Using the hybridoma cells 2D3 which secrete anti-Cry1 monoclonal antibody as DNA source, the variable region genes of heavy chain (V_H) and light chain (V_L) 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 V_L was the main combination domain of scFv-2D3 with Cry1 toxins. Therefore, V_H and V_L were separately expressed and then assayed by indirect ELISA, indicating that both V_H and V_L could recognize the Cry1 toxin, and the activity of V_L was higher than that of V_H, which confirming the molecular docking result. Afterward, double light chain antibodies (V_L-V_L) were designed and produced, and the binding activities of V_L-V_L 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 V_L-V_L-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.

以分泌抗Cry1单克隆抗体的杂交瘤细胞2D3为DNA源，扩增了重链（V _H）和轻链（V _L）的可变区基因，并组装成单链可变片段（scFv）。将scFv-2D3基因克隆到pET-26b载体中进行表达，并通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western blot（WB）鉴定scFv-2D3表达。scFv-2D3的活性通过间接酶联免疫吸附测定（ELISA）确定。结果，成功构建并表达了scFv-2D3，并具有针对Cry1毒素的特异性识别活性。同源建模和分子对接结果表明，V_大号是scFv-2D3与Cry1毒素的主要结合域。因此，V _H和V _L分别表达，然后通过间接ELISA检测，表明V _H和V _L都可以识别Cry1毒素，并且V _L的活性高于V _H，这证实了分子对接结果。此后，设计并生产了双轻链抗体（V _L -V _L），OD ₄₅₀显着增强了V _L -V _L对Cry1Aa，Cry1Ab和Cry1Ac的结合活性。与scFv-2D3相比，该值分别提高了1.18、1.44和1.22倍。对于基于V _L -V _L的双抗体夹心ELISA（DAS-ELISA），七种Cry1毒素的检出限（LOD）和定量限（LOQ）分别为10.5-16.0和12.8-21.5 ng mL ^-1，低于scFv-2D3（14.8–29.2和39.0–50.3 ng mL ^-1）。该结果有助于开发高通量和高灵敏度的Cry1毒素免疫检测技术。