Development of an indirect enzyme-linked immunosorbent assay and lateral-flow test strips for pefloxacin and its analogues in chicken muscle samples

Figures & data

Table 1. Cross-reactivity of PEF-mAb with different FQ compounds.

Compound	IC50 (ng/mL)	CR%	Compound	IC50 (ng/mL)	CR%
Pefloxacin (PEF)	0.218	100.00	Besifloxacin	N/A	<0.01
Norfloxacin (NOR)	0.178	122.64	Garenoxacin	N/A	<0.01
Enoxacin (ENO)	0.214	102.11	Gemifloxacin	N/A	<0.01
Ciprofloxacin (CIPRO)	0.197	111.07	Nalidixic acid	N/A	<0.01
Ofloxacin (OFL)	0.337	64.84	Cinoxacin	N/A	<0.01
Fleroxacin (FLE)	0.173	126.55	Trovafloxacin	N/A	<0.01
Sarafloxacin (SARA)	0.280	77.96	Nadifloxacin	N/A	<0.01
Prulifloxacin (PF)	0.233	93.63	Pazufloxacin	N/A	<0.01
Lomefloxacin (LOM)	1.858	11.76	Oxolinic acid	N/A	<0.01
Pipemidic acid	12.518	1.74	Flumequine	N/A	<0.01
Gatifloxacin	55.040	0.40	Enrofloxacin	N/A	<0.01

Table 2. Recovery results of nine FQ compounds from fortified chicken muscle.

Compound	Spiked level (µg/kg)	Intra-assay (n = 3)		Inter-assay (n = 5)
		Recovery (%)	CV (%)	Recovery (%)	CV (%)
	1.00	96.80	1.08	93.41	4.26
PEF	0.40	105.58	8.95	104.34	7.35
	0.20	94.90	12.17	94.08	10.35
	1.00	94.89	2.74	88.83	2.96
NOR	0.40	107.23	13.59	108.78	11.77
	0.20	111.47	11.30	113.79	9.09
	1.00	99.50	3.76	100.93	2.92
ENO	0.40	90.21	8.87	86.81	5.30
	0.20	85.56	7.24	88.47	6.77
	1.00	96.40	3.01	99.27	4.82
CIPRO	0.40	99.94	5.92	102.07	8.25
	0.20	83.67	6.52	87.43	7.16
	1.00	110.56	3.47	111.86	5.79
FLE	0.40	100.32	11.96	82.42	8.25
	0.20	98.68	8.56	97.27	6.44
	1.00	72.46	6.12	74.47	6.56
OFL	0.40	85.09	10.85	81.71	12.20
	0.20	62.42	7.65	63.50	9.37
	1.00	91.26	4.77	91.06	4.39
SARA	0.40	82.25	10.89	79.87	9.82
	0.20	72.87	8.23	70.80	7.10
	1.00	97.58	8.11	101.66	7.86
PF	0.40	94.76	6.15	98.80	6.78
	0.20	86.30	5.05	86.01	5.38
	4.00	100.98	7.69	102.22	6.00
LOM	2.00	98.82	5.68	98.16	5.08
	1.00	79.93	8.91	82.22	11.43

Figure 1. The schematic diagram of PEF- immunogen synthesis.

Figure 2. Illustration of typical strip test results. (A) negative test, (B) positive test and (C and D) invalid tests.

Figure 3. Characterization of PEF-EDEA-BSA conjugate by UV spectrophotometric.

Figure 4. Effects of (a) pH variation, (b) salt concentration, (c) methanol concentration and (d) acetonitrile (ACN) concentration on ic-ELISA performance.

Figure 5. The PEF Inhibition standard curve under optimised conditions (pH 8.0, 1.6%NaCl, 5% MeOH and, 0.0% ACN in PBS assay).

Figure 6. Lateral flow test strips of nine FQ compounds in 0.01 M PBS assay (A1) PEF, (A2) NOR, (A3) CIPRO, (A4) ENO, (A5) FLE, (A6) PF, (A7) OFL, (A8) SARA and (A9) LOM. Various analyte concentrations; (1 = 0, 2 = 0.1, 3 = 0.25, 4 = 0.5, 5 = 1.0, 6 = 2.5, 7 = 5, 8 = 10, 9 = 25, and 10 = 50 ng/mL). *The visual cut off value.

Figure 7. Detection of nine FQ compounds in chicken muscle using Lateral flow test strips. (B1) PEF, (B2) NOR, (B3) CIPRO, (B4) ENO, (B5) FLE, (B6) PF, (B7) OFL, (B8) SARA and (B9) LOM. Spiked amounts; (1 = 0, 2 = 0.625, 3 = 1.25, 4 = 2.5, 5 = 3.125, 6 = 5, 7 = 6.25, 8 = 12.5, 9 = 25, 10 = 50 and 11 = 100 µg/kg). *The visual cut off value.