Abstract:
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, and 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 IC
50 of the competition inhibition standard curves established based on Z6-SpyCatcher and Z6-SpyTag were 0.18 and 0.20 ng/mL, respectively, and their sensitivities were significantly higher than that of the IC
50 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 IC
50 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.