Construction, Expression, and Identification of Double Light Chain (VL-VL) Antibody from a Unique Bt Cry1-Specific Monoclonal Antibody

Abstract

Using the hybridoma cells 2D3 which secrete anti-Cry1 monoclonal antibody as DNA source, the variable region genes of heavy chain (VH) and light chain (VL) were amplified and assembled as single-chain variable fragment (scFv). The scFv-2D3 genes were cloned into pET-26b vectors for expression and scFv-2D3 expression was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot (WB). The activity of scFv-2D3 was determined by indirect enzyme-linked immune sorbent assay (ELISA). As a result, scFv-2D3 was successfully constructed and expressed, with specific recognition activities to the Cry1 toxins. Homologous modeling and molecular docking results showed that VL was the main combination domain of scFv-2D3 with Cry1 toxins. Therefore, VH and VL were separately expressed and then assayed by indirect ELISA, indicating that both VH and VL could recognize the Cry1 toxin, and the activity of VL was higher than that of VH, which confirming the molecular docking result. Afterward, double light chain antibodies (VL-VL) were designed and produced, and the binding activities of VL-VL against Cry1Aa, Cry1Ab, and Cry1Ac were significantly enhanced with OD450 values improved 1.18, 1.44, and 1.22 times, respectively, compared with scFv-2D3. For VL-VL-based double antibody sandwiched ELISA (DAS-ELISA), the limits of detection (LOD) and limits of quantification (LOQ) were 10.5–16.0 and 12.8–21.5 ng mL−1 for seven Cry1 toxins, respectively, which were lower than scFv-2D3 (14.8–29.2 and 39.0–50.3 ng mL−1). The results were beneficial to developing high-throughput and high-sensitive immune-detecting technology for Cry1 toxins.