Sensitive and highly specific detection of Cronobacter sakazakii based on monoclonal sandwich ELISA

Figures & data

Table 1. The bacterial strains tested in this study.

Microorganisms	Source	Medium
C. sakazakii	ATCC 29544	TSA agar, 37°C
C. sakazakii	ATCC 29004	TSA agar, 37°C
C. sakazakii	IQCC 10403.20	TSA agar, 37°C
C. muytjensii	ATCC 51329	TSA agar, 37°C
Salmonella spp.	CICC 21490	Nutrient agar, 37°C
Enterohemorrhage E. coli	CMCC 44752	Nutrient agar, 37°C
Enteropathogenic E. coli	ATCC 8739	Nutrient agar, 37°C
Entroinvasive E. coli	CMCC 44708	Nutrient agar, 37°C
Listeriosis spp.	CICC 21633	Nutrient agar, 37°C
S. aureus	ATCC 29213	Nutrient agar, 37°C

ATCC: American Type Culture Collection, T. Manassas, Virginia.

CMCC: National Center for Medical Culture Collections, Beijing, China.

CICC: China Center of Industrial Culture Collection, Beijing, China.

IQCC: China Academy of Inspection and Quarantine of food safety of Microbial Culture Collection Management Center.

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

	Capture mAb
Detection mAb	K4	K5	K6	K7	K9	K12	K13	K15	4C10	3C11	G4
K4-HRP		0.77	0.86	0.74	1.11	19.07	22.12	10.47	1.00	1.12	1.24
K5-HRP	1.93		1.59	1.78	1.16	2.54	14.36	7.55	1.09	1.00	1.13
K6-HRP	4.31	1.39		1.41	1.25	13.65	12.50	7.48	1.33	0.91	0.92
K7-HRP	0.12	1.02	1.12		1.02	5.70	4.60	1.06	0.94	0.84	0.96
K9-HRP	4.27	1.06	1.56	1.19		5.48	4.13	1.78	1.16	1.06	1.00
K12-HRP	3.55	0.95	0.84	0.36	0.95		5.89	1.87	0.93	0.98	1.12
K13-HRP	0.95	1.15	1.00	2.46	1.04	11.76		2.69	0.97	1.00	1.13
K15-HRP	0.93	1.07	1.04	4.02	1.00	13.13	14.44		1.12	1.35	1.24
4C10-HRP	0.72	0.74	0.84	0.94	0.98	0.96	0.91	0.93		0.91	0.93
3C11-HRP	1.09	0.91	1.22	1.00	1.06	1.31	1.17	1.20	1.02		1.04
G4-HRP	15.34	1.09	1.20	1.08	1.02	1.15	1.11	1.06	1.02	0.98

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

Table 3. The strains tested by optimized sandwich ELISA (n = 8).

		Sandwich ELISA
Microorganism		OD450 value	P/N value
C. sakazakii	ATCC 29544	1.801 ± 0.11	12.77 (+)
C. sakazakii	ATCC 29004	1.794 ± 0.091	11.54 (+)
C. sakazakii	IQCC 10403.20	1.782 ± 0.11	11.72 (+)
C. muytjensii	ATCC 51329	0.280 ± 0.014	1.99 (–)
Salmonella spp.	CICC 10420	0.183 ± 0.012	1.30 (–)
E. coli spp.	CICC 21530	0.164 ± 0.0082	1.16 (–)
E. coli spp.	ATCC 25922	0.155 ± 0.0079	1.10 (–)
E. coli spp.	CMCC 44737	0.195 ± 0.097	1.38 (–)
Listeriosis spp.	CICC 21633	0.185 ± 0.022	1.31 (–)
S. aureus	ATCC 29213	0.189 ± 0.011	1.34 (–)

Note: P/N value, the positive/negative value, which was the ratio of optical density value of the testing sample to the negative control sample. Values were calculated according to the formula P/N > 2.1. (+) means positive, (–) means negative.

ATCC: American Type Culture Collection, T. Manassas, Virginia.

CMCC: National Center for Medical Culture Collections, Beijing, China.

CICC: China Center of Industrial Culture Collection, Beijing, China.

IQCC: China Academy of Inspection and Quarantine of food safety of Microbial Culture Collection Management Center.

Figure 1. The standard curve for C. sakazakii ATCC 29544 in monoclonal sandwich ELISA (n = 8). (A) Standard curve of the concentration ranging from 1 × 10⁴ and 10⁹ cfu/mL. (B) The linear dynamic range of C. sakazakii ATCC 29544 between 1 × 10⁵ and 10⁸ cfu/mL.

Note: The linear regression equation was y = 4.489x – 21.758. The linear correlation coefficient (R²) was 0.990.

Figure 2. SDS-PAGE and immunobloting analysis of extracted proteins extracted from bacteria. (A) SDS-PAGE of soluble proteins extracted from bacteria; (B) Immunoblotting of mAb K13 for soluble proteins extracted from bacteria; (C) Immunoblotting of mAb K15 for soluble proteins extracted from bacteria.

Table 4. The results of detecting C. sakazakii in powdered infant formula (n = 8).

	The result of sandwich ELISA (4h enrichment)
The concentration of spiked C. sakazakii ATCC 29544 (cfu/mL)	OD450 value	P/N value	Conventional detection method
0	0.168 ± 0.0084	11.99 (–)	–
1	0.694 ± 0.0084	4.13 (+)	+
10	1.144 ± 0.0084	6.81 (+)	+
102	1.916 ± 0.13	11.40 (+)	+
103	2.199 ± 0.12	13.09 (+)	+
104	2.626 ± 0.13	15.63 (+)	+

Note: P/N value, the positive/negative value, which was the ratio of optical density value of the testing sample to the negative control sample. Values were calculated according to the formula P/N > 2.1. (+) means positive, (–) means negative.