To improve the immunoassay performance of mycotoxin anti-idiotype nanobody and to realize mimicry of bispecific detected antigens,in this study,anti-idiotype nanobody against zearalenone(ZEN)and deoxynivalenol(DON)were used,and the SpyCatcher/SpyTag tags were fusion-expressed with N28 and Z6,respectively.Based on the in vitro self-assembly,the construction study of bivalent and bispecific anti-idiotype nanobody of N28 and Z6 were carried out.The results showed that the prokaryotic soluble expression of four SpyCatcher/SpyTag labeled fusion anti-idiotype nanobody,N28-SpyCatcher,N28-SpyTag,Z6-SpyCatcher,and Z6-SpyTag were achieved.The IC50 of the competition inhibition standard curves established based on Z6-SpyCatcher and Z6-SpyTag were 0.18 and 0.20 ng/mL,respectively.Their sensitivities were significantly higher than that of the IC50 of monomeric Z6(0.28 ng/mL).The fusion of the SpyCatcher tag with N28 was favorable for the enhancement of the sensitivity of the N28 assay,with the IC50 value decreased from 94.39 to 42.33 ng/mL,whereas the fusion of SpyTag label with N28 inhibited its binding to DON monoclonal antibody.In addition,bivalent and bispecific anti-idiotype nanobody Z6-Z6,N28-28,and N28-Z6,Z6-N28 of Z6 and N28 were successfully prepared based on the in vitro self-assembly property of SpyCatcher/SpyTag under the simple reaction condition of 4℃ for 12 h.The characterization results of the ELISA showed that the bivalent anti-idiotype nanobody retained their binding properties to the corresponding antibodies.The binding properties of the corresponding antibodies,in which N28-Z6 also achieved the binding activity with ZEN and DON antibodies simultaneously,and Z6-N28 only retained the binding activity with ZEN antibody because the binding of SpyTag tag to N28 would hinder its binding to DON antibody.In conclusion,the present study realized a simple and rapid preparation of ZEN/DON bivalent and bispecific anti-idiotype nanobody based on SpyCatcher/SpyTag and suggested that the SpyCatcher tag had a facilitating effect on the immunoassay performance enhancement of ZEN/DON anti-idiotype nanobody.
维普
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