Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of folic acid in energy drinks and milk samples

Figures & data

Figure 1. (A) Principle of the Ic-ELISA analysis; (B) composition of the lateral-flow ICA strip; and (C) lateral-flow ICA strip detection with negative and positive sample.

Figure 2. The UV–Vis spectra of different antigens. (A) Antigens with BSA as carrier protein; and (B) antigens with OVA as carrier protein.

Figure 3. The SDS-PAGE analysis of different antigens. (A) Antigens with BSA as carrier protein: 1. BSA, 2. BSA-ACA, 3. FA-BSA-1, 4. FA-ACA-BSA-2, 5. FA-BSA-3; and (B) antigens with OVA as carrier protein: 1. OVA, 2. OVA-ACA, 3. FA-OVA-1, 4. FA-ACA-OVA-2, 5. FA-OVA-3.

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

	FA-OVA-1		FA-ACA-OVA-2		FA-OVA-3
Antigens	Titer	IC50 (ng/ml)	Titer	IC50 (ng/ml)	Titer	IC50 (ng/ml)
FA-BSA-1	243,000	10.2	27,000	14.3	243,000	2.4
FA-ACA-BSA-2	9000	20.5	27,000	32.8	9000	7.4
FA-BSA-3	81,000	5.3	27,000	9.6	243,000	11.7

Figure 4. The standard curve of developed Ic-ELISA method.

Table 2. The CR value against FA by the Ic-ELISA method.

Analytes	IC50 (ng/ml)	CR (%)
Folic acid	0.12	100
Pterine	200	0.6
Pteroic acid	0.8	15.
Dihydrofolic acid	2.1	5.7
Tetrahydrofolic acid	1.6	7.5
Methotrexate	>2000	<0.1
5-Methyltetrahydrofolic acid	>2000	<0.1
Vb1	400	0.3
Vb2	>2000	<0.1
Vb3	>2000	<0.1
Vb5	>2000	<0.1
Vb6	>2000	<0.1
Vb7	>2000	<0.1
Vb12	>2000	<0.1

Figure 5. The optimization of the lateral-flow ICA strip. (A) Strip with coating antigen at 0.5 mg/ml: 1. GNP-labeled mAb concentration of 8 µg/ml (pH 8.0); 2. GNP-labeled mAb concentration of 10 µg/ml (pH 8.0); 3. GNP-labeled mAb concentration of 8 µg/ml (pH 9.0); 4. GNP-labeled mAb concentration of 10 µg/ml (pH 9.0); (B) strip with coating antigen at 1 mg/ml: (1) GNP-labeled mAb concentration of 8 µg/ml (pH 8.0); (2) GNP-labeled mAb concentration of 10 µg/ml (pH 8.0); (3) GNP-labeled mAb concentration of 8 µg/ml (pH 9.0); and (4) GNP-labeled mAb concentration of 10 µg/ml (pH 9.0); (C) Optimization of two different combinations: (1) coating antigen at 0.5 mg/ml with a GNP-labeled mAb concentration of 10 µg/ml (pH 8.0); (2) coating antigen at 1 mg/ml with a GNP-labeled mAb concentration of 8 µg/ml (pH 8.0); and (D) optimization of resuspension solution for sample pad in 0.01 M PBS with 0.2% Tween-20: (1) 1% PEG 20000; (2) 1% OVA; (3) 1% BSA; and (4) 1% skim milk powder (w/w). N, FA-negative sample (0 ng/ml); P, FA-positive sample (2.5 ng/ ml).

Figure 6. FA detection by lateral-flow ICA strip. (A) The ultrapure water samples: (1) 0 ng/ml; (2) 0.25 ng/ml; (3) 0.5 ng/ml; (4) 1 ng/ml; (5) 2.5 ng/ml; and (6) 5 ng/ml; (B) the milk samples (before detection, samples were diluted 50×): (1) 0 µg/100 ml; (2) 1.25 µg/100 ml; (3) 2.5 µg/100 ml; (4) 5 µg/100 ml; (5) 12.5 µg/100 ml; and (6) 25 µg/100 ml.

Table 3. The sample analysis with Ic-ELISA method and lateral-flow ICA strip (n = 7).

Analysis samples	Microbiological assay method (µg/100 ml)	Ic-ELISA method (µg/100 ml)	ICA strip assay
M1	2.76 ± 0.091^a	2.36 ± 0.061	−^b
M2	3.62 ± 0.124	3.16 ± 0.15	±^c
M3	2.47 ± 0.087	1.81 ± 0.061	−
M4	5.71 ± 0.11	4.77 ± 0.19	±
E1	98.6 ± 2.0	88.1 ± 3.4	+^d
E2	141.1 ± 3.1	131.4 ± 6.8	+
E3	55.2 ± 3.2	52.1 ± 2.2	±
E4	76.8 ± 4.9	75.2 ± 5.2	+

^aMean value ± standard deviation (n = 7).

^bNegative result. The test line is obviously observed.

^cWeakly positive result. Light test line is observed.

^dPositive result. No test line is observed.