Study On Immunoassays For Trichinella Spiralis Antibodies Against The Cystatin-like Protein(CLP)

Trichinella spiralis is a zoonotic food-borne parasitic nematode that completes a complex life cycle in the same host.It was transmited to animals or humans via infectious meat containing muscle larvae(ML).To control transmissions of T.spiralis through the food chain to humans,sensitive and selective multihost sera-diagnosis is urgent needed for monitoring T.spiralis exposure.The CLP gene with high abundance and strong antigenicity was screened from c DNA library of intestinal infectious larva at 6 h after infection.In this study,the soluble recombinant CLP was obtained by prokaryotic expression system and Ni-NTA affinity chromatography combined with one-step on-column renaturation purification strategy.An indirect ELISA was established based on r CLP antigen(r CLP-i ELISA).The suitability of r CLP as a detection target was systematically evaluated using 270 negative sera as well as positive sera from pigs artificially infected with 1000 and 50000 ML per pig.The seroconversion in r CLP-i ELISA was earlier than that in the commercial kit(ES-i ELISA).Monoclonal antibody(Mc Ab)was prepared with conventional methods.Three high-affinity Mc Abs 1H9,6B5 and 7F8 were screened by r CLP-i ELISA,ES-i ELISA and blocking ELISA methods,respectively.Mc Abs 1H9 and 6B5identified the epitope of ³⁹ HEALFSSDLKQESGV ⁵³,while Mc Ab 7F8 identified the epitope of ¹⁷⁸ REALFSSDSKEQSGV ¹⁹².A competitive ELISA based on Mc Ab 1H9and r CLP(r CLP-c ELISA)was proposed for T.spiralis antibodies test in multihost sera.The r CLP-c ELISA were evaluated using pig(n=1316),mouse(n=189)and human(n=169)serum samples.The results showed that the sensitivity and specificity of r CLP-c ELISA were 100%and 99.56%respectively in field swine sera.In the detection of serum from healthy volunteers and trichinellosis patients,it showed a sensitivity of 97.01%with the specificity of 94.12%.The r CLP-c ELISA has no cross-reaction with other parasite or virus infections with potential in the detection of T.spiralis,T.nelsoni and Trichinella T8 infections.To improve the sensitivity of the method,rabbit anti-CLP polyclonal antibody was modified on the surface of magnetic nanoparticles(MNP-PCAB probe)as capture probes.The oligonucleotide(I1 DNA)and Mc Ab 1H9 were modified on gold nanoparticles to construct a dual-functional gold nanoparticles(Au@Mc Ab-I1 probe)as competitive probes.A competition-sandwich biosensor based on dual-functional gold nanoparticles and hybrid chain reaction(Au@Mc Ab-I1-i HCR)was developed for T.spiralis antibodies test.The Au@Mc Ab-I1-i HCR showed high accuracy(AUC=0.9985)in 169 sera from healthy volunteers/trichinellosis patients,with a sensitivity of 97.01%and a specificity of 99.02%,and no cross-reaction in sera of patients infected with other parasites/enteroviruses.There was a strong correlation(R²=0.9149)between Au@Mc Ab-I1-i HCR and r CLP-c ELISA in values of trichinosis patients sera tests.Compared with the ES-i ELISA kit,the Au@Mc Ab-I1-i HCR had a lower minimum detection limit(24.20 ng/m L)with similar detection time.The CLP antibody curves of infected pigs with different doses showed a trend of antibody titer"increased-slightly decreased-continued to increase".To clarify the abnormal antibody response mediated by CLP,BMDCs,CD4+T cells in OT-?m ice and B cells were used as research objects to test the immunoregulatory activity of CLP and its effect on antibody transformation by co-incubation in vitro.The results showed that CLP inhibited BMDCs mediated B cell activation and antibody secretion directed by inhibiting the activation of naive CD4+T cells.The inhibiton effects of CLP provided part of theoretical support for CLP antibody curves measured by the proposed serological antibody assays.