Development of a broad-selective immunoassay for multi-residue determination of type II pyrethroids in West Lake water

Figures & data

Figure 1.  Structure of the tested pyrethroids and relative metabolites.

Figure 2.  Synthetic scheme for the preparation of hapten 2 and hapten 8.

Table 1. Structure of nine haptens.

Hapten^a	Structure
1
2
3
4
5
6
7
8
9
^aHapten 2, 8 and 9 with -CN were designed as immunising haptens, and all the nine haptens were designed as coating antigens.

Table 2. Summary of optical density of titration tests.

Antisera/CAg^a	Pab-2	Pab-8	Pab-9
OVA^b	0.008	0.007	0.008
CAg-1	0.669	0.364	0.493
CAg-2	1.221	1.083	1.339
CAg-3	0.553	0.432	0.231
CAg-4	0.389	0.163	0.105
CAg-5	0.845	0.711	0.678
CAg-6	0.182	0.117	0.130
CAg-7	1.153	0.702	0.454
CAg-8	1.492	1.553	1.236
CAg-9	0.986	1.330	1.619
^aThe data shown are derived from assays individually performed at an antibody dilution of 1:16,000 and CAg concentration of 16 µg/ml.
^bAbsence of the CAg in well but coated just with OVA was set as a control, all the values are the average of three reduplicates.

Table 3. Selected competitive ELISA screening data against type I and type II pyrethroids.

	Tetramethrin	Permethrin	Cypermethrin	Esfenvalerate
CAgs/antibodies	IC50 (µg/L)^a
CAg-2
Pab-2	>1000	789	776	945
Pab-8	>1000	>1000	746	>1000
Pab-9	>1000	>1000	678	>1000
CAg-5
Pab-2	>1000	>1000	568	>1000
CAg-7
Pab-2	>1000	891	56.4	60.2
CAg-8
Pab-2	>1000	>1000	497	611
Pab-8	>1000	812	659	716
Pab-9	>1000	>1000	656	786
CAg-9
Pab-2	>1000	>1000	833	>1000
Pab-8	>1000	>1000	>1000	>1000
Pab-9	>1000	>1000	902	>1000
^aAll the pyrethroids standards were prepared in water containing 10% methanol, and buffer for ELISA was 0.01 M, pH=7.4 PBS, antibody dilution of 1:16,000 and CAg concentration of 16 µg/ml. IC50 values of the four tested pyrethroids are shown in the table.

Figure 3.  Effect of the methanol concentration on ELISA performance of cypermethrin. (Δ) value of IC50 for cypermethrin; (⊕) absorbance in the absence of cypermethrin (Amax); (▪) value of Amax/IC50. Each point represents the average of three replicates.

Figure 4.  Effect of ionic strength on ELISA performance of cypermethrin. Other details as for Figure 3.

Figure 5.  Effect of pH on ELISA performance of cypermethrin. Other details as for Figure 3.

Figure 6.  Standard curves of the seven type II pyrethroids. These linear trendlines are obtained by Microsoft Office Excel 2007 using linear regression analysis. Each point represents the average of three replicates.

Table 4. Cross-reactivities (CRs) of pyrethroids and relative metabolites.

		Standard equation parameters^a
CAg/antibody	Pyrethroids	Slope	Intercept	R ^2	IC50 (µg/L)^b	CR (%)^c
CAg-7/Pab-2	Cypermethrin	19.4	−11.4	0.99	23.2±2.8	100.0
	Deltamethrin	17.8	−7.0	0.98	24.5±1.9	105.6±8.2
	Esfenvalerate	18.7	−10.2	0.99	25.1±1.1	108.2±4.7
	Fenpropathrin	18.1	1.7	0.98	14.5±1.6	62.5±6.9
	Fluvalinate	15.8	−16.2	0.98	66.1±3.4	284.9±14.7
	Flucythrinate	15.5	−16.4	0.98	72.4±2.7	312.1±11.6
	Cyhalothrin	16.2	−12.3	0.99	46.8±1.7	201.7±7.3
	Phenothrin				>500	>2000
	Permethrin				>500	>2000
	Tetramethrin				>1000	>5000
	Allethrin				>1000	>5000
	Bifenthrin				>1000	>5000
	3-PBA				>1000	>5000
	Cypermethric acid				>1000	>5000
	Fenpropathric acid				>1000	>5000
	Deltamethric acid				>1000	>5000
	Cyhalthric acid				>1000	>5000
	Fenvalerate acid				>1000	>5000
^aThe standard equation was defined as: y=aln(x) + b (obtained from Microsoft Office Excel 2007, using linear regression model).
(B-B0/B0)% was set as y, and logarithm of concentration of pyrethroid (µg/L) was set as x.
^bIC50 values were obtained under optimal conditions. Values are the means and standard deviations of three experiments.
^cCR=[IC50 (other compounds)/IC50 (cypermethrin)]×100%.

Table 5. Recoveries of single residue of cypermethrin, deltamethrin and esfenvalerate in West Lake water samples calculated by the standard curve of cypermethrin.^a

Standard curve for calculation	Analyte^b	Spiked (µg/L)	Measured values (µg/L)^c			Average values (µg/L)^d	Average recoveries (%)^e	CV (%)
Cypemethrin	Control	0	0.19	0.22	0.26	0.22±0.04
	cyp	5	4.13	4.27	4.32	4.2±0.1a	84.8±2.0	2.3
	del		4.25	4.52	4.16	4.3±0.2a	86.2±3.7	4.3
	esf		4.47	4.31	4.54	4.4±0.1a	88.8±2.4	2.7
	cyp	20	19.67	19.92	18.70	19.4±0.6a	97.2±3.2	3.3
	del		19.69	19.85	18.33	19.3±0.8a	96.5±4.2	4.3
	esf		19.06	19.66	17.41	18.7±1.2a	93.6±5.8	6.2
	cyp	50	50.11	49.32	51.26	50.2±1.0a	100.4±2.0	2.0
	del		50.20	48.72	51.31	50.1±1.3a	100.1±2.6	2.6
	esf		50.01	48.21	52.19	50.1±2.0a	100.3±4.0	4.0
^aIn this table recovery data was calculated using the standard curve of the pyrethroid as show below.
^bControl means the corresponding sample spiked no pyrethroid, and use the same treatment to examine the sample background value. Cyp, del and esf stand for cypermethrin, deltamethrin, esfenvalerate, respectively.
^cData were derived from determinations of three samples at each spike level, and each measured values have already deduct the control values.
^dData are composed of mean average detection values and standard deviation, the bold letter behind means 5% significance level (analysis by Duncan's new multiple range method) among cypermethrin, deltamethrin and esfenvalerate at each spiked concentration.
^eData are composed of mean average recovery values and standard deviation.

Table 6. Recoveries of single residue of cypermethrin, deltamethrin and esfenvalerate in West Lake water samples calculated by the standard curve of deltamethrin.^a

Standard curve for calculation	Analyte	Spiked (µg/L)	Measured values (µg/L)			Average values (µg/L)	Average recoveries (%)	CV (%)
Deltamethrin	Control	0	0.31	0.22	0.26	0.26±0.05
	cyp	5	4.76	4.84	4.47	4.4±0.3a	88.2±7.0	7.9
	del		4.87	5.02	4.75	4.2±0.3a	84.0±5.1	6.0
	esf		4.91	4.87	4.68	4.4±0.2a	87.4±3.7	4.2
	cyp	20	20.31	21.22	18.23	19.9±1.6a	99.6±7.7	7.7
	del		20.34	20.51	19.75	20.2±0.4a	101.0±2.0	2.0
	esf		19.74	21.53	19.33	20.2±0.8a	101.0±5.8	5.8
	cyp	50	50.23	49.86	49.82	50.0±0.2a	99.9±0.5	0.5
	del		51.53	50.92	48.45	50.3±1.6a	100.6±3.3	3.2
	esf		50.47	50.61	54.02	51.7±2.0a	103.4±4.0	3.9
^aOther details as for Table 5.

Table 7. Recoveries of single residue of cypermethrin, deltamethrin and esfenvalerate in West Lake water samples calculated by the standard curve of esfenvalerate.^a

Standard curve for calculation	Analyte	Spiked (µg/L)	Measured values (µg/L)			Average values (µg/L)	Average recoveries (%)	CV (%)
Esfenvalerate	Control	0	0.25	0.18	0.24	0.22±0.04
	cyp	5	4.64	4.15	3.93	4.2±0.4a	84.8±7.3	8.6
	del		4.05	4.35	4.14	4.2±0.2a	83.6±3.1	3.7
	esf		4.17	4.69	4.01	4.3±0.4a	85.8±7.1	8.3
	cyp	20	20.22	19.77	17.28	19.1±1.6a	95.5±7.9	8.3
	del		19.73	19.85	19.88	19.8±0.1a	99.1±0.4	0.4
	esf		19.7	19.52	20.09	19.8±0.3a	98.9±1.5	1.5
	cyp	50	50.21	49.82	49.76	49.9±0.2a	99.8±0.5	0.5
	del		51.52	50.92	55.79	52.7±2.7a	105.5±5.3	5.1
	esf		49.83	50.61	51.91	50.8±1.1a	101.5±2.1	2.1
^aOther details as for Table 5.

Table 8. Recoveries of multiple analytes in West Lake samples.^a

Sample	Standard curve for calculation^b	Spiked level (µg/L)^c	ELISA values (µg/L)	Recovery (%)^d	CV (%)
West Lake water	Del	cyp 5, del 5	9.7±1.0	96.8±9.7	10.0
		cyp 5, esf 10	15.1±1.3	100.8±8.8	8.7
		del 5, esf 10	15.1±1.2	100.5±7.8	7.8
		cyp 5, del 5, esf 10	20.3±1.9	101.6± 9.4	9.3
	Cyp	del 10, cyp10	19.7±2.1	98.3± 10.6	10.8
		del 10, esf 20	30.0±3.5	99.9±11.6	11.6
		cyp 10, esf 20	31.1±2.1	103.6±7.1	6.9
		del 10, cyp 10, esf 20	38.9±3.9	97.3±9.8	10.0
	Esf	esf 20, cyp 10	29.3±2.1	97.7±7.0	7.2
		esf 20, del 20	38.3±0.8	95.6±2.0	2.1
		cyp 10, del 20	29.9±3.1	99.6±10.3	10.3
		esf 20, cyp 10, del 20	48.5±3.7	96.9±7.3	7.6
^aOther details as for Table 5.
^bMeans the recovery values are calculated from the stand curve of cypermethrin, deltamethrin and esfenvalerate in every four combinations test, respectively.
^cNumbers behind the tested pyrethroids are the spiked concentrations for each analyte.
^dRecoveries are calculated as: average detection values divided by the total spiked concentration.