Simultaneous detection of OTA and AFB₁ in cereals based on bispecific monoclonal antibody using quantum dot nanobead lateral flow immunoassay

Figures & data

Figure 1. Process of BsMAb generation by hybrid-hybridoma technology. (a) OTA hybridoma cells were mutagenized with different concentration gradients of 8-AG. (b) AFB₁ hybridoma cells were mutagenized with different concentration gradients of 5-BrdU. (c) Tetrasomal hybridoma cells were obtained by fusing the OTA-HGPRT^– cell and AFB₁-TK^– cell.

Scheme 1. Schematic illustration of the BsMAb-based QB-LFIA for the simultaneous detection of OTA and AFB₁. (A) Composition of test strip. (B) Structure of BsMAb. (C) Principle of the BsMAb-based QB-LFIA. (D) Interpretation of different test results.

Figure 2. Characterization of BsMAb. (A) Standard curve for BsMAb detection of OTA. (B) Standard curve for BsMAb detection of AFB₁. (C) Subtype of BsMAb. (D) SDS-PAGE of BsMAb.

Figure 3. Characterization of QB and QB-BsMAb probe. (A) TEM image of individual QB at high magnification. (B) High-resolution TEM image of the QB. (C) Hydrodynamic diameter variations of QB and QB-BsMAb probe. (D) Zeta potential values of QB and QB-BsMAb probe.

Figure 4. Optimization of the key parameters of the BsMAb-based QB-LFIA. (A) Labelling pH (4.0, 4.5, 5, 5.5, and 6.0). (B) Amount of BsMAb (9.0, 10.0, 11.0, 12.0, and 13.0 µg). (C) Amount of EDC (10, 20, 30, 40, and 50 µg). (D) Volume of QB-BsMAb probe (1.0, 1.5, 2.0, 2.5, and 3.0 µL). (E) Concentration of OTA-BSA (0.5, 0.6, 0.7, 0.8, and 0.9 mg/mL). (F) Concentration of AFB₁-BSA (0.6, 0.7, 0.8, 0.9, and 1.0 mg/mL). (G) Immunological kinetic curve of OTA. (H) Immunological kinetic curve of AFB₁.

Figure 5. Standard curves of the BsMAb-based QB-LFIA for the simultaneous detection of OTA and AFB₁ in PBS and cereal samples. (A) PBS spiked with OTA. (B) Soybean spiked with OTA. (C) Corn spiked with OTA. (D) Rice spiked with OTA. (E) PBS spiked with AFB₁. (F) Soybean spiked with AFB₁. (G) Corn spiked with AFB₁. (H) Rice spiked with AFB₁.

Table 1. Calibration curves of the BsMAb-based QB-LFIA for the analysis of OTA and AFB₁.

Analyte	Matrix	Linear equation	R^2	IC50 (μg/kg)^a	Linear range (μg/kg)
OTA	Soybean	y = −1198.0649 lg (x) + 1207.0191	0.9881	0.73	0.1–5
	Corn	y = −1568.7037 lg (x) + 2144.3751	0.9823	1.14	0.5–10
	Rice	y = −1436.5792 lg (x) + 2208.9969	0.9937	1.69	1–10
AFB1	Soybean	y = −505.9941 lg (x) – 148.8251	0.9904	0.01	0.0025–0.25
	Corn	y = −748.8796 lg (x) – 527.4102	0.9997	0.03	0.01–0.1
	Rice	y = −750.7227 lg (x) – 265.9100	0.9873	0.07	0.0025–0.25

^a IC₅₀, half-inhibitory concentration.

Figure 6. Specificity and stability of the BsMAb-based QB-LFIA. (A) Specificity of the BsMAb-based QB-LFIA. Stability results of (B) OTA and (C) AFB₁.

Table 2. Results of recovery experiment (n = 3).

Sample	Spiked (μg/kg)		Measured (μg/kg)		Recovery (%)^a		CV (%)^b
Soybean	OTA	AFB1	OTA	AFB1	OTA	AFB1	OTA	AFB1
	1	0.025	1.1144	0.0262	111.44	104.99	5.16	5.00
	2.5	0.05	2.9764	0.0434	119.06	86.81	3.41	5.63
	5	0.1	5.1129	0.1043	102.26	104.29	1.09	2.50
Corn	OTA	AFB1	OTA	AFB1	OTA	AFB1	OTA	AFB1
	1	0.025	1.2935	0.0205	129.35	82.02	10.41	5.59
	2.5	0.05	2.4705	0.0650	98.82	130.05	1.32	4.23
	5	0.1	5.1668	0.1253	103.34	125.30	1.84	1.86
Rice	OTA	AFB1	OTA	AFB1	OTA	AFB1	OTA	AFB1
	1	0.025	0.8020	0.0201	80.20	80.50	2.41	5.97
	2.5	0.05	2.4776	0.0520	99.10	104.03	10.31	2.37
	5	0.1	5.3390	0.1141	106.78	114.09	4.53	1.62

^a Recovery = (detected concentration/spiked concentration) × 100%.

^b CV, coefficient of variation.

Figure 7. Standard curves of the OTA-based QB-LFIA and AFB₁-based QB-LFIA in PBS and cereal samples. (A) PBS spiked with OTA. (B) Soybean spiked with OTA. (C) Corn spiked with OTA. (D) Rice spiked with OTA. (E) PBS spiked with AFB₁. (F) Soybean spiked with AFB₁. (G) Corn spiked with AFB₁. (H) Rice spiked with AFB₁.

Table 3. Contrast of sensitivity in the proposed method for OTA and AFB₁ detection compared with traditional OTA-based QB-LFIA and AFB₁-based QB-LFIA.

Sample	BsMAb-based QBs-LFIA				OTA-based QBs-LFIA		AFB1-based QBs-LFIA
	OTA detection		AFB1 detection		OTA detection		AFB1 detection
	IC50^a (μg/kg)	Fmax^b /IC50	IC50^a (μg/kg)	Fmax^b /IC50	IC50^a (μg/kg)	Fmax^b/IC50	IC50^a (μg/kg)	Fmax^b/IC50
PBS	1.83	1666.67	0.03	38,219.46	4.35	794.8	0.08	22,391.96
Soybean	0.73	3629.22	0.01	91,017.81	3.21	729.30	0.05	48,733.33
Corn	1.14	3364.12	0.03	41,219.81	2.73	865.27	0.07	39,177.9
Rice	1.69	2118.82	0.07	21,186.83	1.47	1745.30	0.09	20,944.91

^a IC₅₀, half-inhibitory concentration.

^b Fmax is the fluorescence intensity under negative control.