Development of antibody based biosensor kit for rapid detection of tuberculosis by using urine samples

Amrita Masanta1, Shikha Singh1*

1. Centre for Biotechnology, Siksha ‘O’ Anusandhan (Deemed to be University), Bhubaneswar, Odisha, India.
1*corresponding author: Rama Devi Women’s University, Bhoinagar, Bhubaneswar, Odisha, India.
*email: shikhsingh@gmail.com

Samples Total Proteins

Introduction
Healthy Control -0.055±0.505
 Tuberculosis (TB) stands one of the most infectious
and highly contagious disease ever. Mild infection 0.765±0.631
 Mainly cause by Mycobacterium tuberculosis, remains Acute infection 5.777±1.785
dormant state for many years.
Chronic infection 6.693±1.766
 Infection rate cross 10 million
and mortality rate 1.7 million every
year (WHO GLOBAL TB REPORT Fig: 1 Shows the total antigenic protein content (mg/ml) in normal and negative
2018). urine samples at different phases of TB infection.
 TB has been re-emerged globally
as a result of the evolution of drug and multi drug
resistant strains .
 Developing countries people are facing resource-poor
settings that mainly outbreaks the TB transmission
which results constantly increasing of mortality rate.
 Techniques like flowcytometry, radiometric detection,
latex agglutination etc. hold their own set of
imperfections.
 All above problems permit us to design a robust,
highly sensitive & specific with accuracy diagnostic kit
for initial detection of tuberculosis infection.
Fig.2 Banding pattern of 12% SDS-PAGE
Fig.3 Indirect ELISA shows the
Methodology (A) Proteins indicate from positive urine
deviation in absorbance at 450 nm
samples of phase III; Molecular weight of
as an activity of protein.
standard protein is shown on left margin.

Sample collection (urine) and estimation of

antigens (Mycobacterium proteins) Fig.4 Analysis of
Western blotting of
antibody to pooled
Separation of proteins by SDS-PAGE & antigen mycobacterial
quantification by Indirect ELISA using TB antigenic proteins
infected serum. from various phases
of TB infection.

Immunoblotting and specific bands eluted from gel

and followed by tryptic digestion Work to be done
 Immunoblotting and gel tryptic in -solution digestion .
Peptide analysis by MALDI TOF/MS  Peptide analysis by MALDI TOF/MS.
 Peptide spectra analysis and protein identification.
 Development of diagnostic immuno chromatographic kit using antibody and
its standardization.
Peptide spectra analysis and protein identification
Conclusion Acknowledgements
 Biosensing technology have 1. Department of Science and
Results potential for early detection of Technology, New Delhi, India.
TB infection. 2. Centre for Biotechnology, Siksha ‘O’
 Among the 100 urine samples, 25 samples were  Effective for developing Anusandhan , Bhubaneswar, Odisha,
positive (pulmonary TB), 45 samples were negative countries with resource-poor India.
control & 30 samples were healthy control and protein settings.
estimation was carried , shown in fig.1.  It can be cost effective , high References
 SDS-PAGE was done with all urine samples, banding sensitivity, reliability (no false P. Nahid, M. Pai and P. C. Hopewell,
pattern was shown in fig.2. +ve), portability &disposability. Proc. Am. Thorac. Soc., 2006, 3, 103.
 Identification of antigenic protein was done by ELISA  A progress towards TB J. C. Palomino, A. Martin, M.
and activity of protein was shown in fig. 3. elimination can be possible by Camacho, H. Guerra, J. Swings and F.
 Immunoblotting was performed and the response of biosensors. Portaels, Antimicrob. Agents
Ab-Ag was seen against ranging from MW of 10-120 Chemother., 2002, 46(8), 2720–2722.
kDa shown in fig.4.
 The Ab response was high against the antigens with
MW of 35 kDa, 66 kDa, 110 kDa and seen additional
bands of reactivity, including 14 kDa, 16 kDa, 190 kDa
and 110 kDa. “INSPIRE Fellowship Review Meet” organized by Kalinga Institute of
Industrial Technology (KIIT), Bhubaneswar, Odisha, India, Pin: 751024 6th,
7th and 8th June 2019 .