Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/5695—Mycobacteria
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/902—Oxidoreductases (1.)
  • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

• Tuberculosis caused by Mycobacterium, tuberculosis, remains a ixiajor threat to human population, roughly responsible for 2 - 3 millions deaths every year worldwide (1 - 3).
• the secret of the pathogen's success is its ability to escape the host immune system and remain undetected in lungs for decades. Only in 10% of the infected people, the number being higher in immuno-compromised patients, TB erupts as a full-blown disease (4). Delay in diagnosis and treatment impedes the downstream management and control of the disease. With the increasing emergence of multi drug resistant strains and co-infection with HIV the problem is getting further compounded (5 - 7).
• the Mycobacterium, tuberculosis genome carries two isoforms of isocitrate dehydrogenase, M.tb ICD-I and M.tb ICD-2. Multiple sequence alignment revealed a closer similarity of M.tb ICD-I to eukaryotic NADP + dependent ICDs, while M.tb ICD-2 groups with bacterial ICDs (manuscript in preparation).
• ICDs immunogenic markers for tuberculosis through detection of anti-Mtb ICD antibody in sera of different well characterized categories of TB patient through enzyme linked immunosorbent assays.
• the ORFs corresponding to M.tb ICD-I (Rv3339c, 1.230 kb) and M.tb ICD-2 (Rv0066c, 2.238 kb) were PCR amplified from the genomic DNA of H37Rv. BamHl and HmdIII restriction sites were incorporated in the 5' end of forward and reverse primers respectively for both M.tb ICD-I and M.tb ICD-2.
• the primers and parameters for thermal cycle amplification have been tabulated in table 1.
• the amplicons carrying the full length M.tb ICD-I and M.tb ICD-2 were cloned at the BamHl and HmdIII sites of the expression vector pRSET-A (Invitrogen, USA) with six histidine sequence tag at N-terminal.
• the generated constructs 'setAicdl' and 'setAicd2' were further transformed into the BL21 (DE3) strain of E.coli.
• the clones were confirmed by sequencing using the T7 promoter primer, on an ABI prism 377 DNA sequencer (PE Biosystems, USA).
• the genes were over-expressed in the pRSET-A/ E.coli BL-21 (DE3) expression system.
• the over-expressed his-tagged recombinant protein was purified by Ni 2+ -nitrilotriacetate affinity chromatography.
• the cells transformed with the constructs were grown in Terrific Broth (TB) containing ampicillin (lOO ⁇ g /ml) to an OD ⁇ oo of 0.4 to 0.5 at 37°C, cooled to 27°C, induced with 0. ImM isopropyl /?-D-thiogalactoside and grown overnight at 27°C.
• the cells were lysed by sonication, followed by centrifuging at 13000 rpm for 30 minutes at 4°C.
• the clear lysate was loaded onto Ni 2+ -NTA column, which was then washed with 5OmM NaH2PO 4 , 30OmM NaCl 1 2OmM imidazole, pH 8.
• the protein was eluted in the same buffer supplemented with 20OmM imidazole.
• the proteins were 90-95% pure as seen on 10% SDS-PAGE followed by Commasie Blue staining ( Figure 1).
• the purified recombinant proteins were dialyzed against 2OmM TrisCl, pH7.5 with 10OmM NaCl and 3% glycerol and quantified using Bradford Reagent (20).
• Group 3 included patients with extra ⁇ pulmonary tuberculosis.
• Group 1 and group 2 patients were diagnosed by sputum examination (acid-fast bacillus smear positive and negative) while the extra-pulmonary cases were confirmed by tissue biopsy.
• Clinically healthy donors were M. bovis BCG vaccinated and had no symptoms of TB at the time of sera collection. Randomly picked individuals from the population of healthy controls were subjected to PCR test for TB and were found to be PCR negative.
• Mycobacteria other than M. leprae that are not included in the M. tuberculosis complex are referred to as nontuberculous mycobacteria or NTM (21).
• the non - TB patient category included infected individuals who were tested negative for acid fast bacteria by staining and culture based techniques. These patients were also negative for HIV and HBV. These randomly picked patients were suffering from either pneumonia, lower respiratory infections, septicemia, urinary tract infections, gastrointestinal infections, cirrhosis or fever of unknown origin. The study population had no sex or age bias. This study was approved by the Institutional Ethics Committee.
• Immunosorbent assays Enzyme linked immunosorbent assays (ELISA) were performed to check the B cell immune response in human to the M.tb ICD-I and ICD-2 proteins and control antigen HSP 60 and PPD.
• HSP 60 used was M. tb HSP65/GroEL.
• the 96 well microtitre plates (Corning, Costar, USA) were coated with ⁇ 500ng of either control antigens or recombinant M.tb ICD-I and M.tb ICD-2.
• the plates were incubated overnight at 4°C, washed thrice with phosphate buffer saline (PBS) and blocked with 100 ⁇ l of blocking buffer (2% BSA in PBS) for 2 hour at 37°C. The plates were then washed thrice with wash buffer PBST (0.05% Tween 20 in 1 X PBS).
• the M. tuberculosis infected human sera belonging to different clinical groups were diluted 200 times in blocking buffer (1% BSA in PBS) . 50 ⁇ l of sera were added to antigen coated wells followed by incubation for lhr at 37°C.
• HRP activity was detected using a chromogenic substance o-phenylenediamine tetrahydrochloride (Sigma, USA) in citrate-phosphate buffer (pH 5.4) and H2O2 (Qualigens, India) as 1 ⁇ l/ml.
• the reactions were terminated using IN H2SO4, and the absorbance values were measured at 492 nm in an ELISA reader (BioRad, USA). Each ELISA was repeated at least twice with some randomly picked sera samples tested thrice for confirmation, with and without replicates for each sample within individual ELISA.
• the over-expressed N-terminal His-tagged M.tb ICD-I was purified to 95% homogeneity on a Nickel affinity column ( Figure 1).
• the molecular size of the recombinant ICD-I was determined to be 49.2 kDa.
• the purification was carried out under native conditions from soluble fraction with an yield of 3.25 mg protein per 500 ml of start culture.
• M.tb ICD-2 a 83 kDa protein, was purified to 90-95% homogeneity (Figure 1) with an yield of about 20.4 mg per 1000 ml of start culture.
• M.tb ICD-I and M.tb ICD-2 show high reactivity to patient sera as opposed to BCG-vaccinated healthy controls
• Humoral immune responses directed against the M.tb ICD-I and M.tb ICD-2 were compared between patients- with tuberculosis and BCG- vaccinated healthy controls ( Figure 2A and 2B) .
• the recombinant proteins were used to screen the infected and the healthy sera by ELISA ' using anti-human IgG-HRP as conjugates.
• the sera were also tested against M.tb HSO 60 and the purified protein derivative (PPD) ( Figure 2 C and 2D).
• the immunoreactivity of ICD-I, ICD-2, HSP 60 and PPD were statistically analysed and compared with respect to both infected and healthy sera.
• each category yielded p values less than 0.0001 indicating that M.tb ICD-I can differentiate substantially between BCG-vaccinated healthy population and any category of M.tb infected patients, pulmonary or non-pulmonary.
• M.tb ICD-2 could also distinguish substantially between BCG-vaccinated healthy population and any category of M.tb infected patients.
• M.tb ICDs Immunospecificity of M.tb ICDs
• the potential of M.tb ICDs to specifically distinguish between TB, NTMs and non-TB patient sera was tested by examining the cross-reactivity of the recombinant proteins with NTMs and non-TB patient sera. Thirty NTMs and thirty two non-TB patient sera were tested for their immunogenic response against M.tb ICD-I, M.tb ICD-2 and HSP 60. The data were statistically analyzed to check if ICDs could significantly distinguish between TB infected patients and NTMs or non- TB patients.
• M.tb ICD-I and M.tb ICD-2 were evaluated in terms of their immune features as compared to the control antigens HSP 60 and PPD that are ferquently used for diagnosis of tuberculosis.
• ICDs serve as marker of autolysis (16, 17) and are amongst the secretory proteins released during late logrithmic phase. While earlier efforts have pointed to the antigenic potential of M.tb ICDs (17), the present study is the first systematic investigation of their potential as an immune marker.
• M.tb ICD- I and M.tb ICD-2 can be used for diagnosis o ⁇ M. tuberculosis infection even in areas where BCG vaccination is routinely followed.
• the poor performance of PPD can be attributed to its non-specific immune reaction in BCG-vaccinated healthy controls.
• the extrapulmonary TB cases (Group 3) in the study population did not show any significant humoral response to HSP 60 to distinguish them from healthy controls.
• Group 3 patients mounted a very significant B-cell response to ICD-I and ICD- 2, separating them from BCG vaccinated healthy controls.
• Immuno dominance is a parameter that we defined to compare the antibody titers against the tested proteins, i.e, ICD-I , ICD-2 and HSP 60, in the patient sera.
• ICD-I was most antigenic and mounted a very strong B-cell response in all the patient categories, followed by ICD-2 and HSP 60 ( Figure 3). Having shown that M.tb ICDs elicit a B-cell response much higher than HSP 60, we checked for immune specificity of these proteins.
• tuberculosis Even to this day, largely depends on tuberculin skin test and staining and culture techniques. These methods are slow, cumbersome and lack sensitivity and specificity in BCG vaccinated cases. As more and more recombinant antigens are being tested (25 - 31) serological methods are likely to be favoured over others. ELISA per se is unlikely to replace the current tuberculosis diagnosis, however in combination or parallel with other rapid PCR based diagnostic techniques, ELISA can largely improve the accuracy and rapidity of tuberculosis diagnosis for an effective disease management.
• M.tb ICD-I and M.tb ICD-2 can be further analyzed for their pathogen specific antigenic epitopes. Given their important role in the energy cycle, we are currently evaluating these two enzymes of M.tb as possible drug targets. That such important enzymes can also have strong antigenic attributes which enable them to significantly discriminate between BCG-vaccinated healthy controls and TB patients and at the same time TB from other pathogenic infections is a very exciting and novel proposition possibly pointing to their immunomodulatory function.
• Table 1 PCR primers and thermal cycle parameters for amplification of M. tb ICD- I and ICD-2
• Figure 1 Affinity purification of M.tb ICD-I and M.tb ICD-2. Histidine- tagged recombinant protein was purified by nickel column chromatography under native condition and stained with Coomassie Blue following electrophoresis on 10% SDS polyacrylamide gels. The different lanes are: lanes 1 and 2: M.tb ICD-2; lane M: protein molecular size markers (200 kDa, 1 16 kDa, 97 kDa, 66 kDa, 45 kDa, 31 kDa and 21.5 kDa); lanes 3 and 4: M.tb ICD-I .
• FIG. 2 M.tb ICD-I and ICD-2 show high B-cell reactivity to sera from TB infected patients from different groups as opposed to BCG vaccinated healthy controls.
• the humoral immune responses directed against the recombinant proteins, M.tb ICD-I (2A) and M.tb ICD-2 (2B) and control antigens HSP 60 (2C) and PPD (2D) were compared between different categories of patients and healthy controls.
• Group 1 fresh infections
• Group 2 relapsed infection
• Group 3 extrapulmonary TB. The respective sample numbers and p values are shown.
• M.tb ICDs are more immunogenic than HSP 60.
• the ELISA reactivity to M.tb ICD-I, M.tb ICD-2 and control antigen HSP 60 was compared in different patient groups.
• Horizontal bands represent the mean reactivity or average levels of humoral response in each category.
• M.tb ICDs could significantly distinguish TB-infected sera from NTMs and non-TB patient sera.
• Recombinant M.tb ICD-I and M.tb ICD-2 as well as HSP 60 were tested against sera of NTM (4A) and non- tuberculosis patients (4B). The respective humoral responses were compared to TB - infected sera, the p values for which are given in the figures.
• HSP 60 could not distinguish TB-infected patient from either NTM or non-TB significantly.
• Horizontal bands represent the mean reactivity in each category.
• Table 1 PCR primers and thermal cycle parameters for amplification of M.tb ICD-I and ICD-2
• M.tb icd-2 FP AGCTTggatccATGAGCGCCGAACAGCC 94°C for 2' '2.23Kb
• M.tb icd-2 RP CATGGaagcttTCAGCCTTGGACAGCCT (10 Cycles)

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