Development and performance evaluation of indirect ELISA kit with recombinant leptospira protein as coating antigen

doi:10.3969/j.issn.1674-8115.2021.04.007

Abstract

Abstract: Objective

·To develop an indirect enzyme-linked immunosorbent assay (ELISA) kit for the detection of leptospira-specific antibodies and evaluate its performance.

Methods

·The highly conserved outer membrane protein in the epidemic strains of the main pathogenic leptospira in China was selected and expressed by prokaryotic expression system. The recombinant protein was purified and used as coating antigen for mass spectrometry identification, and its antigenicity was detected. Serum samples from 40 patients with leptospirosis, 50 patients with other infectious diseases and 391 healthy controls were selected as test samples. The positive control, positive reference and high-titer mixed positive serum for internal control of the kit were prepared from the serum of patients with leptospirosis, the negative control and reference for internal control of the kit were prepared from the serum of healthy controls, and the weak positive control (critical reference) and sensitivity reference for internal control of the kit were prepared from high-titer mixed positive serum. One strong positive serum and one weak positive serum selected from the serum of patients with leptospirosis were used as strong positive and weak positive precision reference for internal control of the kit. The detection system of the indirect ELISA kit was established, its preparation process and reaction conditions were optimized, and its precision, sensitivity, specificity and stability were evaluated.

Results

·The purified recombinant outer membrane protein of leptospira was identified as the target protein by mass spectrometry. The results of performance evaluation showed that the intra assay precision and inter assay precision of the indirect ELISA kit were less than 15% and 20%, respectively, and the sensitivity and specificity were 95.0% and 95.0%, respectively. The performance of indirect ELISA kit was still stable after 12 months' storage at 2?8 ℃.

Conclusion

·The specificity, sensitivity and other performance indicators of the leptospira antibody detection kit meet the requirements of the in vitro test kit, which may be suitable for clinical auxiliary diagnosis, sentinel monitoring of leptospirosis and epidemiological investigation.

Key words: leptospira, recombinant protein, indirect enzyme-linked immunosorbent assay, IgG antibody, sensitivity/specificity

CLC Number:

R377⁺5

Jie QIAN, Jian YANG, Yue ZHANG, Rong ZHANG, Xiang-yan ZHANG, Xiao-kui GUO. Development and performance evaluation of indirect ELISA kit with recombinant leptospira protein as coating antigen[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 459-466.

Figures/Tables 11

TrendMD

References 15

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Reference for internal control of the kit	Concentration of coating antigen
Reference for internal control of the kit	1 μg/mL	2 μg/ mL	5 μg/ mL	10 μg/ mL
Positive Reference
Strong positive reference	1.709	1.744	1.798	1.810
Weak positive reference	0.518	0.531	0.547	0.556
Negative reference	0.117	0.126	0.136	0.149

Reference for internal control of the kit	Concentration of coating antigen
Reference for internal control of the kit	1 μg/mL	2 μg/ mL	5 μg/ mL	10 μg/ mL
Positive Reference
Strong positive reference	1.709	1.744	1.798	1.810
Weak positive reference	0.518	0.531	0.547	0.556
Negative reference	0.117	0.126	0.136	0.149

Reference for internal control of the kit	Intra batch precision			Inter batch precision
Reference for internal control of the kit	Average D (450 nm/630 nm)	SD	CV/%	Average D (450 nm/630 nm)	SD	CV/%
Weak positive reference	0.349	0.032	9.18	0.396	0.053	13.38
Strong positive reference	1.518	0.053	3.49	1.614	0.081	5.02

Reference for internal control of the kit	Intra batch precision			Inter batch precision
Reference for internal control of the kit	Average D (450 nm/630 nm)	SD	CV/%	Average D (450 nm/630 nm)	SD	CV/%
Weak positive reference	0.349	0.032	9.18	0.396	0.053	13.38
Strong positive reference	1.518	0.053	3.49	1.614	0.081	5.02

Serum sample	Positive		Negative
Serum sample	True Positve	Faulse Positve	True Negative	Faulse Negative
Leptospirosis patients/n	38	?	?	2
Other infectious diseases patients/n	?	3	47	?
Healthy controls/n	?	2	48	?