| A tick-borne hematozoonosis of the Theileria equi(formerly Babesia equi),which infects the red blood cells and lymphocyte of equine animals(horses,mules,donkeys,zebras,etc.)and is characterized by acute hemolytic anemia.The symptoms of T.equi usually cause anemia,fever,jaundice and other symptoms in horses.The epidemic peak of the Theileriosis disease is mainly in the spring and autumn.Many kinds of tick vectors have been occurred in the Xinjiang(unique geographical environment)which caused frequently transmission and the increasing infection rate of T.equi.At present,there are no effective medicines and vaccines for curing T.equi,and also no vaccine for immunizing horses in advance.Sick horses will become life-long carriers when return to normal condition,causing certain economic losses to the horse-breeding industry.Eighty percent of horses in our district are naturally grazed in grasslands and remote mountainous areas,and the diagnostic techniques are relatively underdeveloped.To confirm a suspected case,we need to "crossing of the Tianshan" to send samples to the laboratory for testing.Therefore,it is necessary to establish a rapid method to detect the T.equi,which can identified acute Theileriosis diseases and recessive state of equine.It will provide a technical support for the integrated control.In order to solve the problems in practical production,the recombinant plasmid pET-28a-EMA1 was constructed.Then the recombinant plasmid of pET-28a-EMA1 was purified and used to immunize BALB/c mice,to screen out a stable secretion of monoclonal antibody.Using pET-28a-EMA1 and monoclonal antibody as the basis,a new method of colloidal gold immunochromatographic strip for the detection of steineriosis has been developed.The method is specific,sensitive,stable,time-consuming,and does not require a professional technician.This method does not required for professional equipments and this method also have other advantages.It can be applied to epidemiological investigation and monitoring at the grass-roots level which have great practical significance to take relevant preventive measures in time.1.In this study,the eukaryotic vector pCMV-N-flag-EMA1 was constructed based on the early cloning and expression of pET-28a-EMA1 recombinant protein.The purified pET-28a-EMA1 protein was used to immunize the mice,and the monoclonal antibodies against EMA-1 protein of T.equi were screened by cell fusion technology.The characteristics of the selected monoclonal antibody screened were analyzed by SDS-PAGE,Western Blot and IFAT.The results showed that a mAb 5H2 with stable secreted the anti-EMA-1 protein was selected.Monoclonal antibody 5H2 presented specific reactions with prokaryotic expression of pET-28a-EMA1,pCMV-N-flag-EMA1 eukaryotic expression plasmids in transfected cells and red blood cells infected with T.equi.The titer reached 1:256 000.The heavy chain is IgG 2b and the light chain is κ chain.2.In this study,a colloidal gold labeled pET-28a-EMA1 protein was used to coat the detection line with a pET-28a-EMA1 protein,and a monoclonal antibody 5H2 coat was used to identify the epitope of the pET-28a-EMA1 protein.At the same time,the parameters were selected and optimized including protein labeled pH,labeled concentration,detection line dot membrane concentration and quality control line dot membrane concentration,respectively.The colloidal gold immunochromatographic strip was developed according to specific,sensitive and intra-batch and inter-batch repeated tests.Then it was applied to clinical samples(N=355)which were collected from 5 experimental sites in Xinjiang,the coincidence rate was compared with commercial ELISA kit.The results showed that the best labeling pH of pET-28a-EMA1 protein was 7.5,the best labeling was 60 μg/mL colloidal gold and the detection line membrane concentration was 0.5 mg/mL.In addition,the quality control line membrane concentration was 1 mg/mL and the direct detection time of the samples was 5～10 min.This method displayed no cross-reactivity with the sera positive for Babesia caballi,and the red bands were still visible when the sera were diluted to l:8.The results of repeated stability were consistent.Furthermore,the infection rates of colloidal gold strip and commercial ELISA kit were 65%(56/92)and 54%(50/92),respectively,and the positive agreement rate of the two methods for detecting the antibody of T.equi was 91.3%.Using the establish ed colloidal gold immunochromatographic test strips of the T.equi,the average positive infection rate was 59%in four clinical samples.The establishment of rapid detection method-colloidal gold immunochromatographic strip is really convenient for detecting horsetail disease in grass-roots,large-scale horse breeding sites,and timely implementing control measures. |
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