Isolation of atrazine nanobodies enhanced by depletion of anti-carrier protein phages and performance comparison between the nanobody and monoclonal antibody derived from the same immunogen.

Nanobody, a single domain antibody, has been shown a great promise for immunoassay (IA) applications. To improve the panning efficiency so as to obtain a valuable nanobody, anti-carrier protein phages in a phage display library were depleted to enhance the selection of nanobodies against the herbicide atrazine by using immunomagnetic beads conjugated with bovine serum albumin (IMB-BSA). The depletion of anti-carrier protein phages from the atrazine phage display library tripled the number of atrazine positive phage clones after four rounds of panning. One of the most sensitive phage clones Nb3 selected from the IMB-BSA depleted library was used to compare the performance with the monoclonal antibody (mAb 5D9) developed from the same immunogen. The Nb3-based IA exhibited similar specificity with the mAb 5D9-based IA, but greater thermostability and organic solvent tolerance. The half-maximum inhibition concentration (IC) of the former was 3.5-fold greater than that of the latter (36.7 ng/mL versus 10.2 ng/mL). Because the Nb3-based IA was more robust than the mAb 5D9-based IA, the method detection limit of the two assays was 7.8 ng/mL of atrazine in river samples. The depletion strategy can increase the chance to acquire high quality nanobody and can be applicable for effective development of nanobodies against other small molecules.