The Preparation And Spray-drying Of Egg Yolk Antibody Against CSFV

Swine fever is still a highly contagious disease of pigs, which bring huge losses inpig industry. Monoclonal antibody and specific antibody isolated from animalhyperimmune serum has better therapeutic effect, but it is not suitable for mass productionbecause high cost and low yields; while egg yolk antibody (IgY) has some advantages,suchas wide sources, low cost, suitable for mass production. In recent years, more and morestudies on the IgY for prevent and control diseases. This experiment mainly researches themethods of preparation of egg yolk antibody against swine fever virus, the ELISA methodfor detect the IgY. At the same time, the spray drying method and the protective agentswill be optimized and screened.Methods:1. Build the ELISA method for detect the IgY against CSFV (Classical swine fevervirus, CSFV)(1) The swine fever vaccine virus was adopted for coating ELISA plate. The primaryantibody was the positive IgY against CSFV extracted by chloroform method. Theoptimized concentration of antigen coated and the working concentration of enzymelabelled antibody are determined by phalanx titration. Compareing the P/N value, anddetermine the incubation time.(2) The positive and negative IgY were extracted from30eggs which were immunized or had not immunized with swine fever vaccine. The ODvalue of the positive and negative IgY were detected by ELISA. The critical value ofnegative and positive were calculated by the statistics method. The sensitivity of ELISAfor detecting IgY was compared with the IHA method. The specificity of ELISA fordetecting IgY agaist CSFV was compared with the IgY agaist avian influenza virus,Newcastle disease virus, swine transmissible gastroenteritis virus and porcine epizooticdiarrhea virus. As the same time, the repeatability of ELISA method for detecting IgYagainst CSFV was assessmented.2. The preparation and detection of yolk antibody against swine fever virus.（1）Fifty200days of age health Hy-line layers were selected for injecting swinefever vaccine in chest muscles. The IgY against CSFV were extracted from the eggs afterimmunization. The titer of the IgY was tested with the established ELISA method.The secondary immune were carried out when the titer fell to the middle value. When the titerreached1:4096level, the eggs were collected for extract antibody by water dilutionmethod.(2) The aseptic test of the extracted IgY was implemented.The IgY solution wascultured in nutrient agar and broth medium respectively,37℃last24h.It would beobserved whether the bacterial growth. Mouses and chickens were injected the extractedIgY for animal safety testing. In the safty testing, the mental state and the pathologicalchanges in heart, liver, spleen, lung, kidney were observed.3. The protective agents were screened and the process was optimized for the IgYsolution spray drying.Glucose, sucrose, skimmed milk, lactose, trehalose was selected as IgY protectiveagent respectively in spray drying. The retained antibody activity level was detected forselecting appropriate concentration of the protective agents. An orthogonal L9(34) testdesign was applied to select the optimum spray drying parameters including inlettemperature, feed rate and protective agent concentration according the level of antibodyactivity and flour yield.The results were as follows:1.(1) The positive value was close to1.000and biggest different with the adjacentholes when the antigen coating concentration was1:1000, HRP-labelled secondaryantibody working concentration was1:6000, and the IgY was diluted to1:1024, thenegative value was less than0.2, and P/N value was max. Optimized experimentalconditions: the ELISA plate was closed30min at37℃with2%gelatin; the bestincubation conditions for IgY and Enzyme labeled antibody were1h and45minrespectively at37℃; the best developing time was20min at room temperature. At thistime, OD450nm value was close to1.0, and the P/N value was biggest.(2) According the statistic analysis, The average OD450nm value（X）of negative IgYagainst CSFV was0.103, the standard deviation (SD) was0.014. The result was judged tobe positive when the OD≥0.145, be negative when the OD<0.131, be suspicious when0.145<OD≥0.131.The titer of IgY against CSFV was27detected by IHA method. The OD450nm valueof the IgY was still positive detected by established ELISA method when the IgY wasdiluted to214. This result showed that the eatablished ELISA method has a high sensitivity.All the OD450nm value was negative when we replaced the IgY against CSFV by theIgY against AIV-H9, NDV, PTGV, PEDV. At the same time, CSFV positive and negativeserum was detected by the established ELISA method. The results showed that establishedELISA method has a nice specificity.5copies of IgY were detected by the ELISA using same batch and different batchenzyme label plates. The results showed that the variation coefficients are less than10%. 2.(1) The IgY titer against CFSV can raised to1:4096after third immuned and raisedto1:8192after the fourth immunization and last about one month.(2)The extracted IgY byWater dilution method showed yellow, uniform and no special smell liquid. There were notbacterial growth when we cultured the IgY liquid in the common nutrient agar andanaerobic broth for24hours at37℃temperature. The mouses and chickens had notabnormal symptom after injected IgY and had not abnormal pathological changes byautopsy.3. When we used the protective agents, the antibody activity retention rate wassignificantly higher than that of control group. At the same concentration, the protectiveeffect of the agents was: glucose> trehalose> sucrose> lactose> skim milk.0.25%of glucose was selected as the single factor for optimize the spray dryingmethod. The feed rate had no significant effect on the antibody activity, but real influenceon flour yield. The antibody activity could maintain to be more than90%when the feedrate between600-1300ml/h, but the flour yield decreased significantly (P<0.05) to31.36%when the feed rate greater than800ml/h. The antibody activity retained more than92%when the inlet temperature between70℃to120℃, but the antibody activity retainedsignificantly reduced (P <0.05) when the inlet temperature above130℃.Flour yield wouldsignificantly increase with the temperature went up (P <0.05).Orthogonal test results: the most powder yield would gain when the spray dryingconditions as follows：150℃，500ml/h and0.25%glucose. The antibody activity wouldmaintain satisfied when it was spray dryed at70℃,700ml/h,0.06%glucose. The factorsinfluenced the flour yield was inlet temperature (78.75)> glucose concentration (9.5)> feedrate (5.5). Activity analysis: three factors affect the activity retention F ratio was less than19, so the difference was not significant.Conclusion:1. The ELISA method for detecting yolk antibody against swine fever was establishedsuccessfully.It was stabilized when the ELISA plates were coated with vaccine virus of CSF.theestablished ELISA method was high sensitivity, specificity, repeatability.2. The yolk antibody against CSFV was prepared successfully.High titer IgY against CSFV was obtained when the layers were vaccinatedcontinuously with inactivated swine fever virus. The obtained IgY was safe, sterile.10-folddilution IgY was still able to neutralize a dose of swine fever vaccine virus.3. The spray drying method for yolk antibody was established and optimized.By orthogonal experiment: three levels of feed rate and glucose concentration had nosignificant difference on the antibody activity and flour yield. The inlet temperature hadsignificant influence on flour yield (P <0.05), but no significant effect on activity. The antibody titer was about100%and flour yield was about82.5%when the glucoseconcentration as0.06%,the inlet temperature as150℃and the feed rate as700ml/h.