Development of a polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay to detect dufulin residue in water, soil and agricultural samples

Figures & data

Figure 1. Structures of dufulin and hapten for dufulin (DHS)

Figure 2. UV scanning spectrums of hapten–DHS (a), hapten–DHS–BSA conjugate (b), BSA (c), OVA (d) and hapten–DHS–OVA (e).

Table 1. Summary of titres^a of antisera and competitive inhibition of DFL.

Immunoge^b	Rabbits no.	Boosting					IC50 (ng ml^−1)
		1st	2nd	3rd	4th	5th
DHS–BSA	1#	1:4000	1:10000	1:40,000	1:80,000	1:160,000	133
	2#	1:4000	1:20000	1:80,000	1:160,000	1:240,000	128

^aThe titre of antiserum is defined as the antiserum dilution that gave 2.1 times absorbance of the control serum.

^bDHS–OVA as coating antigen for antisera immunized by DHS–BSA, 2 μg ml⁻¹ for both coating antigens, 1/10,000 dilution for goat anti-rabbit HRP-IgG.

Figure 3. Optimization of parameters of ic-ELISA: (a) the influence of different contents of methanol in PBS buffer; (b) the influence of different pH value of assay buffer; (c) Effect of ionic strength in PBS buffer and (d) ELISA standard curve for DFL by indirect competitive assay.

Table 2. The antibody specificity and CR.

Competitors	IC50 (ng ml^−1)	CR (%)	Structure
Dufulin	48.5	100
Phoxim	>10^4	/
Parathion	>10^4	/
Fenthion	>10^4	/
Fenitrothion	>10^4	/
Chlorpyrifos	>10^4	/
6-aminohexanoic acid	4.8 × 10^3	1.01

Note: / means no inhibition (CR lower than 0.5%).

Table 3. Recoveries of spiked real samples determined by ELISA and HPLC (n = 3)^a.

Sample	Spiked level (μg g^−1)	IC-ELISA		HPLC
		Recovery (%)	RSD (%)	Recovery (%)	RSD (%)
Water	1.0	85.5	5.3	88.3	3.8
	0.1	81.8	6.9	96.5	4.5
	0.05	93.4	2.9	84.5	3.0
	0.01	79.1	5.5	78.2	4.9
Soil	1.0	92.3	9.5	83.5	7.2
	0.1	87.7	5.6	87.6	3.5
	0.05	89.5	6.9	92.1	3.2
	0.01	99.0	12.1	70.2	10.8
Rice	1.0	98.8	10.7	70.6	5.5
	0.1	105.1	5.2	82.2	3.8
	0.05	99.2	9.7	72.1	6.1
	0.01	89.5	8.4	ND^b	/
Tobacco	1.0	98.8	15.8	98.5	9.7
	0.1	101.3	18.6	112.0	13.2
	0.05	119.2	9.5	101.8	10.1
	0.01	138.8	14.2	ND	/
Tomato	1.0	70.7	9.6	77.3	10.5
	0.1	81.5	5.4	68.5	4.8
	0.05	89.2	8.2	72.3	5.9
	0.01	95.1	10.0	ND	/
Cucumber	1.0	102.0	11.1	75.9	8.3
	0.1	93.5	7.2	70.1	5.5
	0.05	103.3	13.9	77.7	9.9
	0.01	99.8	9.4	ND	/

Note: / means no value of relative standard deviation (RSD).

^aData obtained from three determinations.

^bND: not detected.