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Research Proposal in response to DBT Call for Proposal in the
area of Tuberculosis
Multi protein-Epitope Based SERS
immunoassay for Mycobacterium
Tuberculosis
Submitted By
Prof Chandrabhas Narayana
Chemistry and Physics of Materials Unit,
Jawaharlal Nehru Centre for Advanced Scientific Research
Bangalore
In collaboration with
1. Dr N Jayasuryan
Microtest Innovation Pvt Ltd, ITPL, Bangalore
2. Dr Mynnedu Prasad
Dept of Microbiology, LRS Institute Of TB and Respiratory Diseases,
New Delhi
2
PROFORMA – I
PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON RESEARCH
AND
DEVELOPMENT, PROGRAMME SUPPORT
(To be filled by the applicant)
PART I: GENERAL INFORMATION
1. Name of the Institute/University/Organisation submitting the Project Proposal:
Chemistry and Physics of Materials Unit, Jawaharlal Nehru Centre for Advanced
Scientific Research, Jakkur P.O. Bangalore 560064.
2. State: Karnataka . 3. Status of the Institute: Academic, Deemed University
(Please see Annexure-I)
4. Name and designation of the Executive Authority of the Institute/University
forwarding the application:
A. N. Jayachandra,
Senior Administrative Officer
Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O.
Bangalore 560064.
5. Project Title: Multi Protein-Epitope based biosignature and SERS immunoassay
for Mycobacterium Tuberculosis
6. Category of the Project (Please tick) : R&D/ Programme Support
7. Specific Area (Please see Annexure - II): Section 2.8 (Vaccines and Diagnostics)
8. Duration : 3 Years
9. Total Cost (Rs.) 206.68 Lakhs
10. Is the project Single Institutional or Multiple-Institutional (S/M) ? : M
11. If the project is multi-institutional, please furnish the following:
Name of Project Coordinator: Dr Chandrabhas Narayana
Affiliation : Professor
Address : Chemistry and Physics of Materials Unit, Jawaharlal Nehru Centre for
Advanced Scientific Research, Jakkur P.O. Bangalore 560064.
3
12. Scope of application indicating anticipated product and processes
The time is ripe to move from single markers for correlates of detection/protection to a
biosignature comprising a well-defined set of robust indicators in TB that can accelerate
rapid screening and early selection of potential drug and vaccine candidates. Sensitive
assays for many possible antigens which could be detected in biological systems are still
found wanting. Project proposes to develop a prototype for high sensitivity quantitative
assay which has implementations in: diagnostics, treatment and drug development. An
ELISA based quantitative assay will be developed for a cocktail of selected highly
specific antigens which represent TB infection. Surface Enhanced Raman Spectroscopy
(SERS) will be used to enhance the sensitivity of ELISA and hence it will improvise the
detection of antigens from mycobacterium from clinical samples. Sensitivity and lower
threshold of detection is very critical for TB diagnosis since the amount of antigenic
load in an infected individual is very miniscule. The multiplex SERS-ELISA developed
will be validated for its utility in measuring these antigens in biological fluids from
patient’s samples. Simple, portable field usable equipment can be developed if prototype
works, which will be very useful in diagnosis of TB.
Anticipated Products and Processes:
1) Cloning, expression and purification of TB antigens for R& D use.
2) Production and purification of Monoclonal Antibodies for the proposed
antigens
3) SERS enhanced highly sensitive ELISA based detection assay for proposed
antigens
4) Validation of SERS-ELISA with patients samples for detection of proposed
TB antigens in biological fluids
4
13. Project Summary:
Long-term Objectives of the Proposal:
a) Development of a sensitive Antigen Capture Assay for Mycobacterium
tuberculosis.
b) Enhancement of the sensitivity of the ELISA using Surface Enhanced Raman
Spectroscopy (SERS).
c) Development of field-usable Raman equipment for Point-of-care diagnostics of
Tuberculosis.
Specific Aims:
1. Cloning, expression and purification of TB antigens for R&D use.
2. Production and purification of Monoclonal Antibodies for the proposed antigens.
3. SERS enhanced highly sensitive ELISA based detection assay for proposed
antigens.
4. Validation of SERS-ELISA with patients samples for detection of proposed TB
antigens in biological fluids.
Research Design:
Specific aim 1: Cloning, expression and purification of TB antigens for R& D use:
Selection of Antigens: Antigens of potential diagnostic value will be selected based on
the available information as published literatures as well as genomic information. Some
of them are listed below:
BIOLOGICAL
FLUIDS
ANTIGENS References
CSF * Ag85A (3804c), Ag85B
(1686c)
* 65kD hsp
Wiker and Harboe., 1992
Deng. Set al., 2011
Sputum * Ag85complex
* LAM
Wiker and Harboe., 1992
Periera et al., 2000
Serum * 38kDa*, 16kDa
* Mtb81 (Rv1837c)
Young et al., 1986
Hendrickson et al., 2000
Urine * MT_1721, MT-1694, LAM Kashino.SS et al 2008,
Napolitano et al., 2008,
Periera et al., 2000
Pleural fluid * 43kDa
Ascitic Fluid * Ag85B (1686c), Ag85C
* 43kDa
Wiker and Harboe., 1992
Xiu-Yun He et al., 2011
Additional Unknown Antigens coming from Proteomic analysis of PPD (Preliminary
work done)
5
The listed antigens were identified in the biological fluid of infected individuals. We
would clone and express about ten of the above mentioned antigens and explore their
potential in a diagnostic ELISA format.
The methodology would involve, Polymerase Chain reaction to amplify the various
genes from the Mycobacterium tuberculosis genome, Cloning of the amplified genes
into bacterial expression vectors, over-expression of the genes using the IPTG inducible
system, purification of the expressed proteins using Ni-NTA and various other column
chromatographic techniques.
Specific Aim 2) Production and purification of Monoclonal Antibodies for the
proposed antigens:
The purified antigens would be used to immunize BALB/c mice. The titers of the
antibody produced would be estimated, following which the mice splenocytes will be
fused with a myeloma fusion partner for the production of hybridoma. The hybridoma
would be screen for the antibody production against the proposed antigens. Monoclonals
identifying different epitopes on the antigen will be fine mapped using a panel of
overlapping peptides for the respective antigens. Monoclonals with higher affinity to
the antigens will be scaled up and antibodies will be ammonium sulphate precipitated
and purified on protein A affinity column procedures. The antibody with extremely good
affinity and recognizing different regions of the antigens will be used for capture and
detection antibodies. The detection antibodies will be conjugated with appropriate
enzymes for the ELISA or would be conjugated to different Raman reporter moieties,
for use in SERS.
Specific Aim 3: SERS enhanced
highly sensitive ELISA based
detection assay for proposed
antigens.
Paramagnetic beads with Fe2O3 core
and a SiO2 shell, which is
functionalized and conjugated to
capture antibodies, would be used to
capture the antigens from the
biological fluids. The beads are then
concentrated using magnetic field and
the remaining solution is decanted.
Detector antibody with Raman
reporter molecules are then added
followed by additional of gold nano
particles. The Raman signal is captured by the instrument. Multiple antigens can be
detected at the same time by using multiple capture antibodies and multiple detector
antibodies, conjugated to unique spectrally non-overlapping Raman moieties. This
multiplex SERS-ELISA thus has enhanced sensitivity and specificity.
Specific Aim 4: Validation of SERS-ELISA with patients samples for detection of
proposed TB antigens in biological fluids.
6
The optimized SERS-ELISA protocol would then be used to detect TB antigens
in biological fluids of TB patients. The performance of the assay will be compared with
the commercially available kits. The prototype Raman instrument would be tested in the
clinical set-up and the detection parameters and the procedures would be optimized.
Potential of the research for technological innovation:
The technological innovation is to combine the antigen capture ELISA with Surface
Enhanced Raman Spectroscopy. Paramagnetic beads conjugated to monoclonal capture
antibodies targeting different antigens of Mycobacterium tuberculosis would be used for
capturing TB antigens in biological fluids and the sensitivity of detection would be
enhanced a 1000-fold using Raman reporter moieties and gold nano particles.
Summary of the results:
1) We would have cloned, expressed and purified various antigens of
Mycobacterium tuberculosis.
2) A panel of monoclonal antibodies would be developed towards the proposed
antigens, which can be of use in R&D and for development of diagnostic kits.
3) SERS-ELISA, combining the enhanced sensitivity of SERS and the
dependability of ELISA would augment the sensitivity and specificity of
detection.
4) Point-of-care diagnostic field equipment based on Raman spectroscopy is
envisaged.
7
PART II: PARTICULARS OF INVESTIGATORS
(One or more co-investigators are preferred in every project. Inclusion of co-
investigator(s) is mandatory for investigators retiring before completion of the project)
Principal Investigator:
14. Name: Chandrabhas Narayana
Date of Birth: 08-09-1965
Sex (M/F): M
Designation: Professor , Chemistry and Physics of
Materials Unit, JNCASR, Bangalore
Department: Chemistry and Physics of Materials Unit
Institute/University: Jawaharlal Nehru Centre for Advance Scientific
Research,Bangalore
Address: Jakkur P.O., Bangalore INDIA
PIN: 560064
Telephone: +91 (80) 2208 2810
Fax: +91 (80) 2208 2766
Email: [email protected]
Co-Investigator
15. Name: Dr. N. Jayasuryan
Date of Birth : 03-08-1959
Sex(M/F) : M
Designation : Director
Department : Microtest Innovation Pvt Ltd
Address: G-04, Discoverer Block, Whitefield Road, ITPL.
PIN: 560066
Telephone : 9845004989 Fax ..............…............. E-mail: [email protected]
Number of research projects being handled at present: 2
Co-Investigator
16. Name: Dr Vitthal Myneedu Prasad
Date of Birth: 4th
May 1958 Sex (M/F) : M
Designation : Sr Microbiologist and HOD
Department : LRS Institute of Tuberculosis and Respiratory Diseases
Sri Aurobindo Marg, Near Qutub Minar, New Delhi-110030
Telephone : Fax ..............….....
E-mail: [email protected]
8
PART III : TECHNICAL DETAILS OF PROJECT
(Under the following heads on separate sheets)
16. Introduction (not to exceed 2 pages or 1000 words)
16.1 Origin of the proposal
1. Two-thirds of the world population is infected with Mycobacterium tuberculosis
and the fact that early diagnosis can control further spread of the disease.
2. There is no simple rapid and field applicable point of care diagnosis available for
the disease
3. Available techniques of diagnosis are time consuming, not cost effective as well
as it requires trained individuals to handle it.
4. Immunodiagnostics are the most rapid form of diagnostics available with
sensitivity and specificity.
5. Sensitivity and lower threshold of detection is critical for antigen detection in TB
because very less number of the proliferating organism is present in different
organs and the amount of antigen generated by them is very miniscule.
6. The analysis of Mycobacterium tuberculosis proteome revealed antigens widely
recognized by serum samples from different geographical location, ethnicity and
disease progression.
16.2 (a) Rationale of the study supported by cited literature
1) Even after several decades of research, there is no easy definitive diagnostic
test available at primary health centers for TB diagnosis, excepting the gold
standard of a century old smear examination procedure.
2) A plethora of antigens is available in Mycobacterium tuberculosis genome with
potential to be developed into a diagnostic assay.
3) Mass spectrometric analysis of PPD has shown huge number of antigens which
shows specificity to Mycobacterium tuberculosis, but yet to be analyzed for
their bioavailability to assess their potential for diagnosis.
4) Antigen detection if achieved will have definitive advantage than any other
methodology because uniformly it could be detected in people who have active
tuberculosis.
5) Field usable simple gadgets can be developed for detecting the disease at a
point-of-care diagnostics.
6) Immune response detection in terms of antibody detection has several
problems and this can be surmounted by antigen detection.
7) We already have developed a similar technology for HIV-1 detection (patent
office of Republic of South Africa - Patent Number 2009/03128 in 2009) and
hence we have a strong hold in implementing the same for TB diagnostics.
9
(b) Hypothesis
New reagents both purified antigens and antibodies would provide the means to
sensitive and specific assay system for the diagnosis of the disease. A biosignature
comprising cocktail of important biomarkers will make the assay highly specific.
Surface Enhanced Raman Spectroscopy (SERS) technology would enhance the
detection of antigens which are immunoreactive but present in trace amounts. The
enhancement of ELISA sensitivity by SERS is about a 1000-fold. Antigens which are
specific to Mycobacterium tuberculosis and present in body fluids would be preferred
target for specificity in diagnosis. Combination of antigens would enhance the
sensitivity and specificity of the assay system. Our group has successfully applied this
technology for HIV.
(c) Key questions.
1. Can we combine the robustness of ELISA and sensitivity of SERS technology
and to improve sensitivity of detection and build a diagnostic assay for TB?
2. By including novel antigen(s) which has a possibility to be detected in
biological fluids, can we develop a newer diagnostic kit?
3. Can detection of multiple antigens and synergistic accumulation of signals
from multiple Raman reporters enhance the specificity and sensitivity of
detection?
4. Can we detect in biological specimens like sputum and serum, circulating
antigens which will give a definitive diagnosis for smear negative, culture
positive cases?
5. Can we develop field-usable portable Raman equipment for point-of-care
diagnosis of tuberculosis infection?
16.5 Current status of research and development in the subject (both
international and national status)
Although molecular diagnostics are simple and rapid in approach its usability remains
inaccessible to remote areas and middle income group. Analysis of the genome of M.
tuberculosis and M.bovis (vaccine strain) has revealed certain regions of difference
(RD). Quanti-FERON-TB Gold (Cellestis, Carnegie, Australia) (Chegou et al., 2009)
and T-Spot TB test (Oxford Immunotec, Oxford, United Kingdom), which is based on
RD1 antigens ESAT-6 and CFP-10, have been evaluated in clinical studies and showed
better performances than that of TST (Meier et al., 2005). A card based antigen MPT-64
detection from culture supernatant is available for routine diagnosis from BD bioscience
and Standard Diagnostics, INC.
Novel diagnostic tests have emerged as promising alternative to the previously available
tests. These include, IGRA (Interferon gamma release assay) like QuantiFERON-TB
Gold assay and T-SPOT®.TB test. IGRAs are in vitro blood tests that are based on
interferon-gamma (IFN- γ) release after stimulation by TB specific antigens (e.g. ESAT-
10
6 and CFP-10) (Diel et al., 2009). However, IGRA assay meets gold standard for active
state of TB but not for LTBI (Walzl et al.,2011). IGRA yields discordant results
depending upon age, BCG vaccination and migration (Machado et al., 2009). Xpert
MTB/RIF is a highly sensitive, rapid nucleic acid amplification test that identifies
targeted nucleic acid sequence in TB genome by real time PCR and can detect
rifampicin resistant TB. But Xpert MTB/RIF is expensive and cannot detect LTBI.
Over the last few years, there has been a great deal of effort in various laboratories to
develop SERS techniques for use in biomedical diagnostics, pathogen detection, gene
identification, gene mapping, and DNA sequencing. There have been attempts by
various groups to develop techniques to identify and detect the reactions of individual
molecules in living cells with improved spatial and temporal resolution. Raman imaging
is emerging as a rapid and nondestructive analytical tool that yields highly compound-
specific information for chemical analysis and has great potential for high-throughput
analysis and direct imaging. Recent reports have shown an increasing trend in applying
Raman spectroscopy in cellular and tissue imaging studies (Peltier et al., 2002; Van de
Poll et al., 2002; Timlin et al., 2000; Schaeberle et al., 1999). The development of SERS
techniques for biological analysis and medical diagnostics has been investigated some of
the laboratories with varying amounts of success (Dinh et al., 1988; 1998; 2002; Stokes
et al., 2000; Allain et al., 2002; Culha et al., 2003; ). In addition, SERS studies on living
cells using gold nanoparticles (Kneipp et al., 2002) and coherent anti-Stokes Raman
scattering (CARS) for cellular imaging have been reported (Zumbusch et al., 1999). In
the national scenario, SERS has been used to demonstrate the potency of the substrates
developed either as thin films or as nanoparticles. A vast literature exists on the
synthesis of nanoparticles for SERS applications, but seldom any of these techniques
have been used and demonstrated for applications of bio-diagnostics or Raman imaging.
We strongly believe that we are one of the only a few groups in India working on bio-
diagnostic applications of SERS in biological systems.
16.6 The relevance and expected outcome of the proposed study
Point of care diagnostics is not available for TB diagnosis. With nearly two-
thirds of the Indian population being affected, there is a strong need for rapid diagnosis.
The outcome of the proposed study is a field-usable Raman instrument which combines
ELISA and Surface enhanced Raman Spectroscopy, thus enhancing the sensitivity,
specificity and rapidity of diagnosis. Multiple antigens can be captured simultaneously
on paramagnetic beads with capture antibodies, and they can be differentially recognized
using non-overlapping, spectrally distinct Raman reporter, which enhance the signal of
ELISA by a 1000-fold. To our knowledge, we would be the first people in India to use
this technology for TB detection and this has a possibility of developing new IPR and
rapid diagnosis is the need of the hour.
16.7 Preliminary work done so far
11
1. Identification of proteins present in PPD from Mycobacterium tuberculosis as a
collaborative project by Microtest Innovations Pvt Ltd
We carried out a proteomic profiling of Connaught Tuberculin (CT68), prepared from a
human strain of M. tuberculosis cultured using a protein-free medium, using high
resolution Fourier transform mass spectrometry (LTQ-Orbitrap Velos). Mass
spectrometry-derived data was searched using - Sequest algorithm embedded in the
Proteome Discoverer 1.3.0.339 software (Thermo Scientific, Bremen, Germany) against
a protein database of M. tuberculosis from NCBI RefSeq. Search of 5,205 MS/MS
spectra were resulted in 1,146 peptide-spectrum matches, which corresponded to 695
unique peptides. Representative MS/MS spectra are provided in Figure 1. Based on
these 695 unique peptides, we identified 265 proteins of M. tuberculosis in Connaught
Tuberculin (CT68).
Figure 1: Representative MS/MS spectra for proteomic profiling of Connaught
Tuberculin (CT68), prepared from a human strain of M. tuberculosis.(Manuscript
under preparation)
2. Development of monoclonals and p24 capture assay at Microtest innovations pvt
ltd.
Microtest Innovation is exploring diagnostic possibilities & its implication in
understanding more about the disease process in our country. Main area of expertise is
immuno diagnostics, genetic diagnostics, recombinant protein expression, purification,
refolding & molecular biology. The expertise MTI has in immuno biology & molecular
biology are being capitalized in development of newer assays. MTI has excellent
expertise in the entire spectrum of antibody generation & characterization starting from
12
high precission monoclonal, polyclonal &
recombinant antibodies. MTI with collaborating
team has excellent expertise in the area of
virology, one of them being HIV which also
includes mapping B cell epitope of some of the
important antigens of HIV to develop
appropriate Immuno diagnostics in this area.
While infectious disease being the priority, MTI
team is critically looking in other areas of
medicine like cardiovascular disease,
endocrinology & cancer. MTI is developing
different research assays with acute sensitivity
and specificity. Contract research is another area
of operation. Microtest is also building a good
Bioinformatic and structural biology team to
cater to all our needs in the development
process.
Immuno Diagnostics- Production, purification and characterization of Monoclonal
Antibodies:
Monoclonal antibodies have been generated to p24 (from HIV) and have been purified
and characterized. The epitope recognizing these antibodies have been determined. This
includes
Generation and characterization of functional monoclonals
Gene cloning and expression
Scaling up and purification of monoclonals
Characterization in terms of mapping of epitopes
Identifying different classes of antibodies
Detecting affinity constant of monoclonals
Generating labeled antibodies
Identifying functional antibodies
13
Development of Functional Monoclonal Antibody for Taq Polymerase:
Anti Taq Polymerase antibody was developed which binds to an antigen and has an
effect that can be demonstrated in laboratory tests.
14
3. Surface-enhanced Raman spectroscopy (SERS)
We have now developed magnetic core gold or silver shell nanoparticle which support
the adsorption or chemically linking the antibodies on them. The reagents can then be
immobilised easily within an ependoff by placing the tube in magnetic field. The key to
this technique is that the nanoparticles are paramagnetic as well as they are not bigger
than a few hundred nanometers. Since we can decant the supernatant after immobilizing
the reagents, we remove the washing cycle all together as well as we do the whole
reaction in solution. This reduces not only the time but also increases the sensitivity of
the detection.
In order to validate their novel approach to SERS immunoassay, we have tested the
method on the well-known HIV kit, where we used the Raman tag as the OPD, which
upon oxidation produces an azo compound, which has a very strong Raman signature.
Using this we have been able to increase the sensitivity of the ELISA by 2 orders of
magnitude as shown in the Figure 1.
0.0
0.5
1.0
1.5
2.0
10 1 0.1 0.01 0.001 0.0001 No Ag
Antigen concentration (ng/ml)
Ab
s (
49
5 n
m)
0.0
0.5
1.0
1.5
2.0
10 1 0.1 0.01 0.001 0.0001 No Ag
Antigen concentration (ng/ml)
Ab
s (
49
5 n
m)
500 1000 1500
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
0.0001 ng/mL0.001 ng/mL
0.01 ng/mL
0.1 ng/mL
1 ng/mL
10 ng/mL
control
Inte
nsi
ty (
a.u
.)
Raman shift (cm-1)
• NIH kit sensitivity is 1 ng/ml in ELISA (Rs. 4,000)
• Assay sensitivity is enhanced by 100-fold by SERS ) 0.01 ng/ml
0.0
1.0
2.0
3.0
4.0
10 1 0.1 0.01 0.001 0.0001 No Ag
Antigen concentration (ng/ml)
SERS
Absorbance
ELISA only
ELISA-SERS
Comparison of ELISA vs ELISA-SERS using
low-sensitivity HIV-1 p24 kit
15
We were able to determine the presence of this marker in solution at a concentration as
low as 1-10 picograms per millilitre. This is about 100 to 1000 times more sensitive than
ELISA and the assay time took less than 1 h, including the optical detection steps.
Their proof of principle not only bodes well for developing the approach for other
biomarkers but could also have clinical application for assaying for TB.
17. Specific objectives (should be written in bulleted form, a short paragraph indicating
the methods to be followed for achieving the objective and verifiable indicators of
progress should follow each specific objective)
Objective 1
Cloning, Expression and Purification of proposed antigens
Method(s) to be followed:
The genes related to these antigens will be PCR amplified, cloned and expressed using
IPTG induction protocol. The recombinant proteins will be purified by a combination of
standard methods of column chromatography and salt precipitation etc.
Variable indicators of progress:
1. Recombinant expressed TB antigens and expression vectors.
2. Highly purified antigens
Objective 2
Development of highly specific Monoclonal antibodies for the proposed antigens.
Method(s) to be followed:
1. Bioinformatics analysis for epitope sequences for generation of highly specific
monoclonal antibodies.
2. Custom synthesis of peptides.
3. Immunization in Balb/C mice for antibody production.
4. Generation and Screening of Hybridomas for the proposed antigens.
5. Purification and validation of monoclonal antibodies.
Variable indicators of progress:
Purified and characterized monoclonal antibodies that recognize the recombinant and
native proteins from Mycobacterium tuberculosis
Objective 3
Development of Antigen Capture ELISA
Method(s) to be followed:
16
Monoclonal antibodies to the proposed antigens would be generated, following which
the higher affinity antibodies would be used as Capture and/or detector antibodies.
Using these antigen capture assay/ Sandwich ELISA would be developed.
Variable indicators of progress:
1. Two different monoclonal antibodies for each antigen targeting different epitopes.
2. Optimization of Sandwich ELISA.
Objective 4:
Increasing the sensitivity of the assay using SERS
Method(s) to be followed:
Paramagnetic beads with Fe3O4 core, coated with SiO2, which would be functionalized
and linked with the monoclonal Capture antibodies. The detector antibodies would be
conjugated with different Raman reporter moieties. Addition of gold nanoparticles will
enhance the signal from Raman reporters which can be detected and analyzed using the
equipment.
Variable indicators of progress:
1. Preparation of paramagnetic beads.
2. Conjugation of antibodies to beads.
3. Conjugation of Raman reporter moieties to antibodies.
4. Optimization of detection of complex.
Objective 5
Clinical Validation
Method(s) to be followed:
Culture positive smear negative and smear positive samples would be categorized and
the patients various biological fluids like sputum, serum, urine etc would be tested using
the optimized SERS-ELISA and the results compared and validated using established
methodologies.
Variable indicators of progress:
1. Analysis of sensitivity and sensitivity of our assay with existing methodologies.
2. A field usable prototype Raman equipment.
17
18. WORK PLAN
Specific aim 1: Cloning, expression and purification of TB antigens for R& D use:
a) Selection of Antigens: Antigens of potential diagnostic value will be selected
based on the available information as published literatures as well as genomic
information. The already identified secreted antigens which are of diagnostic
importance is listed as a table below
BIOLOGICAL FLUIDS ANTIGENS
CSF
* Ag85A (3804c), Ag85B (1686c)
* 65kD hsp
* 43kDa antigen
Sputum * Ag85complex
* LAM
Serum * 38kDa*, 16kDa
* Mtb81 (Rv1837c)
Urine * MT_1721, MT-1694, LAM
Pleural fluid * 43kDa
Ascitic Fluid * Ag85B (1686c), Ag85C
* 43kDa
The listed antigens were identified in the biological fluid of infected individuals. We
would clone and express about ten of the above mentioned antigens and explore their
potential.
b) Cloning and expression of the selected antigens:
Polymerase Chain Reaction (PCR):
PCR amplification of the genes encoding the proteins using gene specific 5’ and 3’
primers. PCR will be carried out by standard methods of amplification.
Cloning in expression vectors for recombinant expression:
The PCR fragments will be gel purified using the PerfectprepR gene clean up kit from
Eppendorf per the manufacturer’s instructions and taken for digestion. The PCR
fragments will be digested with appropriate enzymes selected for cloning into pET21B
for E.coli expression. The transformation and selection of clones from all the expression
systems can be done by using the standard molecular biology protocols.
Sequencing of the clones:
All the genes cloned in pET21B for E.coli expression will be completely sequenced by
dideoxy chain termination method (outsourced to MWG Biotech) before proceeding
with expression and purification.
Protein expression and purification:
The confirmed clones will be checked for expression by IPTG induction protocol which
can be further scaled up after expression optimization at different temperature, time
scale and IPTG concentrations. The purification of the recombinant protein from the
18
expression systems would be by a combination of standard methods of column
chromatography and salt precipitation etc. and would have to be optimized individually
for each of the proteins. The genes are cloned into pET21b (+) backbone and the genes
would have a C-terminal 6X-Hisdine Tag. The proteins expressed can purified by the
standard Ni-NTA technology.
Specific Aim 2) Production and purification of Monoclonal Antibodies for the
proposed antigens:
a) Immunization
A group of young BALB/c mice, six to eight weeks old will be primed subcutaneously
with 50 µg of protein/mouse emulsified in CFA (Complete Freund’s adjuvant). This will
be followed by a few boosters of the antigen in IFA (Incomplete Freund’s Adjuvant).
Antigen specific antibody titers will be evaluated in ELISA. Mice generating highest
immune response will be used for fusion and hybridoma generation. Three days prior to
fusion, selected mice will be given a final booster with soluble antigen without adjuvant
through the tail veil.
b) Fusion and hybridoma selection
One week before fusion the myeloma cell line P3X63/SP20 will be expanded in
complete DMEM medium to obtain 10 x 106 cells on the day of fusion. One day before
fusion, cells will be split into fresh medium. Spleens from the selected mice will be
harvested aseptically, a homogenous single cell suspension of splenocytes will be
prepared and the cells washed with complete DMEM medium without serum. Cell
number will be determined and both the fusion partners will be mixed at equal
proportion. Cell fusion will be performed at 37ºC using 50% PEG according to standard
protocols. Following fusion, cells will be suspended in complete DMEM medium with
20% FCS at a cell density of 2.5 x 106 cells/ml. 100 l of cell suspension will be
dispensed per wells in a number of 96-well plates and the plates will be incubated for 24
hours. On day-2, 100 µl of complete medium supplemented with HAT will be added to
each well. During the subsequent week, every day, half the medium from each well will
be replaced with fresh medium. HAT selection will be imposed for 14 days and on day
15; cells will be fed with HT medium. The subsequent day cells will be fed with
complete medium without HT. During the following days, all the wells with cell
proliferation will be marked and the spent medium will be rapidly screened for antibody
in ELISA and positive wells will be expanded. Screening will be performed using
different recombinant proteins or peptides. Several clones of hybridoma will be
systematically analyzed and stored (frozen) in aliquots.
c) Screening of the hybridoma:
Selected hybridoma will be grown in culture medium at high cell density to generate
adequate concentration of monoclonal antibodies. Antibodies will be purified by
ammonium sulfate precipitation, dialyzed and protein concentration determined.
19
The culture supernatant antibodies will be ammonium sulphate precipitated and purified
on protein A affinity column. This antibody will be further characterized for its class and
sub-class and will be utilized for developing quantitative assay for western blot analysis
and for neutralization and for diagnostic development depending upon the need. We will
use these assays to quantify infected samples. The protein concentration of the purified
antibodies will be evaluated and monoclonal antibodies will be stored in smaller aliquots
at -20ºC.
d) Fine mapping of the monoclonal antibodies:
Epitopes of the antigen identified by the monoclonal antibodies will be characterized
using the synthetic peptides. A panel of overlapping peptide will be designed,
synthesized and will be used to fine map the monoclonals. Antibodies recognizing
different epitopes will be used as antibody pairs in the capture assay.
e) Develop quantitative assays using these reagents:
The antibody with extremely good affinity and recognizing different regions of the
antigens will be used for capture and detection antibodies. The detection antibodies will
be conjugated with appropriate enzymes for the ELISA and they would be conjugated to
different RAMAN reporter moieties, for use in SERS. Standard would be made using
recombinant proteins and these assays will be validated for antigen detection in
biological fluids.
Specific Aim 3: SERS enhanced highly sensitive ELISA based detection assay for
proposed antigens.
a) Enhanced detection of OPD derivative:
Conversion of o-phenylenediamine to azoaniline by peroxidase is a common
reaction of routine ELISA. Azoaniline is a highly reactive Raman reporter. We
tested if the formation of azoaniline could help enhance the sensitivity of the
HIV-1 p24 ELISA if
SERS of this compound,
instead of the color, is
determined. Using two
different commercial p24
kits of different
sensitivity, we
demonstrated that by the
application of silver
nanoparticles and SERS
detection, the detection
sensitivity of both of the kits could be enhanced by two orders of magnitude as
compared to colorimetry (Figure-1). This result has direct implication on the
novel Raman reporter dyes we would like to develop through the present project.
b) Preparation of paramagnetic beads:
20
We have prepared several Fe2O3 based
and silica and PEG-capped
paramagnetic beads of uniform size
(Figure-2:). These beads will be
instrumental in extracting TB antigens from
the biological fluids. One such preparation
is shown. These paramagnetic beads can be
functionalized on the outer silica, which
will allow conjugation of the monoclonal
capture antibody.
c) Optimization of SERS-ELISA:
The paramagnetic beads coupled to capture antibody would react with the
biological fluid containing the TB antigens and the antigens would be get bound
to the beads. The beads, along with the sample are incubated for a small period
of time and the tubes are then exposed to magnetic field. Unbound antigens and
samples are eliminated by just decanting the sample tube. The bound beads are
then disbursed in the releavant solution and the second antibody, specific to a
different epitope of the antigen are added. The detector antibody used in this
reaction is already conjugated to a Raman reporter moiety. After the incubation
period, the sample tube is exposed to magnetic field and the unbound samples
are eliminated. Gold nanoparticles are then added to the reaction and the signal
that comes from the Raman reporter are then recorded in the instrument. Signal
is obtained only when all the three components are present together, namely, the
Capture antibody, the antigen and the detector antibody. The basic principle is
represented in a schematic below:
21
The strengths of this approach are:
1) There are no special washing steps required.
2) The time duration for the assay is about an hour only.
3) Combination of beads conjugated to different monoclonals can be used to
capture multiple antigens, which gives the strength of multiplexing.
4) Different Raman reporters can be conjugated to different detector antibodies,
thereby, enhancing the multiplexing and help in distinguishing the antigen
present.
5) The assay has a very high signal to noise ratio.
6) The assay is a 1000-fold more sensitive than the conventional ELISA.
Specific Aim 4: Validation of SERS-ELISA with patients samples for detection of
proposed TB antigens in biological fluids.
Firstly, ELISA would be optimized for the different monoclonal pairs and their
corresponding purified recombinant antigens. The ideal capture and detection antibody
pair would be identified using a matrix analysis and affinity based titrations. The affinity
of the antibody antigen reactions would be evaluated using competitive ELISA, in the
presence of chaotropic agents. Once the antigen-capture assay is optimized, the
sensitivity of the ELISA will be enhanced using SERS technology. The optimized
SERS-ELISA protocol would then be used to detect TB antigens in biological fluids of
TB patients. The performance of the assay will be compared with the commercially
available kits. The prototype Raman instrument would be tested in the clinical set-up
and the detection parameters and the procedures would be optimized.
Alternate strategies (if the proposed experimental design or method does not work
what is the alternate strategy):
We have previously optimized the capture and detection of HIV-1 single
stranded DNA detection using this SERS technology. ( A high sensitivity assay for
molecular typing of biological sample, probes and a kit therof - Inventors: Ranga
Udaykumar, Chandrabhas Narayana and N. Jayasuryan - patent granted by patent
office of Republic of South Africa - Patent Number 2009/03128 in 2009.)
We would employ the similar strategy for detection of M. tuberculosis genetic material
in the sample using the same technology.
22
The methodology followed is outlined below:
a) Capture and detection of HIV single-stranded DNA to solid substrate: The
objective of this experiment is to ensure the specificity of the capture and detector
probes for the viral nucleic acid. Single-stranded DNA of the viruses representing
the viral LTR was generated in the laboratory using asymmetric PCR. Three
capture probes (CP-1, specific probe, should capture viral DNA; CP-2 and CP-3
mutant probes, should not capture the viral DNA) were chemically cross-linked to
glass surface. Different amount of viral DNA were captured and d etected with a
detector probe conjugated to Rhodamine-6-G. Subtype-specific Raman signals
were detected only with CP-1 but not with other two probes confirming the assay
specificity. Sensitivity of the assay is approximately 104 molecules with a 1
second acquisition time (Figure-3). Sensitivity of the assay could be enhanced by
scanning the entire spot, using high power lasers, increasing acquisition time and
other molecular manipulations to improve the detection probe.
b) Capture and detection of HIV
RNA to solid substrate: Full-length
viral RNA, isolated from 500 μl of
culture supernatants, was directly
captured and detected in this
experiment using a strategy similar to
‘a’ above. Viral RNA was specif
ically detected by CP-1 but not CP-2
23
at two different template concentrations (Figure-4). This result ascertained that
our novel strategy can detect the viral RNA without amplification.
Similar approach can be used for the
detection of M. Tb DNA from the
biological samples. Considering the
genome of M. Tb is so vast in
comparison with HIV-1 genome, it will
be easier to design multiple specific
windows in the genome of M. Tb, which
are highly suitable for the capture and
hence the detection.
Connectivity of the participating institutions and investigators (in case of
collaborating / multi- institutional projects only)
Microtest Innovation
Pvt Ltd.
• Cloning, expression and purification of Antigens
• Generation of monoclonal antibodies and component Development
• Setting up quantitative ELISA
JNCASR
• Subject matter expert on SERS. Enhancing the sensitivity of ELISA kit by SERS
(Clinical Group at
LRS)
• Providing Clinical Samples for Assay Development
• Assay Validation
JNCASR will
Supervise and
coordinate the project
24
References:
1. Allain,L.R., T. Vo-Dinh. (2002) Anal. Chim. Acta 469, 149 410.
2. Chegou,N.N. Gillian F Black, Martin Kidd, Paul D van Helden and Gerhard
Walzl.(2009), BMC Pulmonary Medicine 9:21.
3. Culha, M., D. L. Stokes, L. R. Allain, T. Vo-Dinh. (2003) Anal. Chem. 75, 6196
410.
4. Danielle R. Napolitano, Nira Pollock, Suely S. Kashino, Virmondes Rodrigues,
Jr.,3 and Antonio Campos-Neto. (2008) Clinical and Vaccine Immunology, Apr.,
p. 638–643.
5. Diel, R., et al., Chest, (2009). 135(4): p. 1010-8.
6. Dinh, T.V., L. R. Allain, D. L. Stokes. (2002) J. Raman Spectrosc. 33, 511 410.
7. Dinh, T.V. (1998) Trends Anal. Chem. 17, 557 410.
8. Dinh,T.V., A. Alak, R. L. Moody. (1988) Spectrochim. Acta B 415, 605.
9. Hendrickson, R.C., John F. Douglass, Lisa D. Reynolds, Patricia D. Mcneill,
Darrick Carter, Steven G. Reed, AND Raymond L. Houghton. (2000) Journal of
Clinical Microbiology, June, p. 2354–2361.
10. Kashino.SS et al (2008) Clin Exp Immunol. Jul;153(1):56-62.
11. Kneipp,K., A. S. Haka, H. Kneipp, K. Badizadegan, N. Yoshizawa, C. Boone,
K. E. Shafer- Peltier, J. T. Motz, R. R. Dasari, M. S. Feld. (2002) Appl.
Spectrosc. 56, 150 410.
12. Machado, A., Jr., et al.,. (2009) Int J Tuberc Lung Dis,. 13(4): p. 446-53.
13. Meier T, Eulenbruch HP, Wrighton-Smith P, Enders G, Regnath T (2005). Eur.
J. Clin. Microbiol. Infect. Dis. 24 (8): 529–36.
14. Pereira arias-bouda, Lan N. Nguyen, Ly M. Ho, Sjoukje Kuijper, Henk M.
Jansen and Arend H. J. Kolk. (2000) Journal of clinical microbiology, June, p.
2278–228.
15. Schaeberle, M.D., H. R. Morris, J. F. Turner, P. J. Treado. (1999) Anal. Chem.
71, 175A 410.
16. Shafer-Peltier., K.E., A. S. Haka, M. Fitzmaurice, J. Crowe, J. Myles, R. R.
Dasari, M. S. Feld: (2002) J. Raman Spectrosc. 33, 552 410.
17. Stokes,D.L T. Vo-Dinh: (2000) Sens. Actuat. B Chem. 69, 28 410.
18. Timlin,J.A., A. Garden, M. D. Morris, R. M. Rajachar, D. H. Kohn. (2000)
Anal. Chem. 72, 2229 410.
25
19. Van de Poll, S.W.E., T. C. Bakker Schut, A. Van der Laarse, G. J. Pupples.
(2002) J. Raman Spectrosc. 33, 544 410.
20. Walzl, G., et al., 2011. Nat Rev Immunol. 11(5): p. 343-54.
21. Wiker, H.G and Harboe M. (1992). Microbiological reviews, Dec. 1992, p. 648-
661.
22. Young,D., L. Kent, A. Rees, J. Lamb, AND J. Ivanyi. (1986) Infection and
Immunity, oct., p. 177-183.
23. Zumbusch,A., G. R. Holtom, X. S. Xie. (1999) Phys. Rev. Lett. 82, 4142 410.
26
19. Timelines: (Please provide quantifiable outputs)
Period of study Achievable targets
6 Months
1. Procurement of chemicals, consumables, equipments.
2. Identification of epitopes and Peptide synthesis and purification.
3. Cloning and expression of Antigens.
4. Development of SERS molecules with high SERS cross section
12 Months
1.Cloning and Expression of Antigens
2. Purification of antigens.
3. Immunization and evaluating the antibody titers.
4. Development of magnetic and noble metal nanoparticles for various
laser excitations, optimization, characterization.
18 Months
1. Hybridoma Screening for Monoclonals.
2. Purification of Monoclonal antibodies.
3. Development of both solution-based and substrate based assays for
SERS detection.
4. Development of SERS technique to detect the antigen-antibody
complex (biosystems) in very low concentrations.
24 Months
1. Development of ELISA using combinatorial approach including all
the antigens.
2. Validation with patient samples in the clinical set-up.
30 Months
1. Development of Raman system for optimum detection with
combination of lasers and microscope objectives.
2. Applying SERS to enhance the sensitivity to standardized ELISA
protocol.
3. Standardization of Protocol and Validation of ELISA kit as per ICH
guidelines.
36 Months
1. Screening clinical samples and Validation.
2. Development of Raman system for optimum detection with
combination of lasers and microscope objectives.
3. Development of prototype of the assay with the Raman system
developed for diagnostics.
20. Name and address of 5 experts in the field Sr.No. Name Designation Address
27
PART IV: BUDGET PARTICULARS
Jawaharlal Nehru Center For Advanced Scientific Research
Budget (In Rupees)
A. Non-Recurring (e.g. equipments, accessories, etc.)
S.
No.
Item Year 1 Year 2 Year 3 Total
1.
Laser source 488 nm Rs. 4
lakhs
-NA- -NA-
2. Laser source 785 nm Rs. 8
lakhs
-NA- -NA-
3. Ultra long working distance
Microscope Objectives
(20x and 2 No. and 50x and 2
No.)
Rs. 8
lakhs
-NA- -NA-
4. Raman Edge Filter sets Rs. 5
lakhs
-NA -NA-
5. Microscope stage Cryostat
(From Room temperature to 4 K
and 1 No.)
Rs. 25
lakhs
-NA- -NA-
Sub-Total (A): 50 Lakhs
B. Recurring
B.1 Manpower (See guidelines at Annexure-III)
S.
No.
Position
No.
Consolidated
Emolument
Year 1 Year 2 Year 3 Total
1. JRF Rs. 16000 + Rs
250 Medical (for
Year 1 and
2)+30%HRA
7,48,800 7,48,800 8,42,400 23,40,000
Sub-Total (B.1) =23.4 Lakhs
B.2 Consumables
S.
No.
Item
Quantity Year 1 Year 2 Year 3 Total
10.00 10.00 10.00 30.00
Sub-Total (B.2) =30.00 Lakhs
Other items Consolidated
Emolument
Year 1 Year 2 Year 3 Total
B.3 Travel
1.00 1.00 1.00 3.00
28
B.4 Contingency
1.00 1.00 1.00 3.00
B.5 Overhead
(If applicable)
3.96 3.96 3.96 11.88
Sub-total of B
(B.1+B.2+B.3+B.4+B.5)
71.28
Lakhs
Grand Total (A + B) 121.28
Justification: High end equipments are required for successful implementation of
the project. High grade analytical reagents are required for the synthesis of
paramagnetic particles and Gold Nanoparticles. A minimum of 1 JRF is required
to carry out the work.
29
PART V: EXISTING FACILITIES
(Jawaharlal Nehru Centre for Advanced Scientific Research)
Resources and additional information
1. Laboratory:
a. Manpower
Five Ph.D students, One Research Associate and 2 project assistants
b. Equipments
We have a well-equipped central instrumentation facility at the
university.
Custom built Raman spectrometer
Atomic Force Microscope
Tramission Electron Microscope
Field emission Scanning Electron Microscope
Confocal Microscope
Mass spectrometer
Flow cytometer
ELISA reader
Laminar Hoods
C02 incubators
2. Other resources such as clinical material, animal house facility, glass house.
Experimental garden, pilot plant facility etc.
We have a well equipped animal house in the campus.
30
PART IV: BUDGET PARTICULARS
Microtest Innovations, Pvt Ltd.
Budget (In Rupees)
A. Non-Recurring (e.g. equipments, accessories, etc.)
S.
No.
Item Year 1 Year 2 Year 3 Total
-NA-
Sub-Total (A)
B. Recurring
B.1 Manpower (See guidelines at Annexure-III)
S.
No.
Position
No.
Consolidated
Emolument
Year 1 Year 2 Year 3 Total
1
JRF
Rs. 16000 + Rs
250 Medical (for
Year 1 and
2)+30%HRA
7,48,800 7,48,800 8,42,400 23,40,000
Sub-Total (B.1) =23, 40,000/-
1. Gene synthesis, Cloning and Expression 2. Scaling up, purification and
characterization of recombinant proteins
3. Peptide synthesis and purification
4. An adequate number of overlapping peptides for multiepitope analysis
4. Immunoassays for peptides and proteins
All these would require a minimum of 1 JRF and hence this manpower is justified.
B.2 Consumables
S.
No.
Item
Quantity Year 1 Year 2 Year 3 Total
1.
Chemicals
15.00 10.00 10.00 35.00
Sub-Total (B.2) =35.00
Justification: 1.Molecular biology reagents
2. Immunology reagents
3. Recombinant technology
The budget for consumables proposed in project is minimum. High quality reagents are
extremely critical for this purpose.
Other items Consolidated
Emolument
Year 1 Year 2 Year 3 Total
B.3 Travel
1.00 1.00 1.00 3.00
B.4 Contingency
1.00 1.00 1.00 3.00
31
B.5 Overhead
(If applicable)
-NA
Sub-total of B
(B.1+B.2+B.3+B.4+B.5)
64.4
Lakhs
Grand Total (A + B) 64.4
Lakhs
Note : Please give justification for each head and sub-head separately mentioned in the
above table.
Financial Year : April - March
In case of multi-institutional project, the budget estimate to be given separately for each institution.
PART V: EXISTING FACILITIES
1. Laboratory:
Microtest innovation is a well-equipped laboratory located in the International
Technology Park, the technology hub of government of Karnataka. The company is
undertaking research and development programs in several frontier areas of national
significance, requiring specialization in biotechnology such as immunology, hybridoma
technology, recombinant protein production, vaccine development and protein folding.
2. Manpower
Three PhD, One MD, Four M.Sc Graduates in Biotechnology
Equipments
Sl No Equipment
1 MilliQ water
2 Autoclaves
3 Microfuges
4 PCR machine
5 -20ºC deep freezer
6 Refrigerators
7 Incubator
8 Electrophoresis unit Agarose
9 Semi-preparative and Analytical HPLC
10 SDS PAGE unit
11 Ultra filtration systems
12 Incubator shaker
13 Sonicator
14 ELISA plate reader
15 Laminar Air flow Hood
16 CO2 Incubators
17 Amicon protein concentrator
18 Table top centrifuge
19 Weighing balance
20 Magnetic Stirrer
21 pH meter
22 CO2 incubator
23 Heating Block
24 Hot air oven
25 Western blotting apparatus
32
PART IV: BUDGET PARTICULARS
(LRS Institute of Tuberculosis and Respiratory Diseases)
Budget (In Rupees)
A. Non-Recurring (e.g. equipments, accessories, etc.)
S.
No.
Item Year 1 Year 2 Year 3 Total
-NA-
Sub-Total (A)
B. Recurring
B.1 Manpower (See guidelines at Annexure-III)
S.
No.
Position
No.
Consolidated
Emolument
Year 1 Year 2 Year 3 Total
-NA-
Sub-Total (B.1) =
B.2 Consumables
S.
No.
Item
Quantity Year 1 Year 2 Year 3 Total
1.
Chemicals
5.00 5.00 5.00 15.00
Sub-Total (B.2) =15.00
Other items Consolidated
Emolument
Year 1 Year 2 Year 3 Total
B.3 Travel
1.00 1.00 1.00 3.00
B.4 Contingency
1.00 1.00 1.00 3.00
B.5 Overhead
(If applicable)
-NA-
Sub-total of B
(B.1+B.2+B.3+B.4+B.5)
21.00
Lakhs
Grand Total (A + B) 21.00
Lakhs
Justification:
Antigens and peptides needs to tested against the serum collected from the patients.
Assays need to be validated at each and every step of the project. Hence the budget is
justified.
33
PART V: EXISTING FACILITIES
LRS Institute of TB and Respiratory Diseases is an Autonomous Institute under the
Ministry of Health & Family Welfare, Govt. of India.
The department of Microbiology regularly carries out the following procedures:
i) Specimen Collection
ii) Sputum Smear Microscopy
iii) Culture (Mycobacteria)
iv) Identification Tests for Mycobacteria
v) Sensitivity testing for M. Tuberculosis
vi) BACTEC Tests
The Bacteriology and Mycology
i) Culture & Sensitivity testing of pyogenic and fungii from specimen from ICU
post operative, and other chest diseases patients.
Serology
This section carries out investigations for HIV, HBsAg and Widal test. IHA for
Aspergillosis.
The department is equipped with latest laboratory equipments like fluorescent
microscope, Binocular microscope and BACTEC 460 TB system, ELISA reader etc.
Shortly it will be acquiring gas liquid chromatography system for anaerobic bacteria and
Mycobacteria and PCR system for diagnosis of Mycobacterial diseases. A new lab.
extension was developed in which state of art bio safety laminar flow cabinets (class-I
cabinet) were installed for specifically carrying out tests for tuberculosis and related
mycobacterial diseases.
It has been identified for expansion for conducting research under RNTCP. The
department is identified as National Reference Laboratory (NRL) and will take-up the
responsibilities of External Quality Assurance and Drug Resistance Survillance
activities under RNTCP in designated states. It is recently certified by CDC, Atlanta,
USA, for its quality in Drug Sensitivity Testing for M. tuberculosis. It is also proposed
to establish the bio-safety Lab-III for Molecular biology based tests for Mycobacteria
and for Epidemiological research.
The department is conducting training programmes for WHO fellows, actively
involved in the training of paramedical personnel (Lab. Technician, Sr.TB Lab.
Supervisors, Treatment organisers, Sr.Treatment supervisors ) who visit from other
states of our country, in implementation of strategies under Revised National
Tuberculosis Control Programme (RNTCP).
34
PART VI: DECLARATION/CERTIFICATION
It is certified that
a) the research work proposed in the scheme/project does not in any way duplicate the
work already done or being carried out elsewhere on the subject.
b) the same project proposal has not been submitted to any other agency for financial
support.
c) the emoluments for the manpower proposed are those admissible to persons of
corresponding status employed in the institute/university or as per the Ministry of
Science & Technology guidelines (Annexure-III)
d) necessary provision for the scheme/project will be made in the
Institute/University/State budget in anticipation of the sanction of the
scheme/project.
e) if the project involves the utilisation of genetically engineered organisms, we agree
to submit an application through our Institutional Biosafety Committee. We also
declare that while conducting experiments, the Biosafety Guidelines of the
Department of Biotechnology would be followed in toto.
f) if the project involves field trials/experiments/exchange of specimens, etc. we will
ensure that ethical clearances would be taken from concerned ethical
Committees/Competent authorities and the same would be conveyed to the
Department of Biotechnology before implementing the project.
g) it is agreed that any research outcome or intellectual property right(s) on the
invention(s) arising out of the project shall be taken in accordance with the
instructions issued with the approval of the Ministry of Finance, Department of
Expenditure, as contained in Annexure-V.
h) we agree to accept the terms and conditions as enclosed in Annexure-IV. The same
is signed and enclosed.
35
i) the institute/university agrees that the equipment, other basic facilities and such other
administrative facilities as per terms and conditions of the grant will be extended to
investigator(s) throughout the duration of the project.
j) the Institute assumes to undertake the financial and other management
responsibilities of the project.
36
PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS
Appendix J
BIOGRAPHICAL SKETCH
To be submitted separately for the Project Coordinator and each Key Investigator
Name : Dr Chandrabhas Narayana
Designation :Professor
Name of the Department/Institute/University :
Chemistry and Physics of Materials Unit
Jawaharlal Nehru Centre for Advanced Scientific Research,
Jakkur P.O., Bangalore 560064. India.
Date of Birth : 08-09-1965 Sex (M/F) : M
Education (Graduation onwards)
Sl
No. Institution
Degree
Awarded Year Field of Study
1.
University of Mysore
B.Sc B.Ed
1982-1986
Physics,
Chemistry,
Mathematics,
Psycology,
Sociology,
Science
Methodology
2. University of Mysore
M.Sc and
B.Ed
1986-1988 Physics and
Science
Methodology
3. Indian Institute of
Science, Bangalore
Ph.D 1989-1995 High Pressure
Materials
Research
4 Cornell university,
Ithaca, NY, USA
Post Doc 1995-1998 Metalization of
Hydrogen
Professional Career (Starting with the most recent employment)
Sl
No. Institution Position From (Date) To (Date)
1.
Jawaharlal Nehru Centre
for Advanced Scientific
Research Bangalore
Professor
May 11 Till Date
37
2.
Jawaharlal Nehru Centre
for Advanced Scientific
Research, Bangalore
Dean,
Fellowships
and Extension
Programmes
Oct09 Oct11
3.
Jawaharlal Nehru Centre
for Advanced Scientific
Research, Bangalore
Associate
Professor
May05 May11
4.
Jawaharlal Nehru Centre
for Advanced Scientific
Research, Bangalore
Assistant
Professor
Sept98 May05
Honors/ Awards
MRSI Medal in 2007: This award was given by Materials Research Society of India for
“outstanding contributions in Material Science and Engineering”.
Sir C.V. Raman Young Scientist Award in 2008: This award was given by the Karnataka
Government for outstanding contributions in the area of Material Science and Physical
Sciences.
CNR Rao Oration Award in 2009: This award was given by Jawaharlal Nehru Centre for
Advanced Scientific Research, Bangalore.
Publications (Numbers Only) 75
1. Books: 1 2. Research Papers, Reports : 3.General
articles :
4. Patents : 6 ( 3 Indian, 3 International) 5. Others (Please specify):
Publications:
1. Gayatri Kumari and Chandrabhas Narayana, New Nano Architecture for SERS Applications, Journal
of Physical Chemistry Letters, 3, 1130 - 1135 (2012). , Read abstract.
2. Soumik Siddhanta and Chandrabhas Narayana, Surface Enhanced Raman Spectroscopy of Proteins:
Implications in Drug Designing, Nanomaterials and Nanotechnology, 2, 1 - 13 (2012). , Read abstract.
3. Narendra Kurra, Venkat Srinu Bhadram, Chandrabhas Narayana and G.U. Kulkarni, Field effect
transistors based on graphitized patterns of carbon contamination, ACS Applied Materials and
Interfaces, 4, 1030 - 1036 (2012). , Read abstract.
4. Diptikanta Swain, Venkat Srinu Bhadram, Papia Chowdhury and Chandrabhas Narayana, Raman and
X-ray Investigations of Ferroelectric Phase Transitions in NH4HSO4, Journal of Physical Chemistry A,
116, 223 - 230 (2012).
5. Harikrishnan S. Nair, Diptikanta Swain, Hariharan N., Shilpa Adiga, Chandrabhas Narayana and Suja
Elzabeth, Griffiths phase-like behavior and spin-phonon coupling in double perovskite
38
Tb2NiMnO6,Journal of Applied Physics, 110, 123919 - 123925 (2011). , Read abstract.
6. Supti Das, Venkata Srinu Bhadram, Chandrabhas Narayana and Aninda J. Bhattacharyya, Brillouin
Scattering Investigation of Solvation Dynamics in Succinonitrile-Lithium Salt Plastic Crystalline
Electrolytes, Journal of Physical Chemistry B, 115, 12356 - 12361 (2011). , Read abstract.
7. P. Mandal, Venkat Srinu Bhadram, Y. Sundarayya, Chandrabhas Narayana, A. Sundaresan and C.N.R.
Rao, Spin-Reorientation, Ferroelectricity, and Magnetodielectric Effect in YFe(1-x)Mn(x)O(3)(0.1 <= x
<= 0.40), Physical Review Letters, 107, 137202 - 137205 (2011). , Read abstract.
8. G. V. PAVAN KUMAR, N. RANGARAJAN, B. SONIA, P. DEEPIKA, NASHIOUR ROHMAN and
CHANDRABHAS NARAYANA, Metal-coated magnetic nanoparticles for surface enhanced Raman
scattering studies.,Bulletin of Materials Science, 34, 207 - 216 (2011). , Read abstract.
9. Partha P. Kundu, G. V. Pavan Kumar, K. Mantelingu, Tapas K. Kundu and Chandrabhas
Narayana, Raman and surface enhanced Raman spectroscopic studies of specific, small molecule activator
of histone acetyltransferase p300, Journal of Molecular Structure, 999, 10 - 15 (2011). , Read abstract.
10. Gopal Krushna Pradhan, Chandrabhas Narayana, Mala Narasappaya Rao, Samrath Lal Chaplot,,
Olivier Pages and et al, The Phonon Percolation Scheme for Alloys: Extension to the Entire Lattice
Dynamics and Pressure Dependence, JAPANESE JOURNAL OF APPLIED PHYSICS, 50, 05FE02
(2011). , Read abstract.
11. Gopal K. Pradhan, Anil Kumar, S.K. Deb, Umesh V. Waghmare and Chandrabhas Narayana, Elastic
and structural instability of cubic Sn3N4 and C3N4 under pressure, Physical Review B, 82, 144112 -
144115 (2010). , Read abstract.
12. Awadesh K. Mallik, S. R. Binu, L. N. Satapathy, Chandrabhas Narayana, Md. Motin Seikh, S. A.
Shivashankar and S. K. Biswas, Effect of substrate roughness on growth of diamond by hot filament
CVD,Bulletin of Materials Science, 33, 251 - 255 (2010). , Read abstract.
13. S. Murugavel, C. Vaid, Venkat Srinu Bhadram and Chandrabhas Narayana, Ion Transport
Mechanism in Glasses: Non-Arrhenius Conductivity and Non-Universal Features, Journal of Physical
Chemistry B, 114, 13381 - 13385 (2010). pdf , Read abstract.
14. M. Viswanathan, P.S. Anil Kumar, Venkata Srinu Bhadram, Chandrabhas Narayana, A.K. Bera and
S.M. Yusuf, Influence of lattice distortion on the Curie temperature and spin-phonon coupling in
LaMn0.5Ca0.5O3, Journal of Physics: Condense Matter, 22, 346006 - 346013 (2010). pdf ,
Readabstract.
15. Diptikanta Swain, Venkata Srinu Bhadram, Venakataprasad Bhat, Gopal K. Pradhan, Chandrabhas
Narayana and C.N.R. Rao, Superionic phase transition in KHSO4: A temperature dependent Raman
investigation, Journal of Physical Chemistry A, 114, 10040 - 10044 (2010). pdf , Read abstract.
16. Sudip Mohapatra, Soumik Siddhanta, D. Ravindar Kumar, Chandrabhas Narayana and Tapas K.
Maji, A facile bottom up approach for fabricating multifunctional silver nanorods, European Journal of
39
Inorganic Chemistry, 31 , 4969 - 4974 (2010). , Read abstract.
17. T Bhuvana, Anurag Kumar, Aditya Sood, Roger H. Gerzeski, Jianjun Hu, Venkata Srinu
Bhadram,Chandrabhas Narayana and Timothy S. Fisher, Contiguous Petal-like Carbon Nanosheet
Outgrowths from Graphite Fibers by Plasma CVD, ACS Applied Materials and Interfaces, 2, 644 - 648
(2010).
18. Gopal K. Pradhan, Chandrabhas Narayana, O. Pagès, A. Breidi, J. Souhabi, A. V. Postnikov, S. K.
Deb, F. Firszt, W. Paszkowicz, A. Shukla and F. El Haj Hassan, Pressure-induced phonon freezing in the
Zn1−xBexSe alloy: A study via the percolation model, Physical Review B, 81, 115207 - 115212 (2010).
19. Gopal K. Pradhan, Diptikant Swain, T.N. Guru Row and Chandrabhas Narayana, High-Temperature
Phase Transition Studies in a Novel Fast Ion Conductor, Na2Cd(SO4)2, Probed by Raman
Spectroscopy,Journal of Physical Chemistry A, 113, 1505 - 1507 (2009).
20. R.V.K. Mangalam, Chandrabhas Narayana and A. Sundaresan, Pressure-dependent phase transition
in the ordered BaBi0.7Nb0.3O3 perovskite, High Pressure Research, 29, 272 - 277 (2009).
21. Vishnu Shankar, Saroj L. Samal, Gopal K. Pradhan, Chandrabhas Narayana and Ashok K.
Ganguli, Nanocrystalline NaNbO3 and NaTaO3: Rietveld studies, Raman spectroscopy and dielectric
properties, Solid State Sciences, 11, 562 - 569 (2009).
22. Chandrabhas Narayana, R. G. Greene and Arthur L. Ruoff, Studies on silane to 70 GPa, Journal of
Physics: Conference Series, 121, 042019 - 042023 (2008). , Read abstract.
23. G. V. Pavan Kumar, R. Selvi, H. Kishore, T. K. Kundu and Chandrabhas Narayana, Surface
enhanced Raman spectroscopic studies of coactivator-associated arginine methyltransferase, Journal of
Physical Chemistry B, 112, 6703 - 6707 (2008).
24. R. V. K. Mangalam, Gopal K. Pradhan, Chandrabhas Narayana and A. Sundaresan, Spin state
transition in ferromagnet Sr0.9Ce0.1CoO2.85, Solid State Communications, 146, 110 - 114 (2008).
25. G. V. Pavan Kumar and Chandrabhas Narayana, Adapting a Fluorescence Microscope to Perform
Surface Enhanced Raman Spectroscopy, Current Science, 93, 778 - 781 (2007).
26. C. SubRamaniam, T. S. Sreeprasad, T. Pradeep, G. V. Pavan Kumar, Chandrabhas Narayana, T.
Yajima, Y. Sugawara, H. Tanaka, T. Ogawa and J. Chakrabarti, Metal-semiconductor transition induced
visible fluorescence from single walled carbon nanotube-noble metal nanoparticle composites, Physical
Review Letters, 99, 167404 - 167407 (2007).
27. K. Mantelingu, B. A. Ashok Reddy, V. Swaminathan, A. Hari Kishore, N. B. Siddappa, G.
Nagashankar, G. V. Pavan Kumar, N. Natesh, S. Roy, P. P. Sadhale, Udaykumar Ranga, Chandrabhas
Narayana and Tapas K. Kundu, Nonspecific to specific HATs off: Alters global gene expression and
repress HIV multiplication ,Chemistry and Biology, 14, 645 - 657 (2007).
28. K. Mantelingu, A. Hari Kishore, K. BalasubRamanyam, G.V. Pavan Kumar, M. Altaf, S. Nanjunda
Swamy, Ruthrotha Selvi, Chandrima Das, Chandrabhas Narayana, K.S. Rangappa and Tapas K.
40
Kundu, Activation of p300 histone acetyltransferase by small molecules altering enzyme structure: probed
by surfaced enhanced Raman spectroscopy, Journal of Physical Chemistry B, 111, 4527 - 4534 (2007).
29. G. Kavitha and Chandrabhas Narayana, Pressure-induced structural transitions in n-Pentane: A
Raman study, Journal of Physical Chemistry B, 111, 7003 - 7008 (2007).
30. T. Bhuvana, G. V. Pavan Kumar, G. U. Kulkarni and Chandrabhas Narayana, Carbon assisted
electroless gold for surface enhanced Raman scattering studies, Journal of Physical Chemisry C, 111,
6700 - 6705 (2007).
31. T. Bhuvana, G. V. Pavan Kumar, Chandrabhas Narayana and G. U. Kulkarni, Nanogranular Au films
deposited on carbon covered Si substrates for enhanced optical reflectivity and Raman
scattering,Nanotechnology, 18, 145702 - 145708 (2007).
32. G. V. Pavan Kumar, S. Sruthi, B. Vibha, B. A. Ashok Reddy, T. K. Kundu and Chandrabhas
Narayana, Hot spots in Ag core-Au shell nanoparticles potent for surface enhanced Raman scattering
studies of biomolecules, Journal of Physical Chemistry C, 111, 4388 - 4392 (2007).
33. Neena Susan John, G. U. Kulkarni, Ayan Datta, S. K. Pati, F. Komori, G. Kavitha, Chandrabhas
Narayana and M. K. Sanyal, Magnetic Interactions in Nickel Alkanethiolates, Journal of Physical
Chemistry C (Letters), 111, 1868 - 1870 (2007).
34. Harshad B. Ghodke, Kasinath Vignesh, G. V. Pavan Kumar, Ramya Krishnan, Chandrabhas
Narayanaand Yamuna Krishnan, The I-tetraplex building block: Rational Design and Controlled
Fabrication of robust 1D DNA Scaffolds via non-Watson Crick self assembly, Angewandte Chemie
(International Edition), 46, 1 - 5 (2007).
35. G. Kavitha and Chandrabhas Narayana, Raman spectroscopic investigations of pressure-induced
phase transitions in n-hexane, Journal of Physical Chemistry B, 111, 14130 - 14135 (2007).
36. Mohammed Arif, G. V. Pavan Kumar, Chandrabhas Narayana and T. K. Kundu, Autoacetylation
Induced Specific Structural Changes in Histone Acetyltransferase Domain of p300: Probed by Surface
Enhanced Raman Spectroscopy, Journal of Physical Chemistry B (Letters), 111, 11877 -
11879 (2007).
37. G. V. Pavan Kumar, B. A. Ashok Reddy, Md. Arif, T. K. Kundu and Chandrabhas Narayana, Surface
Enhanced Raman Scattering Studies of Human Transcriptional Coactivator p300., Journal of Physical
Chemistry B, 110, 16787 - 16792 (2006).
38. Md. Motin Seikh, Chandrabhas Narayana, A. K. Sood, P. Murugavel, M.W. Kim, P. A. Metcalf, J. M.
Honig and C. N. R. Rao, A Brillouin study of the temperature-dependence of the acoustic modes across
the insulator-metal transitions in V2O3 and Cr-doped V2O3, Solid State Communications, 139, 466 -
471 (2006).
39. G. Kavitha and Chandrabhas Narayana, Raman Scattering Studies on n-Heptane under High
Pressure,Journal of Physical Chemistry B, 110, 8777 - 8781 (2006).
41
40. Md. Motin Seikh, Chandrabhas Narayana, A. K. Cheetham and C. N. R. Rao, A comparative study of
the electro- and hole-doped compositions of single crystalline Nd1-xCaxMnO3 (x = 0.6 and 0.4), Solid
State Science, 7, 1486 - 1491 (2005).
41. Tokeer Ahmad, G. Kavitha, Chandrabhas Narayana and Ashok K. Ganguli, Nanostructured barium
titanate prepared through a modified reverse micellar route: Structural distortion and dielectric, Journal of
Materials Research, 20, 1415 - 1421 (2005).
42. Md. Motin Seikh, Chandrabhas Narayana, P. A. Metcalf, J. M. Honig and A. K. Sood, Brillouin
scattering studies in Fe3O4 across the Verwey transition, Physical Review B, 71, 174106 - 174110
(2005).
43. Md. Motin Seikh, A. K. Sood and Chandrabhas Narayana, Electronic and vibrational Raman
spectroscopy of Nd0.5Sr0.5MnO3 through the phase transitions, PRamana, 64, 119 - 128 (2005).
44. L. Sudheendra, Md. Motin Seikh, A. R. Raju, Chandrabhas Narayana and C. N. R. Rao, Dielectric
properties of rare earth cobaltates, LnCoO3 (Ln = La, Pr, Nd), across the spin-spin
transition,Ferroelectrics, 306, 227 - 234 (2004).
45. S. Mandal, G. Kavitha, Chandrabhas Narayana and S. Natarajan, Solvothermal synthesis of an open-
framework zinc chlrophosphate [C8N4H26]Zn3Cl(HPO4)3(PO4)], with a layer structure, Journal of
Solid State Chemistry, 177, 2198 - 2204 (2004).
46. Md. Motin Seikh, L. Sudheendra, Chandrabhas Narayana and C. N. R. Rao, A Raman study of the
temperature-induced low-to-intermediate-spin state transition in LaCoO3, Journal of Molecular
Structure, 706, 121 - 126 (2004).
47. Md. Motin Seikh, Chandrabhas Narayana, L. Sudheendra, A. K. Sood and C. N. R. Rao, A brillouin
scattering study of La0.77Ca0.23MnO3; across the metal-insulator transition, Journal of Physics:
Condense Matter, 16, 4381 - 4390 (2004).
48. Pallavi Teredesai, D. V. S. Muthu, N. Chandrabhas, S. Meenakshi, V. Vijayakumar, P. Modak, R. S.
Rao, B. K. Godwal, S. K. Sikka and A. K. Sood, High pressure phase transitions in metallic LaB6: Raman
and x-ray diffraction studies, Solid State Communications, 129, 791 - 796 (2004).
49. G. Kavitha, S. R. C. Vivek, A. Govindaraj and Chandrabhas Narayana, A low-cost Raman
spectrometer design used to study Raman scattering from a single-walled carbon nanotube, Proceedings
Indian Academy of Sciences (Chemical Science), 115, 689 - 694 (2003).
50. Md. Motin Seikh, Chandrabhas Narayana, Sachin Parashar and A. K. Sood, Temperature-dependent
Brillouin scattering studies of surface acoustic modes in Nd0.5Sr0.5MnO3, Solid State
Communications, 127, 209 - 214 (2003).
51. Chandrabhas Narayana, Brillouin scattering studies on charge-ordered manganite, Journal of Indian
Institute of Science, 82, 093 - 103 (2002).
52. L. Sudheendra, Md. Motin Seikh, A. R. Raju and Chandrabhas Narayana, An Infrared spectroscopic
42
study of the low-spin to intermediate-spin state (1A1 - 3T1) transition in rare earth cobaltates, LnCoO3
(Ln = La, Pr and Nd), Chemical Physics Letters, 340, 275 - 281 (2001).
53. P. Murugavel, N. Chandrabhas, A. R. Raju, A. K. Sood and C. N. R. Rao, Magnetic excitations in
charge-ordered Nd0.5Ca0.5MnO3 : A Brillouin scattering study, Europhysics Letters, 52, 461 - 467
(2000).
54. P. Murugavel, Chandrabhas Narayana, A. Govindaraj, A. K. Sood and C. N. R. Rao, Brillouin
scattering from C70 and C60 Films: Comparative study of elastic Properties, Chemical Physics
Letters, 331, 149 - 153 (2000).
55. P. Murugavel, Chandrabhas Narayana, A. K. Sood and C. N. R. Rao, A Brillouin scattering study of
the quasi-one-dimensional blue bronze, K0.3MoO3, Journal of Physics: Condense Matter, 12, (2000).
56. Jon Orloff, Chandrabhas Narayana and A. L. Ruoff, Use of focused ion beams for making tiny
sample holes in gaskets for diamond anvil cells, Reviews of Scientific Instruments, 71, 216 - 219
(2000).
57. A. L. Ruoff, T. Li, A. C. Ho, M-F. Pai, H. Luo, R. G. Greene, Chandrabhas Narayana, J. C. Molstad,
S. S. Trail, F. J. DiSalvo and P.E. van Camp, Jr., Sevenfold coordinated MgSe: Experimental internal
atom position determination to 146 GPa, diffraction studies to 202 GPa, and theoretical studies to 500
Gpa, Physical Review Letters, 81, 2723 - 2726 (1998).
58. Chandrabhas Narayana, Huan Luo, Jon Orloff and A. L. Ruoff, Solid hydrogen at 342 GPa: no
evidence for an alkali metal, Nature, 393, 46 - 49 (1998).
59. Chandrabhas Narayana, V. J. Nesamony and A. L. Ruoff, Phase transformation of BeS and equation
of state studies to 96 Gpa, Physical Review B, 56, 14338 - 14343 (1997).
60. A. K. Sood, N. Chandrabhas, D. V. S. Muthu and A. JayaRaman, Phonon Interference in BaTiO3 :
High Pressure Raman Study, Physical Review B, 51, 8892 - 8895 (1995).
61. N. Chandrabhas and A. K. Sood, Raman Study of Pressure-induced Phase Transitions in
RbIO4,Physical Review B, 51, 8795 - 8800 (1995).
62. N. Chandrabhas, A. K. Sood, D.V.S Muthu, C. S. Sundar, A. Bharathi, Y. Hariharan and C. N. R.
Rao, Pressure-induced Amorphization in solid C70 : Raman and Photoluminescence Study, Physical
Review Letters, 73, 3411 - 3414 (1994).
63. N. Chandrabhas, A. K. Sood, D. SundaRaman, S. Raju, V. S. Raghunathan, G. V. N. Rao, V. S.
Sastry, T. S. Radhakrishnan, Y. Hariharan, A. Bharathi and C. S. Sunder, Studies on Structure and
Vibrational Properties of Carbon Tubules, PRamana - Journal of Physics, 42, 375 - 385 (1994).
64. A. K. Sood, N. Chandrabhas, D. V. S. Muthu, Y. Hariharan, A. Bharathi and C. S. Sunder, Pressure-
induced Band gap Reduction, Orientational Phase Transition and Re- versible Amorphization in C70
43
Crystals: Photoluminescence and Raman Study, Philosophical Magazine B, 70, 347 - 358 (1994).
65. M. N. Shashikala, N. Chandrabhas, K. Jayaram, A. JayaRaman and A. K. Sood, High Pressure
Raman Spectroscopic Study of LiCsSO4: Pressure-Induced Phase Transitions and
Amorphization, Journal of Physics and Chemistry of Solids, 55, 107 - 112 (1994).
66. D. V. S. Muthu, N. Chandrabhas, A. K. Sood, K. Venkatesan, P. Venugopalan and A. JayaRaman, A
High Pressure Raman Study of K and T Forms of Octachloro Cyclic Phosphazene Tetramer
P4N4Cl8,Journal of Raman Spectroscopy, 23, 611 - 614 (1993).
67. N. Chandrabhas, K. Jayaram, D. V. S. Muthu, A. K. Sood, R. Seshadri and C. N. R.
Rao, Orientational Phase Transitions in C70: A Raman Spectroscopic Investigation, Physical Review B
(Rapid Communication), 47, 10963 - 10966 (1993).
68. M. N. Shashikala, N. Chandrabhas, K. Jayaram, A. JayaRaman and A. K. Sood, Pressure-Induced
Phase Transitions in LiRbSO4: A Raman Spectroscopic Study, Journal of Raman Spectroscopy, 24,
129 - 132 (1993).
69. N. Chandrabhas, M. N. Shashikala, D.V. S. Muthu, A. K. Sood and C. N. R. Rao, Pressure-Induced
Orientational Ordering in C60 Crystals as revealed by Raman Spectroscopy, Chemical Physics
Letters, 197, 319 - 323 (1992).
70. A. K. Sood, N. Chandrabhas, D. V. S. Muthu, A. JayaRaman, N. Kumar, H. R. Krishnamurthy, T.
Pradeep and C. N. R. Rao, Pressure-Induced Shift of the Photoluminescence Band in Single Crystals of
Buck-minster Fullerene C60 and its Implications for Superconductivity in Doped Samples, Solid State
Communications, 81, 89 - 92 (1992).
71. N. Chandrabhas, D. V. S. Muthu, A. K. Sood, H. L. Bhat and A. JayaRaman, Raman Study of
Pressure Induced Structural Transitions in CsIO4 to 12 GPa, Journal of Physics and Chemistry of
Solids, 53, 959 - 965 (1992).
72. K. S. Harshavardhan, M. N. Vijayaraghavan, N. Chandrabhas and A. K.Sood, Raman investigations
of diamond films prepared by combustion flames, Journal of Applied Physics, 68, 3303 - 3306 (1990).
73. B. N. Meera, A. K. Sood, N. Chandrabhas and J. Ramakrishna, Raman study of lead borate
glasses,Journal of Non-Crystalline Solids, 126, 224 - 230 (1990).
74. C. N. R. Rao, R. Nagarajan, R. Vijaya Raghavan, N. Y. Vasanthacharya, G. V. Kulkarni, G. Ranga
Rao, A. M. Umarji, P. Somasundaram, G. N. Subbanna, A. R. Raju, A. K. Sood and N.
Chandrabhas, Superconducting cuprates of the series Bi2Ca1-xLnxSr2Cu2O8+d (Ln = rare earth or
Y), Superconductors - Science and Technology, 3, 242 - 248 (1990).
75. R. Vijayaraghavan, A. K. Ganguli, N. Y. Vasanthacharya, M. K. Rajumon, G. U. Kulkarni, G. Sarkar,
D. D. Sarma, A. K. Sood, N. Chandrabhas and C. N. R. Rao, Investigations of novel cuprates of the
TlCa(1-x)LnxSr2Cu2O7-d (Ln = rare earth) series showing electron-or hole superconductivity depending
on the composition, Superconductors - Science and Technology, 2, 195 - 201 (1989).
44
Professional Experience relevant to the Project
Dr. Chandrabhas Narayana is an expert in ultra High Pressure Research (Mbar Pressures)
and Raman spectroscopy (RS). His laboratory in JNCASR is the only laboratory in India
practicing Brillouin Spectroscopy and he has helped in setting up this facility. The work
carried out during and after his Ph.D. has been recognized by both Indian and foreign
scientists alike. During the past 5 years, Dr. Chandrabhas Narayana has used this knowledge
of RS to work in an interdisciplinary area of Physics, Chemistry and Biology and has
developed a new field of research in JNCASR which is fast gaining popularity world over.
He uses an important technique, Surface Enhanced Raman Spectroscopy (SERS), to look at
two aspects in biological systems: (i) small molecule protein interactions and (ii) DNA/RNA
detection of pathogens without polymerase chain reaction (PCR). The success can be seen
from his paper on Raman of p300 which appeared in Journal of Physical Chemistry B
(2006), 110, 16787-16792 has already been sighted more than 22 times in such a short time.
He has demonstrated the effectiveness of SERS in predicting the interaction of the potential
drug molecule derived from anacardic acid (cashewnut shell) with p300 histone
actyltranferase (HAT) region (Journal of Physical Chemistry B 111, 4527-4534 (2007)).
This was subsequently proven to be correct from x-ray studies of synthesized HAT domain
and drug molecule interactions (Nature 451, 846 (2008)). Recently, he has shown that SERS
can be used to study secondary and tertiary structure changes due to cationic substitution in
proteins, like C-protein. This work is under review now. This would be a new tool in the
hands of Biologists to study the drug-protein interactions and protein structural changes.
Another major achievement of his has been the demonstration of (in the case of HIV) the
viral RNA detection without PCR amplification using SERS. PCR is a well known
technique and a very useful one, but requires sophisticated laboratory (recent incident of the
pandemic SWINE FLU is one such example). It is a challenge for the molecular biologists
to find non PCR based techniques. Dr. Chandrabhas Narayana along with Dr. Udaykumar
Ranga (a highly trained virologist at JNCASR) came up with an assay (recently entered the
national phase: International Patent Number: WO2008062479-A2; WO2008062479-A3;
National Patent Number IN200602161-I4) which can detect about a few thousand copies of
viral RNA without PCR amplification. The implication of this is a massive cost reduction, a
micro array kind of system, normal clinical laboratories can carry out this test, possible
mobile testing, use in various other viral pathogen like Dengue, Chickenguniya, detection,
use in seed industries and agriculture like the propagation of markers into future generation
etc. In order to develop a cost effective diagnostics, Dr. Chandrabhas Narayana has been
working with interested companies to miniaturize Raman spectrometer (National Patent
Numbers: IN200800787-I4; US Patent: US20090244534-A1). He has recently synthesized
improved Raman reporters as well as nanoparticle to detect a few copies of viral RNA. He
has been working on the cutting edge research which would soon set a new trend in medical
diagnostics.
(b) Summary the most significant recent work:
Dr. Chandrabhas Narayana’s research has shown the potential of Surface Enhanced Raman
Spectroscopy to understand the drug-protein interactions and in diagnostics, namely,
detection of viral or bacterial DNA/RNA without polymerase chain reaction. To make the
diagnostics reach clinical laboratories, he has developed Raman instrumentation.
(c) Names of the industries in which the technology (ies) has (have) been used:
The products have not yet been commercialized, but discussions are on with various
industries and possibilities of startup companies are also being explored. One of the
multinational companies, TISMO Technologies Solutions (P) Ltd. (Bangalore branch) is
already working on the prototype of miniaturized Raman Spectrometer based on the patent
of Dr. Chandrabhas NaNarayana (Indian patent IN200800787-I4 and US Patent
US20090244534-A1).
45
Ongoing Research Projects
Sl
No. Title of Project Source of Funds Amount
Duration
(from – to -- )
1 Multiplexed immune and
DNA-based diagnosis of
tuberculosis
Swedish Research
Links programme
Rs.
18,00,000/=
(Indian side
funding)
December
2010 –
December
2013
Completed Research Projects (State only major projects of last 3 year)
Sl
No. Title of Project Source of Funds Amount
Duration
(from – to -- )
1 HIV-1 viral RNA
diagnostics
DST Nano Science
and Nano
Technology (India
Rs.
40,00,000
2007
2 Lattice dynamical and
structural study of Be
based II-VI
semiconductor alloys at
ambient and high
pressures''
Indo-French Centre
for Promotion of
Advanced Research
Rs.
10,00,000/
2004
3 Application of surface-
enhanced Raman
spectroscopy to study the
role of Tat protein in
HIV-1 pathogenesis''
Inter-departmental
Collaborative
Project (Jawaharlal
Nehru Centre for
Advanced
Scientific Research
10,00,000/ 2005
It is certified that the above particulars submitted are true and correct.
Place: Bangalore Signature :
Date : 30.04.2012
46
Name : Jayasuryan Narayana
POSITION TITLE: Director
Date of Birth: 3rd
August, 1959
Address : Director
Microtest Innovations Pvt. Ltd.,
International Tech Park,
White Field Road,
Bangalore – 560066,
India.
Phone: +91 80 41262037
Mob : +91 9845004989
E-mail: [email protected]
Education (Post-Graduation onwards & Professional Career)
Education : University of Bombay 1992 Ph.D.
Sl No. Institution
Place
Degree
Awarded
Year Field of Study
1 University of Bombay Ph.D. 1992 Biochemistry
A. Position and Honors
Position and Employment (Starting with the most recent employment)
Sl No. Institution
Place
Position From (Date) To (date)
1 Microtest Innovations Pvt
Ltd Director Present
Director 2002 Present
2 International Center for
Genetic Engineering and
Biotechnology, New Delhi,
India
Post Doc
1992 1995
Honors/Awards
1987-1988: Awarded Fellow of National Science Foundation (USA) to work
under Dr. Rosalyn. S.Yalow, Nobel Laureate in Medicine,
Soloman. A. Berson Research Laboratory, Veterans
Administration Hospital, New York.
1987-1990: Senior Research Fellow, Indian Council of Medical Research,
India.
1991-1995: Post Doctoral Research, at the International Center for Genetic
Engineering and Biotechnology under UNIDO.
1996-2001: Consultant and Advisor in various biotechnology companies and
research laboratories for development of various biotechnology
programs, All Indian Institute of Medical Sciences, New Delhi
2002- Present: Director, Microtest Innovations Pvt.Ltd, International Tech
Park, Bangalore.
47
Professional Experience and Training relevant to the Project
After trained with Dr. Rosanlyn Yalow, who laid the fundamental basis for
modern immunodiagnostics (for which she was awarded noble prize in medicine in
1977), my doctoral and post–doctoral, research was in the area of basic and applied
aspects of infectious diseases. I have worked in the area of Tuberculosis, Viral Hepatitis
and HIV and have extensive experience in diagnostic development (both Molecular and
Immuno diagnostic). I have also been involved with developing assay systems for
conducting large population-base studies in a cost-effective manner by developing
indigenous technology. Currently, as the Director of Microtest Innovation Pvt Ltd. (a
Biotechnology R&D Company), my focus is on developing novel indigenous diagnostic
tests. Accordingly, Microtest Innovations as a company is dedicated to R&D in the area
of biology and medicine with special emphasis on disease diagnostics. In the past seven
years of its existence, the team in Microtest Innovations has been inventing and
innovating appropriate and affordable diagnostics for existing and emerging health
problem through and by collaborative indigenous scientific and technological effort. Our
motto is to lay a strong foundation in basic research to build a product line in novel
diagnostics and diagnostic components.
B. Publications (Numbers only)
Books : Research Papers, Reports : .General articles :
Patents : .Others (Please specify):
Selected peer-reviewed publications (Ten best publications in chronological order)
1. Gupta BP, Jayasuryan N, Jaleel SJ. Direct detection of hepatitis B virus from
dried blood spots by polymerase chain reaction amplification. Coo Microbiol, 30 (8),
19136, 1992.
2. Gupta A, Mal TK, Jayasuryan N, Chauhan. VS assignment of disulphide bonds
in the X-protein (HBx) of hepatitis B virus. Biochemi Biophys Res Commun, 212
(3), 919-24, 1995.
3. Kumar V, Jayasuryan N, Kumar R. A truncated mutant (residues 58-140) of the
hepatitis B virus X protein retains transactivation function. Proc Natl Acad Sci USA,
93 (11), 5647-52, 1996.
4. Kumar V, Jayasuryan N, Reddi H, Sabal D, Panda SK. A monoclonal antibody
against the X protein of hepatitis B VffuS : fine mapping of its epitope and
application in a quantitative ELISA of the X protein in sera of hepatitis B patients.
Hybridoma, 17 (12), 157-64, 1998.
5. Kochupillai N, Jayasuryan N, Godbole MM, Pandav CS. Benefit and cost of
application of Radioimmuno assay in tuberculosis and iodine deficiency disorders:
two major problems of developing countries. Symposia of the giovanni lorenzini
foundation, Volume 18.1 Cost / benefit and predictive value of radio immuno assay.
Elsevier Publication.
6. Udaykumar Ranga, Raj Shankarappa, Nagadenahalli B. Siddappa, Lakshmi
Ramakrishna, Ramalingam Nagendran, Marthandan Mahalingam, Anita Mahadevan,
48
Narayana Jayasuryan, Parthasarathy Satishchandra, Susarla K. Shankar, and
Vinayaka R. Prasad J, Tat Protein of Human Immunodeficiency Virus Type 1
Subtype C Strains Is a Defective Chemokine. J.Virol. 2004. 78: 2586-2590.
7. Nagadenahalli B. Siddappa,Prashanta K.Dash, Anita Mahadevan, Narayana
Jayasuryan, Bethany Hoffman, Randy Keefe, Kadappa S. Satish, Bhuthiah Satish,
Kuttan Sreekanthan, Ramdas Chatterjee, Kandala Venu, Parthasarathy
Satishchandra,Vasanthapuram Ravi,Susala K. Shankar, Raj Shankarappa, and Uday
Kumar Ranga, Identification of Subtype C Human Immunodeficiency Virus Type 1
by Subtype-Specific Polymerase Chain Reaction and Its Use in the characterization
of Viruses Circulating in the Southern Parts of India. Journal of Clinical
Microbiology, June 2004, p. 2742–2751.
8. Nagadenahali Byrareddy Siddappa , Prashanta Kumar Dasha, Anita Mahadevan,
Anita Desaib,Narayana Jayasuryan, Vasanthapuram Ravi b, Parthasarathy
Satishchandrad, Susarla K. Shankar and Udaykumar Ranga, Identification of unique
B/C recombinant strains of HIV-1 in the Southern state of Karnataka, India. AIDS
2005, Vol 19 No.13, 1426 – 1429
9. Nagadenahalli Byrareddy Siddappa, Mohanram VenkatRamanan, Prasanna
Venkatesh, Mohanbabu Vijayamma Janki3, Narayana Jayasuryan, Anita Desai,
Vasanthapuram Ravi and Udaykumar Ranga, Transactivation and signaling
functions of Tat are not correlated: biological and immunological characterization of
HIV-1 subtype-C Tat protein, 18 August 2006 Retrovirology.
10. Nagadenahalli Byrareddy Siddappa, Venkatesh Prasanna Kashi , Mohanram
VenkatRamanan,Anangi Balasiddaiah,Narayana Jayasuryan,Anita
Mahadevan,Anita Desai,Kadappa S Satish Susarla K,Shankar,Vasanthapuram Ravi
and Udaykumar Ranga. Gene Expression Analysis from Human Immunodeficiency
Virus Type 1 Subtype C Promoter and Construction of Bicistronic Reporter Vectors,
AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 23, Number 10,
2007.
C. Research Support
Ongoing Research Projects
Sl
No.
Title of Project Funding Agency Amount Date of
sanction and
Duration
1 Mechanisms of Chikungunya virus
disease: mouse model, virulent and
fitness determinants and the
development of novel therapeutics
Department of
Biotechnology,
Govt. of India
3 years
May 2010
Completed Research Projects (State only major projects of last 3 years)
Sl
No.
Title of Project Funding Agency Amount Date of
completion
1 Development of a Cost-
effective Viral –Load
Pharmaceuticals Research
and development Support
Jan 2005 to
Jan 2008
49
Assay and its Commercial
Application in
Fund Programme
(PRDSF),Department of
Science and Technology
2 Functional analysis of the
NF-kB polymorphism in
the long terminal repeat of
HIV-1 subtype-C viruses
Department of
Biotechnology, Govt. of
India
Mar 2005 to
Feb 2008
Place: Bngalore Signature of Investigator
Date: 30-04-2012
50
Curriculum Vitae
Dr. Myneedu Vithal Prasad.
D-3, Staff Quarters,
LRS Institute of TB & RD
New Delhi-110030
26529974(H)
26517829 Ext. 204 (R) 207(O)
9871102482
Educational Qualifications:
Undergraduate Studies
S.S.C.(1973) :Government Model Higher Secondary School for Boys, Kurnool, A.P. India.
Intermediate (1975): Government Junior College, Kurnool, A.P. India.
Professional Qualifications:
MBBS (1981): Kurnool Medical College, A.P. India.
MD (1988): All India Institute of Medical Sciences, New Delhi, India.
Internship/Residency Experience:
Internship (compulsory): 1 year (07.22.1981 to 07.21.1982), Government general Hospital,
Kurnool, A.P. India.
Senior House Physician: 04.01.1983 to 06.27.1983, Department of Skin and STD,
Government General Hospital, Kurnool, A.P. India.
Junior Resident: 1 year (07.04.1983 to 07.03.1984, Departments of Dermatology & STD
and Medicine, Dr. RML Hospital, New Delhi, India
Junior Resident: Post Graduate Residency Program and Senior Research Fellow 07.04.1984
to 06.30.1988, Department of Microbiology, AIIMS, New Delhi, India.
Special Training programs attended:
1. August 15- 17, 2005, SAARC Regional Training for Laboratory Trainers on
Imparting Training on Quality Assurance in Sputum Microscopy In Tuberculosis.
SAARC TB Center and Government of Sri Lanka, Colombo, Sri Lanka.
2. December 1st 2002 to February 28
th 2003 – Fogarty Fellowship AIDS and
Tuberculosis International Training at New York University School of Medicine,
New York and National TB Center, New Ark, NJ, USA.
3. July 25th
and 26th
2002 – ISO 9000 for Hospital and Health Care Services, National
Institute for Standardization and Quality Management, Bureau of Indian Standards,
New Delhi, India.
4. September 7th
to October 12th
1996 – Modern Mycobacteriology, Centers for
Disease Control and Prevention, Atlanta, GA. USA and NY State Department of
Health Laboratories.
5. September 11th
& 12th
1996 – Radiation Safety in the Laboratory, Centers for
Disease Control and Prevention, Atlanta, GA. USA.
6. September 10th
1996 – Bio Safety in the laboratory (9396), Centers for disease
Control and prevention, Atlanta, GA.USA.
51
7. July 19th
to 30th
.1993 – Revised Strategy for Controlling Tuberculosis, National TB
Institute, Bangalore, India.
8. December (4weeks) 1992 – Training in Mycobacteriology, Supra National
Reference Laboratory, Tuberculosis Research center, Chennai, India.
Professional Experience
Senior Microbiologist(SAG) 02. 02. 2008 to date ,Department of Microbiology,
LRS Institute of TB and respiratory Diseases, New Delhi, India
Senior Microbiologist (Specialist Grade I, Senior Scale):02.12.1998 to 1.2 2008,
Department of Microbiology, LRS Institute of TB and Respiratory Diseases,
New Delhi-30, India.
Bacteriologist/Microbiologist (Specialist II Senior Scale): 02.12.1994 to
02.11.1998, Department of Microbiology, LRS Institute of TB and Respiratory
Diseases, New Delhi-30, India.
Bacteriologist (Specialist Grade II): 02.12.1990 to 02.11.1994, Department of
Microbiology, LRS Institute of TB and Respiratory Diseases, New Delhi-30,
India.
Senior Demonstrator: 08.03.1989 to 02.11.1990, Department of Microbiology,
UCMS and GTB hospital, GTB Enclave, Delhi-92, India.
Senior Research Fellow: 12.11.1988 to 08.02.1989, Department of
Microbiology, Institute of Pathology (ICMR) New Delhi, India.
Job Profile of the present post:
Post: Senior Microbiologist (Senior Administrative Grade) of Microbiology and
National Reference Laboratory , LRS Institute Of TB and Respiratory Diseases, /
Head, Department Institute: 520 bed Tertiary care and Post Graduate teaching facility for TB and Respiratory
Diseases.
Department Team: Microbiologists – 8, Senior Residents 2,Technical officer- 1, Senior
Laboratory Technicians (contractual) – 5, Medical laboratory Technicians -7, Laboratory
assistants -2, Laboratory attendants – 5.
Functions:
Plan and organize all the functions of the Level IV Mycobacteriology Laboratory and the
Department of Microbiology and Dept of Molecular Medicine and Biotechnology
1. To assist Revised National TB Control Program (RNTCP) as a National Reference
Laboratory (NRL) for External Quality assurance in Smear Microscopy and Drug
Resistance Surveillance.
2. Conduct training Programs for TB Laboratory Technicians, Senior TB laboratory
Supervisors and other Para Medical personnel under RNTCP.
3. To participate in the teaching activities of post graduate course in the institute.
4. To participate in the operational research in the institute.
52
5. To participate in the External quality control program of the Supra National
Reference laboratory of the region.
6. To plan and monitor infection control activities in the institute.
Membership of Professional Bodies:
1. Permanent Registration with Andhra Medical council, Hyderabad, A.P., India.
2. Registered with Delhi Medical council, New Delhi, India.
3. Life Member – Indian Association of Medical Microbiologists – Delhi Chapter.
4. Life Member – Hospital Infection Society of India.
5. Life Member - Geriatrics Society of India.
6. Life Member – Bio materials and Artificial Organs of India.
7. Member – National Laboratory Committee, Central TB Division, Ministry of
Health and family Welfare, Government of India.
8. Member – Expert Group, DOTS – PLUS Committee, Central TB Division, Ministry
of Health and Family Welfare, Government of India.
9. Member - Doctoral Committee, Department of Microbiology, AIIMS, New Delhi,
India.
10. Reviewer- Indian Journal Of Tuberculosis
11. Reviewer- The Indian journal Of Chest and Allied Diseases
Research Experience:
MD Thesis: “Detection of circulating serum antibodies by CIEP in the serological
diagnosis of Typhoid Fever”.
Guide – M.Sc. (Biosciences) Thesis – In vitro effect of Nitrogen Laser on the
standard strain of M.tuberculosis (H37 RV), Jamia millia Islamia University, New
Delhi, India.
Guide - M.Sc. (Microbiology) Thesis – Profile of Bacteria and Fungi in children
with respiratory tract infections at LRS institute, New Delhi, India. Thesis
submitted to Bundelkhand University, Jhansi, India. (2001).
Co-guide – M.Biotech Thesis entitled ‘Study of M.bovis infections in cultured
strains of human strains of mycobacterium’. Thesis Submitted to AIIMS, New
Delhi, India. (2001)
Co Investigator – Department of Biotechnology funded project entitled “Efficacy
and safety of Immune modulator (Mycobacterium w) as an adjunct therapy in
category II pulmonary TB. (Double Blind, randomized, placebo controlled, multi
centric clinical trial). (2005).
Administrative Experience:
1.Head, Department Of Microbiology:
The department grew from a simple smear microscopy center (level I Lab) to NRL (Level
IV Lab) under RNTCP.
Dept is recognized for conducting Doctoral Programme (PhD) of IGNOU, New Delhi,
India
Dept is recognized for conducting post doctoral Training in Mycobacteriology
2. Chairman, Hospital Infection control committee LRS Institute since 2000:
Works under taken:
Planned and implemented infection control strategies in the institute.
53
The department got upgraded by installing Bio Safety Level III Facilities.
Developed facilities for safe disposal of infectious materials.
Developed plans for separate wards for MDR-TB patients.
3. Chairman, Estate committee of the institute since 2002.
4. Public Grievances officer, since 2004.To 2006
5. Appellate Authority, Under Right to Information Act 2005. Till 2006.
Achievements:
Started laboratory services in the institute after taking over as In-charge of laboratory.
Over the period the lab grew and Microbiology, Pathology, Biochemistry and Mol.
Medicine divisions were established as full-fledged Departments.
Department of Microbiology is recognized as a National reference Laboratory for TB under
RNTCP programme of India. It is also recognized and approved by IGNOU for doctoral
programme in Mycobacteriology and Microbiology. It is externally quality tested by WHO
Supra National reference lab of Institute of Tropical Medicine Antwerp Belgium. It is
accreditated for testing for TB and DST for all anti TB drugs by conventional as well as by
MGIT method.
It is one of the few labs which have got state of the art BSL III lab facilities in the country.
Research Papers Published
1. Rajnish Gupta, M.sircar, A.Jaiswal, V.K.Arora, K.Gupta, P.Visalakshi and
V.P.Myneedu. A thyroid Tubercular abscess and bilateral symmetrical hilar
lymphadenopathy; a rare association. Ind. j Chest Dis. Allied Sci. 2004: 46: 121-
126
2. Manju Sharma, M.Alfamilamo, H.K.Prasad, V.P.Myneedu, N.Nand.
Characterization by single strand conformation polymorphism of mutations in the
rpoB gene of Rifampicin resistant M.tuberculosis strains from Vancouver, Mexico
city and New Delhi. J Asso Phy of Ind., 2000, 48: 568 -572.
3. Sunil Bhatt,Subrato Mukherjee, V.P.Myneedu,R.C.Jain, R.Sarin, Unsupervised
intermittent short course chemotherapy with intensive health education .Ind J Tub.,
1998, 45: 145 – 151.
4. R. Rajalingam, N.K. Mehra, R.C. Jain, V.P.Myneedu, J.N.Pande. Polymerase chain
reaction based sequence specific oligonucleotide hybridization analysis of HLS
class II antigens in Pulmonary Tuberculosis relevance to chemotherapy and disease
severity. J inf. Diseases 1996, 173: 669 – 676.
5. M.M.Puri, Myneedu.V.P. Jain.R.C., Nitrogen and Helium-neon Laser therapy in the
treatment of Drug resistant Pulmonary tuberculosis cases. Laser Therapy,1995,
7:123
6. Rupak Singla, V.P.Myneedu, M.M.Puri, A.Jaiswal, R.C.Jain,. A study of
Ethionamide, cycloserine, Isoniazide, sodium PAS, and Kanamycin in the re
treatment of drug failure pulmonary TB cases. Ind J. Tub, June 1995, 42: 23 – 26.
54
7. Ruth Stavrum Vithal Prasad Myneedu, Virendra K. Arora, Niyaz Ahmed, Harleen
M.S. Grewal In –Depth Molecular Characterization of Mycobacterium tuberculosis
from New Delhi – Predominance of Drug Resistance Isolates of the ‘Modern’ (Tb
D12) Type PLoS One.2009; 4(2):e4540. Epub 2009 Feb 23.
8. Ajay Wanchu., Yuxin Dong., Sunil Sethi, V.P. Myneedu, Arthur Nadas, Zhentong
Liu, John Belisle, Suman Laal A. Biomaekers for Clinical and Incipient
Tuberculosis: Performance in a TB-Endemic Country. PLoS One.2008 Apr 30; 3
(4):e2071.
9 H.Syre, V.P Myneedu, V.K. Arora, and H.M.S. Grewal Direct Detection of
Mycobacterial Species in Pulmonary Specimens by Two Rapid Amplifiaction Test,
the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test and the Geno
Type Mycobacteria Direct Test. J.Clin.Microbiol.2009 Nov.,
47(11):36359.Epub2009Sep 30.
10 VisalakshiP, Meharwal S, Myneedu V.P, Behera D (2009) Evaluation of direct
method of drug susceptibility testing of Mycobacterium tuberculosis to rifampicin
and isoniazid by nitrate reductase assay in a national reference laboratory
Diagonistic Microbiology and Infectious Disease 66:148-152
11 .MyneeduV.P ,Visalakshi, Varma.A.K., Behera.D, Bhalla.M, Prevalence of XDR
TB cases – A retrospective study from a tertiary care TB hospital (Accepted for
publication In Ind.J.Tub)