Gururaj V. Kadival

Use of polymerase chain reaction in the diagnosis of abdominal tuberculosis

Background:  Early diagnosis and prompt treatment of abdominal tuberculosis is vitally important as it greatly reduces disease and treatment related morbidity and even mortality in extreme cases. A polymerase chain reaction (PCR) test was evaluated for its feasibility as a diagnostic tool in abdominal tuberculosis (TB) in the Indian scenario.

Detection of antibodies to defined M. tuberculosis antigen (38 kDa) in cerebrospinal fluids of patients with tuberculous meningitis.

Antibodies to the 38 kDa antigen of M. tuberculosis which is serospecific to the tuberculosis complex group of organisms was studied in CSF samples of patients with tuberculosis meningitis. Patients were classified into four groups, viz. post-mortem-proved, culture-proved, clinically suspected and tuberculoma. Anti-38 kDa antibody was detected by ELISA and was positive in 60%, 80% 62.5% and 0%, respectively in the four groups. Controls showed a false-positive detection of 5%. Follow-up of patients was done up to 6 weeks and antibody levels dropped in all the patient groups.

Polymerase chain reaction in the diagnosis of tuberculosis. Comparison of two target sequences for amplification

Amplification of a 340 bp sequence of the 38 kDa protein gene of Mycobacterium tuberculosis by the polymerase chain reaction has been developed. The sensitivity of this PCR was shown to be 10 fg both by agarose gel electrophoresis and Southern blot hybridisation being equivalent to 2-3 organisms and highly specific to M. tuberculosis and excluding even M. tuberculosis H37Ra and Mycobacterium bovis BCG. Sputum samples from patients with pulmonary tuberculosis gave a positivity rate of 45%. PCR was also performed using pt8 and pt9 primers which amplified a 541 bp sequence of IS6110. 41% of the above samples gave positive amplification. Three samples that were positive for 38 kDa sequence were negative for IS6110.

Radioimmunoscintigraphic approach for the in vivo detection of tuberculomas—A preliminary study in a rabbit model

Radioimmunoscintigraphic approach could provide a viable non-invasive alternative for the diagnosis of deeply situated tuberculomas. We have evaluated this in a rabbit model constructed to give a characteristic localized tubercular lesion, with complimentary morphological, histological and antigenic profiles. 131I-anti Myobacterium bovis (BCG) antibody was shown to localize at the lesion, where as 131I-bovine serum albumin and 99mTc-red blood cell scans were negative. The clearance of intradermally-injected tuberculous antigen could be traced into ascending lymph nodes using 131I-anti M. bovis (BCG) antibody.

Use of the Recombinant 38-kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production

The Mycobacterium tuberculosis 38‐kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B‐cell and T‐cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid‐phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme‐linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.

Mycobacterium tuberculosis 38kDa antigen and its encoding gene-experience in diagnostic applications.

ConclusionOur experience has revealed that the detection of 38 kDa antigen or antibody to the antigen in various fluids is useful in diagnosis of various mainfestations of tuberculosis. The PCR developed for 340bp sequence of its encoding gene also shows a high degree of sensitivity and specificity. Thus the 38 kDa antigen/antibody combination or the PCR are ideal for development of kits for diagnosis. These immunoassays to be successful, isolation of the 38 kDa antigen in large quantities is essential. Using recombinant DNA technology and expression inE. coli this has been achieved. However, such recombinant antigen does not have the same immunological properties as the native antigen and hence not suitable in immunodiagnosis. To fully realise the potential of the MoAb defined antigens such as the 38 kDa antigen, 19 kDa antigen and others it is essential to devise alternative vector-host systems that help in glycosylation, do not accumulate as inclusion bodies and can be isolated with less damage.

Study of anti-idiotype antibodies to the monoclonal antibody HGT3a and its relation to the 38 kDa antigen of Mycobacterium tuberculosis.

The hypervariable regions of the immunoglobulins which function as the antigen binding sites are capable of provoking an antibody response and are referred to as anti-idiotypic antibodies. Antisera were raised in rabbits against the idiotypes of a mouse monoclonal antibody HGT3a which binds only to the 38 kDa antigen of the M. tuberculosis complex group of organisms. Idiotype specificity in these antisera was determined by dot ELISA, Western blot and solid phase inhibition assays. In vivo administration of this rabbit anti-idiotypic antibody to Swiss mice evoked an anti-anti-idiotypic antibody response, further confirming the internal antigen mimicry by the anti-idiotypic antibodies of the 38 kDa antigenic epitope and its potential use as a surrogate antigen. Antibody response to the anti-idiotypic antibodies in the sera of patients with pulmonary tuberculosis showed significant correlation with the antibody response to the 38 kDa antigen studied in the same clinical samples indicating a close similarity of the 38 kDa antigen of M. tuberculosis and the rabbit anti-idiotypic antibody produced against MoAb HGT3a.