Development of chemiluminescent enzyme immunoassay for the determination of malachite green in seafood

Figures & data

Figure 1. (a) Effects of coating concentration on the IC₅₀ and RLU_max/IC₅₀ ratio. (b). Effects of antibody dilution on the IC₅₀ and RLU_max/IC₅₀ ratio.

Figure 1. (c). Effects of reaction time on the IC₅₀ and RLU_max/IC₅₀ ratio. (d). Effects of dilution buffer on the IC₅₀ and RLU_max/IC₅₀ ratio.

Figure 1. (e). Effects of ionic strength on the IC₅₀ and RLU_max/IC₅₀ ratio. (f). Effects of buffer pH on the IC₅₀ and RLU_max/IC₅₀ ratio.

Figure 2. Normalised standard curve by CLEIA for MG under optimised conditions.

Table 1. Cross-reactivity of MG antisera with analogues.

Compounds	IC50 (ng/mL)	Cross-reactivity (%)
Malachite green (MG)	0.22	100
Methylene blue (MB)	6.4	3.4
Brilliant green (BG)	8.1	2.7
Crystal violet	21.9	1.0
Leuco malachite green (LMG)	≥2000	≤0.01
Leuco crystal violet (LCV)	≥2000	≤0.01
Astrazon orange (AO)	≥2000	≤0.01

Table 2. Intra-assay and inter-assay precision at different concentration of MG.

	Intra-assay (n = 4)		Inter-assay (n = 4)
MG (ng/mL)	Mean ± SD	CV (%)	Mean ± SD	CV (%)
4.05	4.13 ± 0.21	5.08	3.83 ± 0.24	6.27
1.35	1.38 ± 0.12	8.70	1.33 ± 0.15	11.28
0.45	0.42 ± 0.05	11.9	0.47 ± 0.02	4.26
0.15	0.14 ± 0.011	7.86	0.14 ± 0.013	9.29
0.05	0.05 ± 0.005	10.0	0.05 ± 0.006	12.0

Table 3. Recoveries of MG from spiked fish and shrimp by CLEIA.

		Intra-assay (n = 3)			Inter-assay (n = 3)
Samples	Spiked level (ng/g)	Mean ± SD (ng/g)	Recovery (%)	CV (%)	Mean ± SD (ng/g)	Recovery (%)	CV (%)
Tilapia	0.1	0.088 ± 0.011	88.00	12.50	0.084 ± 0.01	84.00	11.90
	1	0.86 ± 0.08	85.79	9.30	0.82 ± 0.07	82.43	8.54
	3	2.87 ± 0.29	95.67	10.10	2.68 ± 0.23	89.33	8.58
	5	4.61 ± 0.38	92.20	8.24	4.35 ± 0.38	87.00	8.74
Turbot	0.1	0.092 ± 0.01	92.00	11.23	0.089 ± 0.008	89.00	9.29
	1	0.93 ± 0.07	93.12	7.94	0.96 ± 0.07	95.91	6.93
	3	2.85 ± 0.17	95.13	5.91	2.73 ± 0.25	90.87	9.16
	5	4.57 ± 0.44	91.44	9.57	4.72 ± 0.30	94.37	6.27
Salmon	0.1	0.087 ± 0.01	87.26	11.14	0.101 ± 0.007	101.23	7.41
	1	1.04 ± 0.10	103.69	9.17	0.95 ± 0.05	95.24	5.36
	3	2.83 ± 0.16	94.33	5.49	3.07 ± 0.19	102.33	6.33
	5	4.69 ± 0.43	93.82	9.16	4.86 ± 0.49	97.21	10.13
Snakehead	0.1	0.093 ± 0.007	93.17	7.56	0.089 ± 0.007	88.75	8.12
	1	0.92 ± 0.07	91.78	7.68	0.87 ± 0.07	87.24	8.19
	3	3.05 ± 0.27	101.67	8.73	2.91 ± 0.23	97.13	7.98
	5	4.73 ± 0.47	94.57	9.84	4.92 ± 0.45	98.38	9.24
Freshwater grouper	0.1	0.102 ± 0.01	102.17	11.17	0.094 ± 0.01	94.16	10.71
	1	0.86 ± 0.07	86.25	8.17	0.92 ± 0.08	92.36	8.57
	3	2.84 ± 0.25	94.67	8.81	2.92 ± 0.27	97.33	9.13
	5	5.13 ± 0.51	102.56	9.76	4.93 ± 0.45	98.64	9.19
Shrimp	0.1	0.097 ± 0.01	97.00	10.31	0.108 ± 0.01	108.0	9.26
	1	0.87 ± 0.08	86.59	9.20	0.90 ± 0.07	89.80	7.78
	3	2.77 ± 0.25	92.33	9.03	2.75 ± 0.23	91.67	8.36
	5	4.76 ± 0.48	95.20	10.08	4.58 ± 0.51	91.61	11.14

Table 4. Analytical result of indirect competitive CLEIA and HPLC methods (n = 4).

	HPLC			Indirect competitive CLEIA
Spiked level (ng/g)	Mean ± SD (ng/g)	Recovery (%)	CV (%)	Mean ± SD (ng/g)	Recovery (%)	CV (%)
20	16.5 ± 1.9	82.5	11.5	17.8 ± 2.1	89.0	11.8
40	33.8 ± 3.1	84.5	9.2	32.4 ± 3.3	81.0	10.2
80	65.3 ± 2.7	81.6	4.1	70.8 ± 4.6	92.3	6.5