Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of citrinin in cereals

Figures & data

Figure 1. Principle of the lateral-flow immunochromatographic strip detection. (A) Composition of the lateral-flow immunochromatographic strip. (B) Strip detection with negative sample and positive sample.

Figure 2. The UV-Vis spectra of different antigens (CIT-BSA/CIT-OVA).

Table 1. The evaluation of different immunogens and coating antigens.

Antigens	CIT-OVA-A1		CIT-OVA-A2		CIT-OVA-A3
	Titer	IC50 (ng/mL)	Titer	IC50 (ng/mL)	Titer	IC50 (ng/mL)
CIT-BSA-A1	–	–	9000	–	–	–
CIT-BSA-A2	–	–	243,000	–	9000	98.9
CIT-BSA-A3	–	–	27,000	–	243,000	26.7

Figure 3. The optimization of ic-ELISA. (A) The optimization of ic-ELISA method with different methanol content; (B) The optimization of ic-ELISA method with different ionic strength (NaCl content); (C) The optimization of ic-ELISA method with different pH values; and (D) The standard curve established under the optimum condition.

Table 2. The CR value of mAb against CIT by the ic-ELISA.

Analytes	IC50 (ng/mL)	CR (%)
Citrinin	0.761	100
Aflatoxin B1	>1000	<0.1
Aflatoxin M1	>1000	<0.1
Deoxynivalenol	>1000	<0.1
Zearalenone	>1000	<0.1

Figure 4. The analysis of lateral-flow ICA strip by naked eyes. (A) The sensitive analysis with CIT: (1) 5 ng/ml; (2) 2.5 ng/ml; (3) 1 ng/ml; (4) 0.5 ng/ml; (5) 0.25 ng/ml; (6) 0 ng/ml; (B) The CIT-spiked maize sample: (1) 80 ng/g; (2) 40 ng/g; (3) 20 ng/g; (4) 8 ng/g; (5) 4 ng/g; (6) 0 ng/g; (C) The CIT-spiked wheat sample: (1) 80 ng/g; (2) 40 ng/g; (3) 20 ng/g; (4) 8 ng/g; (5) 4 ng/g; (6) 0 ng/g; and (D) The CIT-spiked rice sample: (1) 80 ng/g; (2) 40 ng/g; (3) 20 ng/g; (4) 8 ng/g; (5) 4 ng/g; (6) 0 ng/g.

Figure 5. The analysis of lateral-flow ICA strip by strip scan reader. (A) The standard curve of ICA strip by strip scan reader; (B) The calibration curve of ICA strip by strip scan reader with different sample.

Figure 6. The calibration curve of ic-ELISA with different sample.

Table 3. The spiked sample analysis with ic-ELISA and ICA strip (n = 6).

Samples	Spiked (ng/g)	Result (ng/g)	Recovery (%)	Spiked samples	Spiked (ng/g)	Result (visual)	Result (ng/g)	Recovery (%)
	4	4.72 ± 0.22^a	118		4	-^b	4.83	121
Maize	8	9.85 ± 0.37	123	Maize	20	± ^c	25.5	127
	16	18.49 ± 1.02	116		80	+ ^d	− ^e	−
	4	4.71 ± 0.19	118		4	-	4.74	119
Wheat	8	9.73 ± 0.46	122	Wheat	20	±	24.8	124
	16	19.49 ± 0.97	122		80	+	−	−
	4	4.77 ± 0.29	119		4	-	4.41	110
Rice	8	9.65 ± 0.48	121	Rice	20	±	23.7	119
	16	17.85 ± 0.79	112		80	+	−	−

^aMean value ± standard deviation (n = 6).

^bNegative result. The test line is obviously observed.

^cWeakly positive result. Light test line is observed.

^dPositive result. No test line is observed.

^eWithout result.