Establishment And Application Of Colloidal Gold Immunochromatographic Assay In Diagnosis Of Haemonchus Contortus Infection

Haemonchus contortus is a common digestive tract parasite in ruminants,which causes weight loss,anemia and even death in animals after infection,causing huge losses to the livestock economy,and with the widespread use of benzimidazole,macrolide and other drugs,problems such as drug resistance and drug residues are becoming increasingly serious.Accurate diagnosis of the infection in animals is of great importance for the prevention and control of the disease.Conventional diagnostic methods(such as fecal examination,larval hatching and necropsy)are time-consuming and laborious,have low sensitivity,and require a certain level of professionalism among operators.Therefore,there is an urgent need to establish a simple,sensitive,specific and rapid diagnostic method for the early detection of Haemonchosis.In previous studies,five proteins containing cold shock binding domain protein(CS),hepatocellular carcinoma cell-associated antigen 59(HCA59),methyltransferase type 12protein(Mt12),NADH: ubiquinone oxidoreductase structural domain protein(NDUDC)and Ras-containing structural domain protein(Ras)were found to have early diagnostic potential,and the indirect ELISA method based on these proteins had good sensitivity(87.5-100%)and specificity(90.6-100%).To further improve the early diagnostic ability of these five proteins,one fragment of each protein was screened and named as Hc-CS fragment(Hc-CS fragment,Hc-CSf),Hc-HCA59 fragment(Hc-HCA59 fragment,Hc-HCA59f),Hc-Mt12 fragment(HcMt12 fragment,Hc-Mt12f),Hc-NDUDC fragment,Hc-NDUDCf),and Hc-Ras fragment,Hc-Rasf),in the above order to form the Hc-5C complex protein,and to analyze the early diagnostic potential of this complex protein.Then We established an indirect ELISA and double antigen sandwich colloidal gold immunochromatographic strip assay both based on Hc-5C.1 Antigenic characterization of Hc-5C complex proteinBlast analysis was performed on Hc-CS(Gen Bank accession number CDJ84294.1),HcHCA59(CDJ80864.1),Hc-Mt12(CDJ87424.1),Hc-NDUDC(CDJ88764.1)and Hc-Ras(CDJ86500.1)to screen 1 specific fragment each to form the Hc-5C complex protein.The five fragments contained amino acid residues 9-58,10-61,137-189,8-40 and 28-76 of the original protein,respectively.The first four fragments showed 80-90%,65-88%,66-79% and63-84% similarity to Teladorsagia circumcincta and Dictyocaulus viviparus,respectively,and the fifth fragment showed 50% similarity to Necator americanus and Nippostrongylus brasiliensis;in order,it may contain 3,5,4,4 and 2 B-cell antigenic epitopes and 2,3,2,2and 1 T-cell antigenic epitopes.Hc-5C encodes 251 amino acids,with a relative molecular mass(M.W.)of 28.54 k Da,a theoretical isoelectric point(p I)of 5.97,a Grand Average of Hydropathy of-0.312,and a partial overlap of less than 25% with other nematode proteins.The similarity ranged from 36-88%,and all four secondary structures were present,with theα-helix predominating,probably retaining 59.9% of B-cell antigenic epitopes and 80.5% of T-cell antigenic epitopes and generating new antigenic epitopes.The results of the analysis suggest that the Hc-5C complex protein may have higher potential than the original 5 proteins in the diagnosis of Haemonchosis.2 Hc-5C gene cloning and expressionFive pairs of specific primers were designed based on the Hc-5C gene sequence.p ET-32a/Hc-CS,p ET-28a/Hc-Mt12,p ET-28a/Hc-NDUDC plasmids and H.contortus c DNA were used as templates,and the target product Hc-5C was amplified by Touch down PCR(TD PCR)and overlap extension PCR(SOE PCR).The Hc-5C PCR product was ligated with the p ET28 a vector,transformed into DH5α competent bacteria,and positive single colonies were picked for sequencing.SDS-PAGE showed that the recombinant Hc-5C protein(r Hc-5C)was approximately 32.54 k Da in size and distributed in the inclusion bodies.The western blot showed that r Hc-5C could be recognized by goat sera infected with H.contortus,while negative sera showed no bands,which has the potential to be used for the diagnosis of Haemonchosis.3 Analysis of the early diagnostic potential of Hc-5CHealthy goats were artificially infected with the third stage larvae of H.contortus.Blood was collected and sera were separated at 7,14,21,28,35,42,49,56 and 61 days after infection and before infection(day 0).r Hc-5C was analyzed by Western blot using goat sera from different infection periods as primary antibodies and rabbit anti-goat HRP-Ig G as secondary antibodies.The results showed that r Hc-5C was not recognized by pre-infection sera and could be recognized by sera from goats artificially infected with H.contortus for 7-61 days.Results showed that r Hc-5C had the potential to be used for the diagnosis of early and long-term infection of H.contortus.4 Establishment and optimization of an indirect ELISA method based on Hc-5C recombinant proteinAn indirect ELISA method was developed using r Hc-5C as the encapsulated antigen.The antigen coating amount,primary antibody conditions,secondary antibody conditions,TMB reaction time,and blocking conditions were optimized;the negative and positive threshold values,batch reproducibility and specificity of the ELISA were determined;58clinical samples of goat sera,23 necropsy samples and 35 fecal samples,were tested and compared with the results of necropsy.The results showed that the optimum reaction conditions for this indirect ELISA were: antigen coating volume 40ng/well;primary antibody1:25 dilution,incubation at 37℃ for 0.5h;secondary antibody 1:5000 dilution,incubation at37℃ for 0.5h;4% BSA closed at 37℃ for 2h;TMB color development for 25min;the negative and positive threshold values were 0.242 and 0.286,respectively;the intra-batch coefficient of variation ranged from 2.3 to 8.5%;the inter-batch coefficient of variation ranged from 2.4 to 11.2%;there was no cross-reactivity with positive sera of Toxoplasma gondii and Trichinella spiralis;the compliance rate of clinical samples with necropsy and Mc Master were 78.3% and 82.9%,respectively.The results suggest that r Hc-5C indirect ELISA is feasible for clinical application,but other methods are still needed to improve the compliance rate or further optimize the protein structure to improve the detection effect.5 Establishment of colloidal gold immunochromatographic test strips based on r Hc-5CA double-antigen sandwich type of colloidal gold immunochromatographic test strip was developed using commercial colloidal gold particles labeled with r Hc-5C,and the detection line(T-line)and quality control line(C-line)were prepared by using r Hc-5C and anti-r Hc-5C rabbit polyclonal antibodies to delineate NC film.The amount of colloidal gold conjugate and the amount of T-line antigen coating were optimized;the specificity,reproducibility and sensitivity of the test strips were confirmed;54 clinical samples of goat Serum,19 necropsy samples and 35 fecal samples,were tested and compared with r Hc-5C indirect ELISA and necropsy results.The results showed that the test strips were able to distinguish positive and negative sera for H.contortus after 1:35 dilution of the sera;there was no cross-reactivity with positive sera of T.gondii,T.spiralis,and Fasciola gigantica;the reproducibility was good;the clinical test results showed that the compliance rate with necropsy and Mc Master were 84.2% and 85.7%,respectively.The number of clinical samples tested is small,and the number of samples tested needs to be further expanded in the follow-up work.