title: Serodiagnostic potential of immuno-PCR using a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor in tuberculosis patients.
abstract: A novel indirect immuno-polymerase chain reaction (I-PCR) assay was developed for the detection of circulating anti-Ag85B (antigen 85B, Rv1886c), anti-ESAT-6 (early secretory antigenic target-6, Rv3875) and anti-cord factor (trehalose 6,6'-dimycolate) antibodies from the sera samples of pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients and the results were compared with an analogous enzyme-linked immunosorbent assay (ELISA). We covalently attached the amino-modified reporter DNA to the dithiothreitol (DTT)-reduced anti-human IgG antibody through a chemical linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The detection of cocktail of anti-Ag85B, anti-ESAT-6 and anti-cord factor antibodies was found to be superior to the detection of individual antibodies. The sensitivities of 89.5% and 77.5% with I-PCR and 70.8% and 65% with ELISA were observed in smear-positive and smear-negative PTB cases, respectively with high specificity (90.9%). On the other hand, a sensitivity of 77.5% with I-PCR and 65% with ELISA was observed in EBTB cases. The detection of cocktail of antibodies by I-PCR is likely to improve the utility of existing algorithms for TB diagnosis.
authors.affiliation: Centre for Biotechnology, Maharshi Dayanand University (MDU), Rohtak 124001, Haryana, India.
authors.forename: Netrapal
authors.initials: N
authors.lastname: Singh
authors.affiliation: Department of Biostatistics, All India Institute of Medical Sciences (AIIMS), New Delhi 110029, India.
authors.forename: Vishnubhatla
authors.initials: V
authors.lastname: Sreenivas
authors.affiliation: Department of Statistics, MDU, Rohtak 124001, Haryana, India.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Sheoran
authors.affiliation: Centre for Biotechnology, Maharshi Dayanand University (MDU), Rohtak 124001, Haryana, India.
authors.forename: Suman
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Department of TB & Respiratory Medicine, University of Health Sciences (UHS), Rohtak 124001, India.
authors.forename: Krishna B
authors.initials: KB
authors.lastname: Gupta
authors.affiliation: Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India.
authors.forename: Gopal K
authors.initials: GK
authors.lastname: Khuller
authors.affiliation: Centre for Biotechnology, Maharshi Dayanand University (MDU), Rohtak 124001, Haryana, India. Electronic address: pkmehta3@hotmail.com.
authors.forename: Promod K
authors.initials: PK
authors.lastname: Mehta
mesh_terms: D000217:Acyltransferases / Q000097:blood / Q000276:immunology*; D000293:Adolescent; D000328:Adult; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000097:blood / Q000276:immunology*; D001426:Bacterial Proteins / Q000097:blood / Q000276:immunology*; D003311:Cord Factors / Q000097:blood / Q000276:immunology*; D004229:Dithiothreitol / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry; D008301:Maleimides; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012698:Serologic Tests / Q000379:methods*; D014376:Tuberculosis / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D014397:Tuberculosis, Pulmonary / Q000097:blood / Q000175:diagnosis / Q000276:immunology; D055815:Young Adult
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D003311:Cord Factors; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D007074:Immunoglobulin G; D008301:Maleimides; C025285:N-(4-carboxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester; D000217:Acyltransferases; C119774:antigen 85C, Mycobacterium tuberculosis; D004229:Dithiothreitol
keywords: Antibodies; Antigen 85B; Cord factor; ESAT-6; Immuno-PCR; Tuberculosis