Phage-displayed nanobody based double antibody sandwich chemiluminescent immunoassay for the detection of Cry2A toxin in cereals

Figures & data

Figure 1. (a) Number of phage output in each round of panning. (b) Identification the positive clones binding to Cry2A toxin by phage-ELISA. NC, negative control.

Figure 2. (a) The amino acid sequences of the positive clones. (b) Result of matching between nanobodies and anti-Cry2A PAb detected by sandwich ELISA. NC, negative control.

Figure 3. Optimization of (a) capture antibody (anti-Cry2A PAb) and (b) detection antibody (phage-displayed nanobody P2) concentrations.

Figure 4. Standard curve of phage-displayed nanobody based DAS-CLIA for Cry2A toxin analysis under the optimized conditions.

Figure 5. Cross-reactivity of the DAS-CLIA with other Bt toxins.

Table 1. Recovery analysis of Cry2A in rice, wheat, and corn samples by DAS-CLIA.

Matrix	Spike level (μg/kg)	DAS-CLIA (n = 3^a)
		Mean ± SD (μg/kg)	Recovery (%)	CV (%)
Rice	5	5.3 ± 0.3	106	5.6
	50	48.3 ± 4.5	96.6	9.3
	500	468 ± 36.7	93.6	7.8
	5000	4137 ± 184	82.7	4.4
Wheat	5	5.6 ± 0.5	112	8.9
	50	58.8 ± 4.0	118	6.8
	500	452 ± 31	90.4	6.9
	5000	4425 ± 236	88.5	5.3
Corn	5	5.5 ± 0.4	110	7.3
	50	44.8 ± 3.6	89.6	8.1
	500	475.4 ± 21.4	95.1	4.5
	5000	4267 ± 209	85.3	4.9

^aEach assay was performed thrice on the same day, and each reading was taken by the same person.