Development of rELISA using novel markers for the diagnosis of paratuberculosis

Mukta Jain; Amit Kumar; Rathnagiri Polavarapu; Saurabh Gupta; G K Aseri; Deepansh Sharma; Jagdip Singh Sohal

doi:10.1016/j.jim.2021.113105

Development of rELISA using novel markers for the diagnosis of paratuberculosis

J Immunol Methods. 2021 Oct:497:113105. doi: 10.1016/j.jim.2021.113105. Epub 2021 Jul 21.

Authors

Mukta Jain¹, Amit Kumar², Rathnagiri Polavarapu³, Saurabh Gupta⁴, G K Aseri¹, Deepansh Sharma¹, Jagdip Singh Sohal⁵

Affiliations

¹ Amity Center for Mycobacterial Disease Research, Amity University Rajasthan, Kant-Kalwar, Delhi-Jaipur Highway, NH11C, Jaipur 303 002, India.
² Department of Immunology & Defense Mechanism, Sardar Vallabh Bhai Patel University of Agriculture & Technology (SVPUAT), Modipurum, Meerut 250 110, India.
³ Genomix Molecular Diagnostics (P) Ltd, 5-36/207 Prasanthnagar, Kukatpally, Hyderabad 500 072, India.
⁴ Department of Biotechnology, Institute of Applied Sciences & Humanities, GLA University, 17 KM Stone, NH-2, Post- Chaumuhan, Mathura, U.P. 281 406, India.
⁵ Amity Center for Mycobacterial Disease Research, Amity University Rajasthan, Kant-Kalwar, Delhi-Jaipur Highway, NH11C, Jaipur 303 002, India. Electronic address: jssohal@jpr.amity.edu.

PMID: 34298067
DOI: 10.1016/j.jim.2021.113105

Abstract

Paratuberculosis is one of the complex livestock infections whose control has largely been hampered due to the lack of efficacious diagnostics. Present study optimized plate ELISA assay for the diagnosis and screening of paratuberculosis using recombinant secretory proteins. Five secretory antigens (2677c, 3547c, 4308c, 1693c, and 2168c) were produced in the recombinant system using the E. coli host and used for the optimization of the assay. These proteins were selected because of their prior proven specificity and antigenicity as humoral immunity markers. The assay was first optimized using traditional ELISA reader and then the performance was evaluated using a handheld ELISA reader. Findings were identical in both traditional ELISA reader as well as handheld ELISA reader. Optimized ELISA was found reproducible using different batches of the recombinant antigens as well as in terms of the inter and intra assay %CV values. The present ELISA has a sensitivity and specificity of 91.6% and 100%, respectively. Also, rELISA revealed AUC_ROC and Youden index J of 0.95 and 0.91, respectively. In conclusion, assay conditions of MAP-recombinant protein-based ELISA were optimized and the optimized ELISA ODs can be read using portable handheld ELISA reader. Thereby, opening a future window to develop assay for onsite testing.

Keywords: ELISA; Paratuberculosis; handheld ELISA reader; herd screening assay; recombinant proteins; secretory antigens.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Antibodies, Bacterial / blood*
Antigens, Bacterial / genetics
Antigens, Bacterial / immunology*
Biomarkers / blood
Buffaloes
Cattle
Early Diagnosis
Enzyme-Linked Immunosorbent Assay / veterinary*
Goats
Immunodominant Epitopes
Mycobacterium avium subsp. paratuberculosis / immunology*
Paratuberculosis / blood
Paratuberculosis / diagnosis*
Paratuberculosis / immunology
Predictive Value of Tests
Recombinant Proteins / immunology
Reproducibility of Results
Serologic Tests / veterinary*
Sheep, Domestic

Substances

Antibodies, Bacterial
Antigens, Bacterial
Biomarkers
Immunodominant Epitopes
Recombinant Proteins