Development of a highly sensitive biotin–streptavidin enzyme-linked immunosorbent assay for detecting diethyl phthalate based on a specific polyclonal antibody

Figures & data

Figure 1. The UV spectra of DEP hapten, protein and conjugates; absorbance value at characteristic peak, 336 nm: OD_BSA-DEP = 0.630, OD_{DEP hapten} = 0.138, OD_BSA = 0.005; 360 nm: OD_OVA-DEP = 0.424, OD_{DEP hapten} = 0.020, OD_OVA = 0.154; C_BSA: 0.25g·L⁻¹, C_OVA: 0.28g·L⁻¹, C_hapten: 0.05g·L⁻¹; protein and conjugate were dissolved in PBS buffer; hapten was dissolved in DMF.

Table 1. Optimal concentrations of Bio-pAb-DEP and OVA-DEP.

	The concentration of OVA-DEP (μg·mL^−1)
Dilutions of Bio-pAb-DEP^a	41.88	20.94	8.38	4.19	2.09	1.05
100	1.473	1.332	1.296	1.221	1.269	1.371
200	1.295	1.134	1.052	1.140	1.175	1.198
500	0.806	0.942	0.872	0.891	0.860	0.939
1000	0.534	0.693	0.642	0.731	0.635	0.626
2000	0.363	0.556	0.383	0.506	0.340	0.446
3000	0.306	0.329	0.318	0.436	0.265	0.351
Blank	0.093	0.097	0.090	0.125	0.112	0.095

^a The concentration of Bio-pAb-DEP was 0.971 mg·mL⁻¹.

Figure 2. Suitable operating conditions of the immunoassay method: (a) the blocking reagent, (b) ionic strength in PBS buffer, (c) pH of buffer, (d) concentrations of SA-HRP, (e) incubation time, and (f) the influence of different volume percentages of solvent on PBS buffer.

Figure 3. Standard curve for DEP analyzed by BA-ELISA. The concentrations of DEP were 0.001 μg·L⁻¹, 0.005 μg·L⁻¹, 0.01 μg·L⁻¹, 0.05 μg·L⁻¹, 0.1 μg·L⁻¹, 0.5 μg·L⁻¹, 1 μg·L⁻¹, 2.5 μg·L⁻¹, 5 μg·L⁻¹, 10 μg·L⁻¹, 25 μg·L⁻¹, 50 μg·L⁻¹. The linear range was from 0.021 μg·L⁻¹ to 9.512 μg·L⁻¹. The linear equation was Y = 22.53LogC_DEP + 57.96 (R² = 0.9832, n = 16).

Table 2. The variability of intra-assay and inter-assay test.

	DEP concentration (μg·L^−1)
Object	0.05	0.1	0.5	1	2.5	5
Intra-assay CVs (%)	10.55	9.25	8.65	8.48	6.83	5.64
Inter-assay CVs (%)	12.67	11.38	11.21	9.79	8.98	7.26

Table 3. CR of Bio-pAb-DEP with DEP structural analogs.

Analogs	Structure	IC50(μg·L^−1)	CR (%)
DEP		0.443	100
DMP		9.197	4.82
DPrP		9.028	4.91
DBP		10.357	4.28
DIBP		18.704	2.37
DEHP		14.447	3.06
DINP		32.815	1.35
4-DENP		3.051	14.52
4-DEAP		2.788	15.89

Figure 4. Comparison of data in wine samples by the BA-ELISA and GC–MS analysis. The regression equations was Y = 0.81X + 0.76 (R² = 0.9748).

Table 4. Concentration of DEP in wine samples by BA-ELISA and GC–MS.

		Concentration (mean ± SD) (μg·L^−1) (n = 6)
Samples		BA-ELISA	GC–MS
Beer	B1	5.93 ± 0.068	5.72 ± 0.021
	B2	7.68 ± 0.052	6.15 ± 0.034
	B3	8.07 ± 0.082	7.03 ± 0.019
Red wine	R1	12.82 ± 0.091	12.37 ± 0.028
	R2	13.11 ± 0.097	11.39 ± 0.019
	R3	12.06 ± 0.115	10.96 ± 0.030
White liquor	W1	59.15 ± 0.099	45.88 ± 0.033
	W2	53.93 ± 0.107	48.12 ± 0.046
	W3	47.06 ± 0.091	43.14 ± 0.036
Yellow rice wine	Y1	29.65 ± 0.082	23.77 ± 0.024
	Y2	49.31 ± 0.073	39.98 ± 0.025
	Y3	43.37 ± 0.083	30.56 ± 0.019

Table 5. Recovery of DEP detected by BA-ELISA and GC–MS in spiked wine samples.

Samples		Sample concentration (μg·L^−1)	Spiked level (μg·L^−1)	Average recovery % and CV % (n = 6)
				BA-ELISA	GC–MS
Beer	B1	5.93	1	106.65 ± 6.34	105.48 ± 4.61
			10	101.32 ± 5.81	100.06 ± 3.73
			20	95.14 ± 8.05	90.27 ± 2.69
White liquor	W1	59.15	20	97.18 ± 7.39	107.25 ± 3.67
			50	112.33 ± 8.76	99.94 ± 5.50
			100	89.19 ± 9.43	92.61 ± 4.97