Simultaneous detection of tylosin and tilmicosin in honey using a novel immunoassay and immunochromatographic strip based on an innovative hapten

Figures & data

Figure 1. Chemical structure of TIM, DES, and ATYL.

Table 1. Chemical structure of TYL.

Substance	Tylosin Mol. Form.	R1	R2	R3
TYL A	C46H77NO17	Mycarose	–OCH3	–CHO
TYL B	C39H65NO14	–H	–OCH3	–CHO
TYL C	C45H75NO17	Mycarose	–OH	–CHO
TYL D	C46H79NO17	Mycarose	–OCH3	–CH2OH

Figure 2. Synthesis mechanism of TYL-CMO.

Figure 3. HPLC-MS analysis and identification of TYL-CMO: (a)–(d) the liquid chromatogram spectrum of TYL-CMO; (e) the mass spectrum of TYL-CMO.

Figure 4. The UV–visible spectroscopy of TYL, protein and conjugated antigens: (a) confirmation of immunogen (TYL-CMO-BSA); (b) confirmation of coating antigen (TYL-CMO-OVA).

Figure 5. Optimization of standard dilution buffer for ic-ELISA: (a) NaCl content in PB; (b) pH value; (c) acetonitrile content; (d) standard curve of the TYL and TIM by optimized ic-ELISA.

Table 2. CR of analogues and related compounds.

Compounds	IC50 (ng/ml)	CR (%)
TYL	0.57	100
TIM	2.10	27.57
DES	0.58	97.43
ATYL	0.91	62.42
Avermectin	>100	<1
Ivermectin	>100	<1
Erythromycin	>100	<1
Neomycin	>100	<1
Kanamycin	>100	<1
Streptomycin	>100	<1

Table 3. Recovery determined by ic-ELISA in honey sample (n = 3).

Chemical	Spiked level (ng/g)	Detection level (ng/g)	Mean ± SD (ng/g)	CVs (%)	Average recovery (%)
TYL	1	0.5	0.5560 ± 0.02	3.6	112.00
	2	1	0.9440 ± 0.07	7.4	94.40
	4	2	1.8185 ± 0.15	8.2	90.92
	30	0.6	0.6282 ± 0.06	9.6	104.70
	50	1	1.0994 ± 0.15	13.6	109.94
	80	1.6	1.4502 ± 0.11	7.6	90.64
TIM	2	1	1.0578 ± 0.03	2.8	105.78
	4	2	1.9821 ± 0.09	4.5	99.10
	10	5	4.7794 ± 0.24	5.0	95.59
	30	1	0.9562 ± 0.08	8.4	95.62
	60	2	1.8493 ± 0.19	10.3	92.46
	90	3	2.6812 ± 0.28	10.4	89.37

Figure 6. ICS result in PBS: (a) TYL (1 = 0 ng/ml, 2 = 0.5 ng/ml, 3 = 1 ng/ml, 4 = 2.5 ng/ml, 5 = 5 ng/ml, and 6 = 10 ng/ml); (b) TIM (1 = 0 ng/ml, 2 = 1 ng/ml, 3 = 2.5 ng/ml ppb, 4 = 5 ng/ml, 5 = 10 ng/ml, and 6 = 15 ng/ml).

Figure 7. ICS assay result in honey sample: (a) TYL (1 = 0 ng/ml, 2 = 0.1 ng/ml, 3 = 0.25 ng/ml, 4 = 0.5 ng/ml, 5 = 1 ng/ml, and 6 = 2.5 ng/ml); (b) TIM (1 = 0 ng/ml, 2 = 0.25 ng/ml, 3 = 0.5 ng/ml, 4 = 1 ng/ml, 5 = 2.5 ng/ml, and 6 = 5 ng/ml).