The platform for the production of nanobody-HRP fusion proteins was constructed as previously described [26 (link)]. PCV2-Cap protein nanobody (Nb15) gene fragment was inserted between the HA tag and HRP sequences of the novel vector pCMV-N1-HRP. The positive recombinant plasmids were confirmed by sequencing and named as RANbodies-PCV2-Nb15-HRP. To produce nanobody-HRP fusion protein, the mammalian cell line HEK 293T cells were transfected with RANbodies-HRP-PCV2-Nb15 plasmid using polyetherimide (PEI, Polysciences Inc. Warrington, USA) reagent. After the cells were transfected for 3 days, the medium containing secreted nanobody-HRP fusion protein was harvested and filtered through 0.45 µm pore cellulose acetate membranes for direct use (Scheme 1a ).
To detect the titer of the nanobody-HRP fusion protein in the medium, the filtered medium was directly used for iELISA. Briefly, the wells of the ELISA plate were coated with 400 ng/well purified PCV2-Cap protein overnight at 4 °C, after blocking and washing, a diluted medium was added and incubated. After washing, tetramethylbenzidine (TMB) was added for a colorimetric reaction at RT for 15 min. Finally, the colorimetric reaction was stopped by adding 3 mol/L H2SO4 (50 µL/well), and the OD450nm values were read using an automated ELISA plate reader (Bio-Rad, USA).
To detect the titer of the nanobody-HRP fusion protein in the medium, the filtered medium was directly used for iELISA. Briefly, the wells of the ELISA plate were coated with 400 ng/well purified PCV2-Cap protein overnight at 4 °C, after blocking and washing, a diluted medium was added and incubated. After washing, tetramethylbenzidine (TMB) was added for a colorimetric reaction at RT for 15 min. Finally, the colorimetric reaction was stopped by adding 3 mol/L H2SO4 (50 µL/well), and the OD450nm values were read using an automated ELISA plate reader (Bio-Rad, USA).
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