Development of an enzyme-linked immunosorbent assay for quantification of estriol in milk

Figures & data

Figure 1. Synthetic route for (A) haptens A and (B) haptens B of E3.

Figure 2. (A) Calibration curve and (B) ciELISA calibration curve in the linear range for histamine (n = 3).

Table 1. Cross-reactivity of related compounds in ciELISA under optimised conditions

Compound	Structure	IC50 (ng mL^−1)	CR^a (%)
estriol		2.55	100%
estradiol		>10000	>0.1
oestrone		>10000	>0.1
progesterone		>10000	>0.1
ethinylestradiol		>10000	>0.1

^aCR, cross-reactivity. The percentage of CR was calculated by the following equation: CR (%) = [IC₅₀ (estriol, ng mL⁻¹)/IC₅₀ (cross-reactant, ng mL⁻¹)] × 100.

Figure 3. Analysis of matrix effect by different sample pretreatment: (A) centrifugalising method; (B) zinc acetate -potassium ferrocyanide method; and (C) dilution method.

Table 2. Results of the determination of E3 in spiked milk samples with different pretreatment method under optimised conditions (n = 3).

Sample pretreatment	Spiked (ng mL^−1)	Measured (ng mL^−1)			Average Recovery (%)	CV (%)
centrifugalising method	10.0	6.5	8.9	11.3	88.9	14.7
	20.0	22.2	21.0	21.0	106.7	3.2
	40.0	37.1	45.6	50.8	111.2	16.2
zinc acetate -potassium ferrocyanide method	10.0	9.1	9.0	9.4	91.6	2.7
	20.0	25.5	25.2	24.9	125.8	1.1
	40.0	33.2	32.1	34.5	83.2	3.1
dilution method	10.0	8.9	7.9	8.1	83.2	6.6
	20.0	18.2	19.0	21.7	98.2	9.4
	40.0	45.0	42.4	45.7	110.1	4.2

Figure 4. Correlation of HPLC results with ELISA results.