A sensitive lateral flow immunoassay for the multiple residues of five adamantanes

Figures & data

Figure 1. Schematic illustration of the LFIA for the detection of ADAs.

Figure 2. The synthetic schemes of haptens for AMA. (a) The hapten (AMA-hapten) was synthesized by a nucleophilic substitution reaction. (b) The hapten (RMA-hapten) was synthesized by a succinic anhydride method.

Figure 3. LC–MS analysis of haptens of ADAs. (a) The liquid chromatogram spectrum of AMA-hapten; (b) the mass spectrum of AMA-hapten; (c) The liquid chromatogram spectrum of RMA-hapten; (d) the mass spectrum of RMA-hapten.

Table 1. Titre and specficity of the immunized mice.^a

Immunogens/coating antigens		AMA-hapten-BSA/ RMA-hapten-OVA					RMA-hapten-BSA/AMA-hapten-OVA
Mouse No.		1	2	3	4	5	6	7	8	9	10
Titre	OD at 450 nm	2.141	1.623	2.402	2.293	2.373	2.451	2.728	2.512	2.627	2.935
Specificity^b	AMA 10 ng/mL	59%	56%	19%	57%	35%	<5%	<5%	<5%	<5%	<5%
	RMA 5 ng/mL	54%	57%	25%	60%	41%	<5%	<5%	<5%	<5%	<5%
	SMA 5 ng/mL	52%	60%	37%	65%	58%	<5%	<5%	<5%	<5%	<5%

^aThe data shown are at a coating antigen concentration of 0.3 µg/mL and an serum dilution of 1/3000.

^bThe specificity of each serum was primarily assessed by the [1-B/B₀]*100% values at three different standard solution, which were measured by ic-ELISA

Figure 4. Characterization of the mAb against ADAs. (a) Isotype determination of the anti-ADAs mAb. (b) Affinity testing results of the antibody.

Table 2. The characterization of anti-ADAs mAb.

Chemical compound	Structure	Standard curve and IC50 (ng/mL)	CR (%)
AMA			100
RMA			204
SMA			226
AMK			11
AMM			12

Figure 5. The test results of ADAs with LFIA. (a) Qualitative detection of ADAs in chicken samples (SMA: 0, 0.1, 0.25, 0.5, 1, 2.5 μg/kg; RMA: 0, 0.1, 0.25, 0.5, 1, 2.5 μg/kg; AMA: 0, 0.5, 1, 2, 5, 10 μg/mL; AMM: 0, 5, 10, 20, 50, 100 μg/kg; AMK: 0, 5, 10, 20, 50, 100 μg/kg); (b) Standard curves for detecting chicken samples using LFIA. The X-axis is shown as the logarithmic concentrations of ADAs; and T/T₀ defined as the ratio of the colour density of the spiked sample to that of the blank sample.

Table 3. Recovery and precision of LFIA for testing chicken samples spiked ADAs (n = 5).

ADAs	Spiked level (μg/kg)	Detection level (μg/kg) Mean ± SD	Recovery rate (%)	CV (%)
SMA	0	^aND	^bNC	NC
	0.2	0.22 ± 0.005	110.5 ± 2.3	2.10
	0.5	0.51 ± 0.02	101.4 ± 4.2	4.13
RMA	0	ND	NC	NC
	0.2	0.21 ± 0.006	106.7 ± 3.0	2.79
	0.5	0.51 ± 0.02	101.9 ± 4.8	4.70
AMA	0	ND	NC	NC
	2	2.21 ± 0.06	110.3 ± 3.2	2.94
	5	4.75 ± 0.37	95.0 ± 7.5	7.85
AMM	0	ND	NC	NC
	25	22.34 ± 0.65	89.4 ± 2.6	2.91
	50	50.18 ± 2.46	100.4 ± 4.9	4.89
AMK	0	ND	NC	NC
	25	24.75 ± 0.46	99.0 ± 1.8	1.87
	50	45.76 ± 0.79	91.5 ± 1.6	1.74

^aND, not detectable. ^bNC, not calculated.

Table 4. Comparison of LFIA with ELISA kit for detecting ADAs residues in chicken samples (n = 3).

Sample No.		LFIA (μg/kg)	ELISA (μg/kg)	Difference between LFIA and ELISA (%)
SMA	1	0.17	0.16	7.9
	2	0.45	0.44	2.2
RMA	3	0.27	0.23	16.4
	4	0.54	0.57	5.9
AMA	5	1.69	1.76	3.9
	6	3.54	3.81	7.1
AMM	7	12.17	12.21	0.3
	8	21.29	20.72	2.7
AMK	9	19.03	21.43	11.2
	10	35	33.21	5.4