Development of an immunochromatographic assay for detection of tylosin and tilmicosin in muscle, liver, fish and eggs

Figures & data

Figure 1. Chemical structures of tylosin, tilmicosin and desmycosin–CMO–BSA.

Figure 2. The illustrations of immunochromatographic assay (ICA) results (A) and the visual limit of detection of single antibiotics [(B) desmycosin, (C) tylosin, (D) tilmicosin] with ICA test: upper line, C line; lower line, T line. The standard solutions of desmycosin, tylosin and tilmicosin at each final concentration of 0, 1, 5, 10, 20, 40, 60, 80 and 100 ng/mL (numbers across the top of strips from left to right) were tested.

Figure 3. Standard curves for desmycosin, tylosin and tilmicosin by immunochromatographic assay.

Table 1. Cross-reactivity (CR) of macrolide antibiotics by the ICA based on mAb.

Macrolide antibiotics	IC50 (ng/mL)	CR (%)
Desmycosin	1.6	100
Tylosin	2.9	55.2
Tilmicosin	4.4	36.4
5-O-mycaminosyltylonolide	35.7	4.5
Spiramycin	>10,000	<0.02
Erythromycin	>10,000	<0.02
Kitasamycin	>10,000	<0.02
Azithromycin	>10,000	<0.02
Josamycin	>10,000	<0.02
Oleandomycin	>10,000	<0.02
Roxithromycin	>10,000	<0.02
Avermectins	>10,000	<0.02
Ivermectin	>10,000	<0.02

Note: ICA, immunochromatographic assay; mAb, monoclonal antibody.

Table 2. Recoveries of tylosin and tilmicosin spiked in tissues by the ICA (n=64^a).

		Tylosin spiked		Tilmicosin spiked
Sample	Spiked level (ng/g)	Mean recovery±SD (ng/g)	CV (%)	Mean recovery±SD (ng/g)	CV (%)
Porcine muscle	10	82.3±9.1	11.1	76.3±8.2	10.5
	50	73.6±6.4	8.7	72.6±6.4	8.8
	100	81.3±4.3	5.2	80.8±4.7	5.8
Porcine liver	10	76.3±6.3	11.0	79.5±8.4	10.6
	50	103.2±13.8	13.4	82.4±11.2	13.6
	100	85.4±5.5	6.4	95.7±4.7	4.9
Chicken muscle	10	78.3±9.3	11.9	82.5±11.2	13.6
	50	82.6±5.1	6.2	78.6±9.2	11.7
	100	84.7±5.7	6.7	83.8±4.9	5.8
Chicken liver	10	82.5±12.3	14.9	78.4±8.4	10.1
	50	74.3±6.5	8.8	89.4±8.7	9.7
	100	85.4±9.7	11.4	79.8±10.5	13.2
Fish	10	90.3±12.4	13.7	101.5±8.7	8.6
	25	72.8±10.3	14.1	83.4±10.6	12.7
	50	85.4±7.5	8.8	100.9±8.9	8.8
Egg	10	71.5±9.6	13.4	86.4±9.4	10.9
	25	86.8±10.8	12.4	82.5±7.4	9.0
	50	78.1±3.4	3.8	88.3±5.6	6.3

Note: ICA, immunochromatographic assay; CV, coefficient of variation.

^a The test was repeated eight times with eight replicates per concentration.

Table 3. Results of tylosin or tilmicosin analysis by HPLC and ICA test in animal muscle, liver, fish and egg samples.

		Result of HPLC (ng/g)
Sample		Tylosin	Tilmicosin	Total	Visual results of ICA (n=5)
Porcine muscle	1	12.4		12.4	−,−,−,−,−
	2		37.8	37.8	−,−,−,−,−
	3	65.3		65.3	+,+,+,+,+
	4	18.4	25.2	43.6	+,+,+,+,+
	5		7.5	7.5	−,−,−,−,−
Porcine liver	1	11.7		11.7	−,−,−,−,−
	2	37.7		37.7	−,−,−,−,−
	3		48.2	48.2	+,+,+,+,+
	4				−,−,−,−,−
	5	8.9		8.9	−,−,−,−,−
Chicken muscle	1				−,−,−,−,−
	2	26.6		26.6	−,−,−,−,−
	3				−,−,−,−,−
	4	103.2		103.2	+,+,+,+,+
	5		121.3	121.3	+,+,+,+,+
Chicken liver	1	17.2		17.2	−,−,−,−,−
	2				−,−,−,−,−
	3	78.5	12.7	91.2	+,+,+,+,+
	4				−,−,−,−,−
	5	2.7		2.7	−,−,−,−,−
Fish	1	82.3		82.3	+,+,+,+,+
	2	16.7		16.7	−,−,−,−,−
	3				−,−,−,−,−
	4		12.3	12.3	−,−,−,−,−
	5		7.5	7.5	−,−,−,−,−
Egg	1				−,−,−,−,−
	2	8.6		8.6	−,−,−,−,−
	3	7.8	4.6	12.4	−,−,−,−,−
	4		8.9	8.9	−,−,−,−,−
	5				−,−,−,−,−

Note: ICA, immunochromatographic assay; HPLC, high-performance liquid chromatography.

Table 4. Result analyses of HPLC, ELISA kit and ICA for the detection of tylosin in porcine muscle and liver samples from animal experiments (n=5).

		HPLC	ELISA kit	ICA	ICA
Matrices	Withdrawals (days)	Average±SD (ng/g)	Average±SD (ng/g)	Average±SD (ng/g)	Visual results
Muscle	0	186.4±13.8	172.9±23.5	178.6±14.2	+,+,+,+,+
	1	78.3±13.8	67.5±4.2	72.6±8.7	+,+,+,+,+
	3	<50	<20	22.5±3.8	−,−,−,−,−
Liver	0	437.4±33.2	473.4±48.2	486.1±38.7	+,+,+,+,+
	1	64.7±9.8	58.6±7.3	64.3±5.2	+,+,+,+,+
	3	<50	<20	24.6±3.7	−,−,−,−,−

Note: Results are presented as means (n=5). Limit of detection of tylosin by HPLC method was 50 ng/g. Equation curves and correlation coefficients were as follows: HPLC/ICA (muscle samples: y=1.0805x – 20.988, R²=0.9902); HPLC/ICA (liver samples: y=1.1191x – 20.713, R²=0.9992); ELISA/ICA (muscle samples: y=0.9905x+7.1376, R²=0.9997); ELISA/ICA (liver samples: y=1.008x+8.8876, R²=0.9998). Cut-off values were set at 40 ng/g as positive samples (no red band visible on the test line) and from 0 to 40 ng/g as negative sample (band weaker or similar to negative control) for the semi-quantitative analyses by ICA.

+, positive; −, negative; SD, means of standard deviation; ICA, immunochromatographic assay; HPLC, high-performance liquid chromatography.