Development of a monoclonal antibody-based ELISA to detect Escherichia coli O157:H7

Figures & data

Table 1. Bacterial strains tested in this study.

Microorganism	Source and serotype	Medium
Escherichia coli
Enterohemorrhage E. coli	CMCC44752 (O157:H7) CICC21530 (O157:H7)	SMAC agar, 37°C
Enteropathogenic E. coli	ATCC8739(O146), CMCC44737(O142)	Nutrient agar, 37°C
Entroinvasive E. coli	CMCC44708 (O112)	LB broth, 37°C
E. coli	ATCC25922	LB broth, 37°C
Clinical E. coli strain
O157:H7	J17	Nutrient agar, 37°C
Non O157:H7	SHDEL, SHDHN J20, ENA7A, MTGH32, EWHJ116	Nutrient agar, 37°C
Salmonella spp.	CICC21490	Nutrient agar, 37°C
Staphylococcus aureus	ATCC29213	Nutrient agar, 37°C

Table 2. Sandwich ELISA for pair-wise interaction analysis (P/N value).

	Capture mAb
Detection mAb	1	2	3	4	5	6	7	8	9	10
1-HRP		0.95	1.01	1.12	1.19	0.88	0.79	0.62	0.68	1.47
2-HRP	3.45		2.84	2.57	2.68	3.21	2.96	2.82	3.24	2.94
3-HRP	1.00	0.80		0.89	0.94	0.80	0.90	0.80	0.80	0.83
4-HRP	1.23	1.11	1.31		0.59	0.95	0.66	0.60	0.79	1.03
5-HRP	1.01	0.93	0.89	0.86		0.83	0.97	0.90	0.89	1.21
6-HRP	4.45	3.05	4.02	3.59	2.46		3.20	2.76	2.31	3.82
7-HRP	0.78	0.96	0.94	0.76	0.72	0.76		0.75	1.11	1.03
8-HRP	1.08	1.04	0.96	0.87	1.08	1.09	1.05		0.83	0.71
9-HRP	0.92	1.20	0.94	0.95	1.00	0.69	0.91	0.86		0.82
10-HRP	0.52	1.51	0.91	0.97	0.59	0.92	1.14	0.91	0.75

Note: P/N value was the ratio of optical density value of the sample to the negative control.

Figure 1. Standard curve for E. coli O157:H7 in sandwich ELISA. (A) Standard curve when concentrations of E. coli O157:H7 ranging from 10⁴ to 10⁸ cfu/mL. (B) The linear part of the standard curve for the standard E. coli O157:H7. The linear regression equation was y=2.196x–10.195, and linear correlation coefficient (R²) was 0.9833. The points represent the mean absorbance values obtained from triplicate wells.

Table 3. Detection of E. coli O157:H7 in artificially contaminated green tea samples.

E. coli O157:H7 (cfu/ml)	P/N value from sandwich ELISA	Culturing methods
1	>2.1(+)	+
10	>2.1(+)	+
10^2	>2.1(+)	+
10^3	>2.1(+)	+
10^4	>2.1(+)	+
10^5	>2.1(+)	+
10^6	>2.1(+)	+
10^7	>2.1(+)	+
10^8	>2.1(+)	+

Note: “ + ” symbol represents positive result.