Development and validation of a sensitive enzyme-linked immunosorbent assay for clonidine hydrochloride in pig urine and pork samples

Figures & data

Figure 1. (a) The synthesis procedure of CLO-BSA; (b) Identification of CLO-BSA by SDS-PAGE; (c) The titer curve of the four mice pAb; (d) Sensitivity identification of the No.2 mouse pAb.

Figure 2. (a) The identification of the mAb (34F10) subclass; (b) The sensitivity of 34F10 identified by ic-ELISA.

Figure 3. (a) The checkerboard titration experiment; (b) The standard curve of ic-ELISA.

Table 1. The cross-reaction of mAb with other agonist drugs.

Agonist drugs	Structures	IC50 (ng/mL)	Cross-reactivity (%)
PA		>1.0 × 10^4	<0.002
BA		>1.0 × 10^4	<0.002
CYP		>1.0 × 10^4	<0.002
SAL		>1.0 × 10^4	<0.002
RAC		>1.0 × 10^4	<0.002
CLT		>1.0 × 10^4	<0.002

Figure 4. Elimination of matrix effects in (a) pig urine sample and (b) pork sample; (c) IC₅₀values for pig urine sample and pork sample following different dilutions with PBS; ND: not detected.

Figure 5. Correlation between the analysis of CLO in pig urine via the ic-ELISA and HPLC methods.

Table 2. The recovery experiment of the ic-ELISA for CLO in pig urine and pork samples.

Samples	Intra-assay				Inter-assay
	Spiked (ng/mL)	Found (ng/mL)	Mean RV (%)	CV (%)	Spiked (ng/mL)	Found (ng/mL)	Mean RV (%)	CV (%)
Pork	0.1	0.084 ± 0.01	84.04 ± 3.83	4.56	0.1	0.087 ± 0.01	87.18 ± 6.03	6.91
	0.2	0.197 ± 0.02	98.47 ± 10.04	10.19	0.2	0.195 ± 0.02	97.53 ± 8.52	8.74
	0.4	0.368 ± 0.04	91.94 ± 10.36	11.27	0.4	0.372 ± 0.03	93.06 ± 7.32	7.87
	0.6	0.555 ± 0.04	92.43 ± 6.71	7.26	0.6	0.544 ± 0.02	90.69 ± 2.81	3.1
Pig urine	0.1	0.092 ± 0.01	88.01 ± 9.09	10.33	0.1	0.088 ± 0.01	88.42 ± 8.60	9.72
	0.2	0.173 ± 0.01	86.65 ± 2.15	2.48	0.2	0.187 ± 0.01	93.57 ± 4.28	5.15
	0.4	0.398 ± 0.02	99.55 ± 5.76	5.79	0.4	0.399 ± 0.03	99.81 ± 7.01	7.02
	0.6	0.562 ± 0.05	93.74 ± 9.27	9.89	0.6	0.595 ± 0.04	99.12 ± 5.98	6.03