outbreaks of gastroenteritis and diarrheal during the new century, in which GⅡ-4type Nov was dominant. Now, the Nov of food, feces and water can be detected by RT-PCR method which is repeatable and has high sensitivity but it needs a long time and high equipment requirement. And RT-PCR must be using the mixed primer because of the diversity of strains. Today, RT-PCR has been gradually replaced by fluorescent quantitative PCR, which is more rapid and sensitive, especially using Taqman probe. A single test can provide evidence and quantitative detection. However, it is difficult to be used at the scene and grassroots popularity because of the high equipemt requirements. 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 preparations 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. In2003, de-Almeida CM prepared anti BfpA IgY and used it in immunoadsorption and found it can specifically identify the EPEV. Ohnishi T used IgY and IgG to test HGF and found IgY can increase the sensitivity by10times which can not only detect the HGF of bleed but also the HGF of the normal human’s urine. Juliarena expressed GST-p24fusion protein and immuned laying hens to prepare specific IgY for immunodiagnostic. The result showed IgY can avoid the cross-reaction of conventional mammalian antibody. IgY was used in a series of analysis technology as a detection reagent, and will be expected to apply to the field of diagnostic reagents and paly an important role in the development of diagnostic reagents.In the study, laying Leghorn chicken were immuned by GⅡ.4type (VA387) Nov P particles to prepare the anti-Nov yolk IgY. We prepared the HRP enzyme-labeled antibody based on IgY and establish double-antibody sandwich ELISA system to detect Nov and evaluated it. It laid a foundation for the applications in diagnosis and disease prevention of IgY and provide new ideas for the detection of Nov.Objective: 1To prepare the chicken IgY against Nov.2To study the characteristics of specific anti-Nov antibody IgY.3To establish and evaluate a double-antibody sandwich ELISA system for the detection of Nov.Methods:1The preparation of the IgY(1) GⅡ.4type (VA387) Nov P particles and the same amout of Freund’s adjuvant were emulsified to prepare immune agents. Laying Leghorn chickens were immuned by intramuscular injection in the muscle under the wings several times.(2) The eggs before immunization with one week and the three months after immunization were collected.(3) The anti-Nov chicken IgY were purified by the methods of improved water dilution method and saturate ammonium sulfate twice fractional precipitation(final saturation was55%and33%, respectively).(4) The changes of each cycle of chicken IgY were detected by indirect ELISA.(5) The purity and specificity were identificated by SDS-PAGE and Western blot, respectively.(6) The total protein content of the chicken egg yolk antibody was determinated by Coomassie blue method.(7) IgY solution was diluted to different concentrations and placed in25℃room temperature to evaluate the room temperature stability.Its pH stability and thermal stability were evaluated. The effect of the pepsin and repeated freezing and thawing to the IgY were evaluated.2The establishment and evaluation of double-antibody sandwich ELISA system based on the anti-Nov IgY.(1) The HRP enzyme-labeled antibody was prepared by sodium iodide method. (2) The protein content of the enzyme-labeled antibody was determinated by the ultraviolet absorption method under280nm and its mark rate and the mole ratio were calculated.(3) The double-antibody sandwich ELISA system based on the anti-Nov IgY was established.(4) The conditions of the double antibody sandwich ELISA were optimized.(5) The minimum detection limit, the intra-assay coefficient of variation, the inter-assay coefficient and the cross-reactivity with rotavirus and adenovirus of the double antibody sandwich ELISA were evaluated.(6)The system was compared with the gold standard for clinical application evaluation. The sensitivity, specificity and Youden index were calculated.Results:1The preparation of the IgY(1) The amount of the purified anti-Nov chicken IgY rised rapidly after the first booster immunization until the fifth week after the first time of immunization reached its highest titer was1:25600, after that the titer was decreased slowly to1:12800.(2) The purity was mainly shown two straps of60KD and30KD by SDS-PAGE electrophoresis. The result of Western blot showed the IgY can specific binding to the GⅡ.4type (VA387) Nov P particles.(3) The concentration of the chicken IgY against Norovirus was7.2mg per milliliter egg yolk.(4) The evaluation results of physical and chemical properties of chicken egg yolk antibody showed that the titer of different dilutions of IgY was decreased slightly but still maintained at a high activity after stored at a room temperature of25℃. The antibody activity had little change at60℃and below it for30min, fell slightly at70℃for30min and disappeared at80℃for30min. The activity of IgY was not affected at the range of pH3-12, while the pH≤2or≥12the activity decline rapidly. The activity slightly decreased after the effect of the pepsin with pH≥3, disappeared after the effect of the pepsin with pH<2. The activity of IgY slightly decreased with a lesser extent after freeze-thaw cycles between-20℃and4℃with5times.2The establishment and evaluation of double-antibody sandwich ELISA system based on the anti-Nov IgY.(1) The mark rate of the HPR-enzyme labeled IgY was48.73%and the mole ratio was1.74and the protein content was8.58606mg/ml.(2) The best package diluted and optimal enzyme-labeled antibody dilutions of the double antibody sandwich ELISA were1:80, the optimal blocking solution was2%BSA and the closure time was1h, the preferred enzyme-labeled antibody’s reaction time was30min and the best substrate roletime was15min.(3) The minimum detection limit of the double antibody sandwich ELISA was20ng/ml, the intra-assay coefficient of variation was1.021%, and the inter-assay coefficient was1.150%. And there was lower response rate of the cross-reactivity with rotavirus and adenovirus.(4) The detection rate was no statistical difference(P=0.629>0.05) between the double antibody sandwich ELISA and the gold standard. The consistent coefficients Kappa was0.637, the goodness of fit was general. The sensitivity was82.50%, specificity was81.82%and Youden index was0.6432.Conclusion:1The specific anti-Nov was prepared successfully and the highest titer was1:51200.2IgY has high purity and high specificity to Nov P particles.3Improved water dilution method and saturate ammonium sulfate twice fractional precipitation method to separate and purify the IgY are simple, fast and efficient and the IgY has high activity which is suitable for large scale extraction. 4The IgY obtained is stable to temperature and pH. The activity slightly decreased after the effect of pepsin and repeated freezing and thawing.5The enzyme-labeled antibody based on the IgY was prepared successfully.6The double antibody sandwich ELISA system was established and the optimized conditions are as follows:(1) The best package diluted and optimal enzyme-labeled antibody dilutions of the double antibody sandwich ELISA are1:80.(2) The blocking solution is2%BSA and the closing time was1hour.(3) The reaction time of the enzyme-labeled antibody was30min.(4) The best substrate roletime was15min.7The minimum detection limit of the double antibody sandwich ELISA was20ng/ml, the intra-assay coefficient of variation was1.021%, and the inter-assay coefficient was1.150%. And there was lower response rate of the cross-reactivity with rotavirus and adenovirus.8The detection rate was no statistical difference between the double antibody sandwich ELISA and the gold standard. The goodness of fit was general. The sensitivity was82.50%, specificity was81.82%and Youden index was0.6432.