Design and optimization of Dot-blot ELISA system using specific antigens for simple and rapid diagnosis of glanders

Authors
Sajjad Yazdansetad 1
Nader Mosavari 2
Keyvan Tadayon 3
Iraj Mehregan 1
1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 Department of Tuberculin & Mallein, Razi Vaccine & Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
3 Department of Veterinary Aerobic Bacteria Vaccines, Razi Vaccine & Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
Abstract
Introduction

Glanders is one of the oldest contagious and dangerous zoonotic diseases manifesting ulcerative granulomatous lesions on the skin and mucous membranes. Early methods possessing desirable sensitivity and specificity is important to diagnose the disease considering the just only one case report and preventing disease by identification and eradication. The present study was aimed to design and optimize Dot-blot ELISA system using specific antigens for simple and rapid detection of glanders using equine sera samples.

Materials & Methods

Burkholderia mallei strains were cultured in nutrient broth supplemented with glycerol 4%. Whole cell antigens of bacterial strains were precipitated by trichloroacetic acid (TCA) followed by sonication method. The optimum concentrations of antigen-antibody were determined by checkerboard titration. The nitrocellulose membrane was coated by antigen, then stopped by skimmed milk as a blocker. The suspicious equine serum was added to the membrane following HRP-conjugated antibody, the signals were detected by TMB as a substrate.

Results

4 out of 90 sera samples were positive by Dot-blot ELISA. The specificity and sensitivity of the test were evaluated 96.62% and 100%, respectively. B. mallei culture and isolation from clinical specimens of the horses validated the test.

Conclusion

Our study showed that the Dot-blot ELISA is specific and sensitive in glanders diagnosis and it seems to be practical and efficient test due to the rapid, easy interpretation without any special tools, cost-effective and also field-friendly.
Keywords
Burkholderia mallei
glanders
Dot-blot ELISA
antibody
1.Smith ME, Gossman WG. Glanders and Melioidosis. 2019. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28846298
2. Van Zandt KE, Greer MT, Gelhaus HC. Glanders: an overview of infection in humans. Orphanet J Rare Dis 2013;8:131.
3. Inglis TJJ, Merritt AJ. Burkholderia pseudomallei and Burkholderia mallei. In: Tang YW, Sussman M, Liu D, Poxtron I, Schwartzman J (Eds). Molecular Medical Microbiology. 2nd ed. Academic Press; 2014. p. 769-91.
4. Khan AS, Sage MJ. Centers for Disease Control and Prevention. Biological and Chemical Terrorism: Strategic Plan for Preparedness and Response. Morb Mortal Wkly Rep. 2000;49(RR-4):1-26. Available from: https://www.cdc.gov/MMwr/preview/mmwrhtml/rr4904a1.htm
5. Renart J, Behrens MM, Fernández-Renart M, Martinez JL. 23-IMMUNOBLOTTING TECHNIQUES. In: Eleftherios D, Theodore C (Eds). Immunoassay. San Diego: Academic Press; 1996. p. 537-54. Available from: http://www.sciencedirect.com/science/article/pii/B9780122147302500248
6. Dohre SK, Kamthan A, Singh S, Alam SI, Kumar S. Identification of a new diagnostic antigen for glanders using immunoproteome analysis. Comp Immunol Microbiol Infect Dis 2017;53:26-32.
7. Gerbig Jr DG, Fenk CJ, Goodhart AS. The Dot Blot ELISA: A Rapid & Simple Experiment to Demonstrate Antibody-Antigen Interactions. Am Biol Teach 2000;62(8):583-7.
8. Neubauer H, Sprague LD, Zacharia R, Tomaso H, Al Dahouk S, Wernery R, et al. Serodiagnosis of Burkholderia mallei infections in horses: state-of-the-art and perspectives. J Vet Med B Infect Dis Vet Public Health 2005;52(5):201-5.
9. Naureen A, Saqib M, Muhammad G, Hussain MH, Asi MN. Comparative evaluation of Rose Bengal plate agglutination test, mallein test, and some conventional serological tests for diagnosis of equine glanders. J Vet Diagn Invest 2007;19(4):362-7.
10. Sprague LD, Zachariah R, Neubauer H, Wernery R, Joseph M, Scholz HC, et al. Prevalence-dependent use of serological tests for diagnosing glanders in horses. BMC Vet Res 2009;5(1):32.
11. Jamooli S, Mosavari N, Assmar M. Evaluation of indirect immunofluorescence assay for the diagnosis of bacterial agent of glanders. Iran J Med Microbiol 2016;10(3):1-10.
12. Khan I, Wieler LH, Melzer F, Gwida M, Santana VL d. A, de Souza MMA, et al. Comparative evaluation of three commercially available complement fixation test antigens for the diagnosis of glanders. Vet Rec 2011;169(19):495.
13. Verma RD, Sharma JK, Venkateswaran KS, Batra HV. Development of an avidin-biotin dot enzyme-linked immunosorbent assay and its comparison with other serological tests for diagnosis of glanders in equines. Veterinary Microbiology1990;25(1):77-85.
14. John IM, Kapoor PK, Malik PK. Serodiagnosis of glanders by dot-ELISA using various antigens. Indian J Anim Sci 2010;80(10):1084-6.
15. Elschner MC, Scholz HC, Melzer F, Saqib M, Marten P, Rassbach A, et al. Use of a Western blot technique for the serodiagnosis of glanders. BMC Vet Res 2011;7:4.
16. Nichols WS, Nakamura RM. Agglutination and Agglutination inhibition assays. In: Rose NR, Friedman H, Fahey JL (Eds). Manual of Clinical Laboratory Immunology. 3rd ed. Washington: ASM Press; 1986. p. 49-56.
17. OIE. World Organization for Animal Health [OIE]. Manual of diagnostic tests and vaccines for terrestrial animals. In Paris; 2018. Available from: http://www.oie.int/en/standard-setting/terrestrial-manual/