Identification of a new diagnostic antigen for glanders using immunoproteome analysis

• Published in: Comparative immunology, microbiology and infectious diseases microbiology and infectious diseases, 2017-08, Vol.53, p.26-32
• Main Authors: Dohre, Sudhir K, Kamthan, Aayushi, Singh, Sandeep, Alam, Syed Imteyaz, Kumar, Subodh
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Animals; Antibodies, Bacterial - blood; Antigens; Antigens, Bacterial - blood; Antigens, Bacterial - immunology; Antigens, Bacterial - isolation & purification; Arginine; Arginine deiminase; Burkholderia; Burkholderia mallei; Burkholderia mallei - immunology; Burkholderia mallei - isolation & purification; Chaperonin 60 - blood; Chaperonin 60 - genetics; Chaperonin 60 - immunology; Chromatography; Deoxyribonucleic acid; Diagnosis; Diagnostic software; Diagnostic systems; DNA; DNA-directed RNA polymerase; E coli; Elongation; Elongation factor EF-Tu; Enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Equines; Escherichia coli - genetics; Glanders; Glanders - diagnosis; Glanders - immunology; GroEL; GroEL protein; Horse Diseases - diagnosis; Horse Diseases - immunology; Horse Diseases - microbiology; Horses; Hydrolases - blood; Hydrolases - immunology; Identification; Immunoblotting; Immunoproteins - chemistry; Immunoproteins - isolation & purification; Immunoproteome; Infectious diseases; Malate dehydrogenase; Malate Dehydrogenase - blood; Malate Dehydrogenase - immunology; MALDI-TOF; Peptide Elongation Factor Tu - blood; Peptide Elongation Factor Tu - immunology; Peptide Elongation Factors - blood; Peptide Elongation Factors - immunology; Proteins; Proteomics; Proteomics - methods; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Ribonucleic acid; RNA; Sensitivity and Specificity; Serologic Tests; Translation elongation
• ISSN: 0147-9571; 1878-1667
• DOI: 10.1016/j.cimid.2017.06.007

•Proteomic approach with immunoblotting with equine sera was used to identify new diagnostic targets for glanders.•At least 12 protein antigens e.g. GroEL, EF-TU, EF-TS, arginine deiminase, malate dehydrogenase with diagnostic potential were identified.•GroEL, one of the immunoreactive proteins, was generated as recombinant protein and was evaluated by ELISA with glanders positive and negative serum samples.•The developed ELISA had a sensitivity and specificity of 96% and 98.7% respectively. Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of these proteins, GroEL, was cloned and expressed in E. coli and purified using Ni-NTA affinity chromatography. The recombinant GroEL protein was evaluated in ELISA format on a panel of glanders positive (n=49) and negative (n=79) equine serum samples to determine its diagnostic potential. The developed ELISA had a sensitivity and specificity of 96 and 98.7% respectively. The results of this study highlight the potential of GroEL in serodiagnosis of glanders.