Highly broad-specific and sensitive direct competitive enzyme-linked immunosorbent assay for screening multi-antibacterial synergists: assay optimization and application to animal-derived food

Figures & data

Figure 1. Chemical structures of ASGs and hapten.

Figure 2. Effects of physicochemical factors on analytical characteristics of the competitive standard curve for TMP based on mAb 3B6, 9C9, 5C4 and 14G1 in dcELISA. (a) coating condition; (b) blocking buffer; (c) blocking time; (d) concentration of Tween-20; (e) concentration of BSA; (f) concentration of skim milk; (g) pH; (h) concentration of NaCl; (i) methanol; (j) acetonitrile; (k) reaction time; (l) colour development time.

Figure 3. Cross-reactivity (CR) for five ASGs under optimized conditions in dcELISA. (a) 3B6; (b) 9C9; (c) 5C4; (d) 14G1

Table 1. Cross-reactivity (CR) for five ASGs under optimized conditions in dcELISA.

	3B6		9C9		5C4		14G1
	IC50	CR	IC50	CR	IC50	CR	IC50	CR
ASGs	(ng mL^−1)	(%)	(ng mL^−1)	(%)	(ng mL^−1)	(%)	(ng mL^−1)	(%)
TMP	0.35 ± 0.014	100.00	0.51 ± 0.018	100.00	0.26 ± 0.015	100.00	1.35 ± 0.021	100.00
BQP	0.22 ± 0.011	158.20 ± 1.27	0.22 ± 0.014	223.10 ± 1.28	0.18 ± 0.012	138.20 ± 1.25	0.75 ± 0.011	179.10 ± 1.91
BDP	5.37 ± 0.141	6.52 ± 0.09	0.22 ± 0.013	231.20 ± 1.38	0.20 ± 0.016	125.90 ± 0.93	1.17 ± 0.015	115.50 ± 1.4
OMP	9.24 ± 0.167	3.79 ± 0.08	5.97 ± 0.101	8.60 ± 0.17	3.49 ± 0.145	7.50 ± 0.10	7.31 ± 0.148	18.50 ± 0.14
DVD	0.95 ± 0.034	36.67 ± 0.41	0.98 ± 0.037	51.90 ± 0.48	0.31 ± 0.020	83.87 ± 0.75	0.67 ± 0.013	201.50 ± 1.61

Figure 4. The dcELISA calibration curves for TMP in assay buffer and assay buffe diluted samples of (a) whole milk, (b) skim milk, (c) eggs, (d) chicken, (e) pork. Parallels were found between the standard curve prepared in assay buffer and diluted samples, respectively (n = 3).

Table 2. The recovery and CV of five ASGs from spiked samples using the dcELISA. (n = 3).

ASG	Whole milk			Skim milk			Eggs			Chicken			Pork
	Spiked	Recovery	CV	Spiked	Recovery	CV	Spiked	Recovery	CV	Spiked	Recovery	CV	Spiked	Recovery	CV
	(ng mL^−1)	(%)	(%)	(ng mL^−1)	(%)	(%)	(ng g^−1)	(%)	(%)	(ng g^−1)	(%)	(%)	(ng g^−1)	(%)	(%)
TMP	0.45	97.1 ± 0.014	2.5	0.50	92.8 ± 0.012	10.3	0.30	91.8 ± 0.018	7.4	0.50	95.3 ± 0.018	2.4	0.10	79.5 ± 0.012	5.9
	0.75	88.6 ± 0.018	3.8	1.50	98.2 ± 0.018	5.2	0.60	97.8 ± 0.101	9.4	1.00	93.9 ± 0.123	7.4	0.25	95.6 ± 0.011	9.6
	1.00	90.1 ± 0.151	7.4	4.00	97.7 ± 0.151	2.4	1.20	95.05 ± 0.115	12.1	2.50	98.1 ± 0.145	2.9	1.00	90.1 ± 0.124	9.1
BQP	0.45	100.2 ± 0.019	12.1	0.50	105.4 ± 0.016	11.6	0.30	94.4 ± 0.014	3.8	0.50	82.7 ± 0.110	7.8	0.10	103.5 ± 0.012	5.8
	0.75	92.6 ± 0.013	5.2	1.50	94.9 ± 0.015	6.9	0.60	92.7 ± 0.019	4.8	1.00	95.1 ± 0.102	7.6	0.25	85.4 ± 0.014	8.3
	1.00	96.1 ± 0.123	6.3	4.00	100.1 ± 0.123	9.6	1.20	100.1 ± 0.120	3.8	2.50	98.8 ± 0.156	7.8	1.00	95.4 ± 0.106	3.7
BDP	0.45	95.7 ± 0.015	7.8	0.50	97.2 ± 0.012	3.7	0.30	97.2 ± 0.015	5.2	0.50	88.4 ± 0.134	5.9	0.10	91.1 ± 0.018	3.2
	0.75	98.2 ± 0.019	3.4	1.50	98.3 ± 0.017	9.7	0.60	87.5 ± 0.149	7.3	1.00	93.7 ± 0.102	9.2	0.25	95.1 ± 0.016	9.1
	1.00	98.7 ± 0.169	4.1	4.00	94.8 ± 0.123	10.4	1.20	104.5 ± 0.121	3.4	2.50	101.8 ± 0.116	13.4	1.00	85.6 ± 0.125	6.4
OMP	0.45	83.1 ± 0.018	2.9	0.50	80.6 ± 0.016	5.8	0.30	101.6 ± 0.016	4.8	0.50	100.7 ± 0.123	10.1	0.10	104.1 ± 0.015	6.1
	0.75	86.2 ± 0.015	12.1	1.50	87.9 ± 0.017	3.6	0.60	91.4 ± 0.126	4.6	1.00	95.9 ± 0.118	5.6	0.25	82.8 ± 0.012	7.2
	1.00	91.9 ± 0.178	5.2	4.00	93.9 ± 0.126	11.5	1.20	97.4 ± 0.123	7.5	2.50	95.8 ± 0.106	6.4	1.00	94.5 ± 0.112	5.5
DVD	0.45	93.2 ± 0.016	3.9	0.50	79.6 ± 0.016	3.7	0.30	99.2 ± 0.015	13.1	0.50	99.1 ± 0.198	3.9	0.10	96.1 ± 0.016	9.1
	0.75	94.1 ± 0.014	9.4	1.50	101.5 ± 0.019	10.5	0.60	102.1 ± 0.124	5.5	1.00	96.1 ± 0.178	10.3	0.25	100.4 ± 0.012	4.7
	1.00	87.0 ± 0.141	6.5	4.00	95.9 ± 0.189	5.9	1.20	91.3 ± 0.164	4.0	2.50	96.6 ± 0.165	4.5	1.00	86.4 ± 0.123	10.4