关键词: 磁珠 , Cry1Ac毒素 , 单链抗体 , 双抗夹心ELISA

Abstract: 【Objective】By using the biotin-avidin labeled magnetic bead screening strategy, this paper aims at establishing a new type of double-antibody sandwich ELISA detection method for Cry1Ac toxin, so as to build a new detection mode to analyze the sensitive and rapid Bt toxin detection method. 【Method】 The enrichment of Cry1Ac phage-scFvs was investigated after each round of panning (total four rounds) by using magnetic beads with subtractive panning. Positive clones as specific binding to Cry1Ac were selected from the secondary library, which was gained from the fourth round of panning by the method of monoclonal ELISA, and after the PCR identification and DNA sequencing of these positive clones, post-relevant tests were conducted when confirming the complete insertion of the scFvs. Meanwhile, the positive clone, scFv-A12, with relatively best binding activity, was chosen to replace the host strain HB2151 for the purpose of induced-soluble expression and purification. And the purified scFv-A12 was regarded as “capture antibody”, the anti-Cry1Ac rabbit polyclonal antibody as “detection antibody”, thus to establish the detection method of double sandwich ELISA for Cry1Ac.【Result】With the coating antigen of biotinylated Cry1Ac protein, four rounds of panning were conducted by using the antibody phage library. The results show that, along with the increase of panning rounds, the relative productivity was enhanced, and the data of the fourth round was increased by 42 times compared with that of the first round. The positive standard for monoclonal phage ELISA to identify anti-Cry1Ac phage antibody was decided by the figure gained from dividing test hole OD450 by negative hole OD450, which should be over 2.1. Three hundred bacterial clones were selected randomly from the Petri dish after the fourth panning, in which six positive clones were identified by monoclonal ELISA, being respectively named as hsCry1Ac-C5, hsCry1Ac-D8, hsCry1Ac-C12, hsCry1Ac-A12, hsCry1Ac-F9 and hsCry1Ac-F4. Among them, hsCry1Ac-A12 had a relatively high affinity. After the bacteria PCR detection upon above six positive clones, the results show that the target band was obvious in the place of 935 bp, and with the sequencing and amino acid sequence alignment on the target band, four positive clones with different amino acid sequences were gained. After successfully replaced the host strain HB2151, hsCry1Ac-A12 was cultured at 30℃ with 1 mmol•L-1 IPTG for inducing expression. Through the His Trap HP nickel affinity column purification and the SDS-PAGE identification of its expression product, the molecular weight of hsCry1Ac-A12 protein was approximately 30 kD. After optimizing the antibody concentration by titration test and taking advantage of the single chain antibody as “capture antibody”, polyvalent antibody as “detection antibody”, the detection method of double antibody sandwich ELISA was established. Within the linear range of 1.25-2.43 μg•mL-1, the linear regression equation was Y=0.2522X+1.2832(R2=0.9564), with the limit of detection 1.08 ng•mL-1.【Conclusion】This paper adopted the method of magnetic beads with subtractive panning, the detection method of double-antibody sandwich ELISA for Cry1Ac toxin was established after the expression and purification of the selected clone A12 with best binding activity which provide a new detection mode for detecting the Cry1Ac contained in GMF rapidly and accurately.

Key words: Cry1Ac toxin , single-chain variable fragments , magnetic beads , sandwich ELISA