Generation of panels of anti-idiotypic single-chain variable fragments mimicking Cry2Aa toxin using the chain shuffling technique

Figures & data

Table 1. Sequence of the PCR primers used for construction of chain-shuffled libraries^a.

Primer	Sequence
Primer 1	CAG GAA ACA GCT ATG AC
Primer 2	CGA CCC GCC ACC GCC GCT G
Primer 3	C AGC GGC GGT GGC GGG TCG
Primer 4	CTA TGA GGC CCC ATT CA

^aComplementary sequences for SOE-PCR are in bold.

Figure 1. Analysis of amplified VH, VL and SOE-PCR products by agarose gel electrophoresis. (A) 1:DNA ladder, from top to bottom, 2000,1000,750, 500, 250, 100 bp; 3–4:VL (Tomlinson I); 6:VH (B10); 7: VH (F2). (B) 1: DNA ladder, from top to bottom, 2000, 1000, 750, 500, 250, 100 bp 3: VL (Tomlinson I)-VH (B10); 4: VL (Tomlinson I)-VH (F2); 5: VL (B10)-VH (Tomlinson I); 6: VL (F2)-VH (Tomlinson).

Table 2. Protein sequences of parental clones and mutants derived from chain-shuffled libraries^a.

	CDR-H2	CDR-H3	CDR-L2	CDR-L3
B10 (parental clone)	IAYAGSNTN	KNSSGFD	YAASSL	CQQTSTTPY
2H1	ISSTGAYTSN	KADASFD	YAASSL	CQQTSTTPY
MUT10	IAYAGSNTN	KNSSGFD	YAASSL	CQQYSSNPS
MUT19	IAYAGSNTN	KNSSGFD	YAASDL	CQQTSTTPY
MUT21	IAYAGSNTN	KNSSGFD	YAASSL	CQQGYATPY
F2 (parental clone)	IDSYGTNTD	KAFNSFD	YAASAL	CQQYSSSPS
2F10	IDSYGTNTD	KAFNSFD	YAASDL	CQQYSSNPS
3B1	IDSYGTNTD	KAFNSFD	YAASDL	CQQAAAYPS
3F1	CDRs of 3F1 were identical with F2. Mutation took place in K171E in framework and a missing G120 in linker
3H11	CDRs of 3H11 were identical with F2. Mutation took place in K171E, Q221R, T234S in framework and a missing G120 in linker

^aIn grey is shown the amino acid positions that were mutated during chain shuffling.

Figure 2. Determination of the apparent affinity of mutants and parental clones by competitive phage ELISA (n = 3). B/B₀ stands for the ratio ELISA mean absorbance values of antibody-binding response in the presence of free antigen to the absence of antigen. Various concentrations of free antigen inhibited the binding of phage antibodies with the coating antigen, the signal was detected by using HRP-conjugated anti-M13 monoclonal antibodies. The calibration curves were constructed by using a four-parameter equation by Sigmaplot 12.0.

Figure 3. Determination of binding ability of anti-idiotypic phage antibodies with BBMV of Plutella xylostella larvae by ELISA (n = 3). For each clones, 10⁶ phages/well were added to ELISA plate coated with BBMV and detected by HRP-conjugated anti-M13 monoclonal antibodies. Blank is the mean signal of wells which had been added PBS instead of phage antibodies. BSA is an irrelevant phage antibody from Tomlinson (I + J) library which recognizes BSA.