Development And Application Of Indirect Elisa Kit For The Detection Of Antibody To Porcine Hemagglutinating Encephalomyelitis

Porcine hemagglutinating encephalomyelitis was an acute and infectious disease,which is caused by porcine hmagglutinating encephalomyelitis virus(PHEV).The main victims are young piglets,while adult pigs mostly tend to have stealth infections.The major clinical symptoms of this disease include vomiting,diarrhea,neurological symptoms and so on.Besides,PHEV infected piglets usually demonstrate two kinds of disease subtypes,vomiting consumptive type and encephalomyelitis respectively.In vomiting consumptive type,PHEV mainly infects young piglets which have been born within two days.The mortality rate of piglets is between 20%-80%,with clinical manifestations like nausea,vomiting,diarrhea,difficulty in swallowing.Eventually,the long-term non-meal consumption would lead to weight loss,body dysfunction and death.In terms of the surviving piglets,stunting would occur during the growth of them,finally becoming stiff pigs.It is possible that encephalomyelitis type develops on the basis of vomiting consumptive type,in which PHEV mainly infects piglets within three weeks.The clinical manifestations are increasing body temperature of sick piglets,which is accompanied by nausea,vomiting as well as loss of appetite.In addition,there are obvious neurological symptoms,such as muscle tremor,ataxia,hind limb stiffness or paralysis,water-like limbs,abnormal sensitivity to the external environment and sound stimulation,late onset of eye tremors,inability to stand up,breathing difficulties and eventually death.Additionally,this disease has short onset time with high mortality rate.Only a few sick piglets will recover and the mortality rate will be 20%-100%.Since its first outbreak in Canada in 1952,PHEV is not uncommon around the world,which has been reported in different places from Japan,the United States,and Europe.In 1986,the disease was primarily reported in China.Recently,it seems that the trend is increasing.According to serological surveys,the disease is currently found in the northeast of China,with a relative high infection rate in Jilin and Liaoning province.So it is imperative to figure out effective diagnostic and preventive measures.Now,the detection method for PHEV is still on the basis of laboratory diagnosis,professional experimental instruments and laboratory staffs are highly required.So it is urgent to establish a rapid,convenient,sensitive and specific diagnostic method in order to meet the above requirements.Enzyme-linked immunosorbent assay would be more suitable for our diagnostic work.In our current work,the resuscitation of PHEV recombinant protein N bacteriophage was carried out,which is followed by N protein purification by referring to the optimal conditions for induction.And finally,the N antigen was successfully obtained.Which is followed by the establishment and optimization of an Elisa method.Meanwhile,an indirect antibody detection reagent for porcine hemagglutinating encephalomyelitis virus structural protein N was established,thus providing the basis for the future serological investigations.The details are as follows:1.Antigen preparation and quality detection of PHEV-N recombinant proteinAccording to the optimum induction condition obtained from our laboratory,synthesis of target protein was carried out.Then,His Gravi Trap Kit was used to conduct the purification of target protein.SDS-PAGE electrophoresis was carried out to detect purity and concentration of target protein.Results: LB fluid medium was inoculated with resuscitated bacteria solution in a ratio of 1:100.Then,the solution was put into a 37? shaker to run 200 rmp for 2 hours.Then,100 m M IPTG was added in the solution in a ratio of 1:1000 and 6-hour induction was carried out.After ultrasonication,the solution was purified.SDS-PAGE electrophoresis of purified products was carried out,a clear band at 55 k D only has been shown.It indicates that the purity of the target protein was high.Through protein concentration detection,the mean concentrations of three batches of protein obtained were 34.46mg/m L?31.63 mg/m L and 37.28 mg/m L respectively.2.Establishment and optimization of the Elisa methodThe purified recombinant N protein was coated in the Elisa plate,the Elisa method was then established.Its optimization was carried out and its coincidence rate was compared with that of the HI method.Results: the optimal coating concentration of antigen was 0.0625?g;the optimal dilution rate was 1:200;the optimal coating time of antigen was 2h at 37? or overnight at 4?;the optimal sealing agent was 3% skimmed milk;the optimal sealing time was 2 hours at 37?;the optimum reaction time of serum was 90min;the optimal dilution ratio was 1:10000;the optimal reaction time of enzyme-labeled secondary antibodies was 90 min at 37?;and the optimum reaction time of substrate was 20 min.According to principle of statistics,when the value of serum OD450 nm ? 0.388,it was considered as positive;when the value of serum OD450 nm < 0.317,it was considered as negative,where the value between 0.317 and 0.388 was suspicious.By using the established Elisa method,positive serums of PHEV,PEDV,PRV,CSFV and TGEV were detected at the same time.The results showed that positive serums of PEDV,PRV,CSFV and TGEV were negative for PHEV antibody,indicating good specificity of this experiment without cross reaction.Meanwhile,its sensitivity was also determined.The results showed that when the dilution ratio of standard positive serum reached 1:6400,the result was still positive,indicating good sensitivity.In addition,the coincidence rate of methods of Elisa and HI was 75%,showing that Elisa was more sensitive than HI.3.Development and application of indirect Elisa antibody detection kit of porcine hemagglutinating encephalomyelitis virus(PHEV)Based on the established Elisa method,control serum was identified and Elisa plates were prepared with purified recombinant protein.Three batches of Elisa antibody detection kits were prepared and their repeatability within a batch and among batches,storage life,sensibility and specificity etc.were examined.Then,they were applied for the detection of clinical samples.The results were as follows:According to determination experiments of three batches of kits within a batch and among batches,we have found that the variation coefficients of the same batch of kits were between 3.43% and 7.56%,which were less than 10%.Four samples of serum of different antibody titer were detected randomly with kits in the three batches,where variation coefficients were from 1.09%-2.60%,which were less than 3%,indicating stable detection results and good repeatability of kits within a batch and among batches.Through respective sensitivity tests of the three batches of kits,we have found that when the dilution ratio of serum reached 1:6400,it was still positive.After that,positive serums of PHEV,PEDV,PRV,CSFV and TGEV were detected.The results showed that positive serums of PEDV,PRV,CSFV and TGEV were negative for PHEV antibody.Meanwhile,the storage life of kits was tested and the kit can be stored at-20? for six months.In the end,assembled indirect Elisa antibody detection kits of PHEV were used to test 633 serum samples from Shandong,Jilin,Liaoning(Yingkou region)and Tianjin etc.The investigation showed that positive rates in all regions were more than 50 %.In conclusion,this study successfully prepared a PHEV antibody detection kit with high sensitivity,specificity and repeatability and it can be applied to detection of PHEV immune antibodies and epidemiological survey.