Identiﬁcation of proteins from tuberculin puriﬁed protein derivative (PPD)
, Jane Newcombe
, Tom A. Mendum
, Odir A. Dellagostin
, Johnjoe McFadden
Centro de Biotecnologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil
School of Biomedical and Life Sciences, University of Surrey, Guildford, GU2 7XH Surrey, UK
Received 2 December 2008
Received in revised form
10 July 2009
Accepted 10 July 2009
Mycobacterium bovis AN5
Mycobacterium avium D4
The tuberculin puriﬁed protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis,
however it is poorly deﬁned. Most mycobacterial proteins are extensively denatured by the procedure
employed in its preparation, which explains previous difﬁculties in identifying constituents from PPD to
characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based charac-
terization of PPD from several different sources by LC-MS/MS, which combines the solute separation
power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify
proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identiﬁed
among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of
the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%)
but there was a preponderance of proteins involved in lipid metabolism. We identiﬁed a group of 21
proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In
addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This
study provides a better understanding of the tuberculin PPD components leading to the identiﬁcation of
additional antigens useful as reagents for speciﬁc diagnosis of tuberculosis.
Ó 2009 Elsevier Ltd. All rights reserved.
Tuberculosis continues to be a worldwide problem for both
humans and animals.
Human tuberculosis is predominantly
caused by Mycobacterium tuberculosis. Bovine tuberculosis, caused
mainly by Mycobacterium bovis is an important cause of economic
losses and can be a zoonotic infection.
An important control
strategy for the prevention of this disease is the use of an effec-
tive vaccine. The M. bovis bacillus Calmette-Guerin (BCG) vaccine,
an attenuated strain of M. bovis, has been widely used for control
of human tuberculosis despite controversy over its protective
In cattle, BCG has been used in a series of trials with
various degrees of protection against M. bovis challenge.
However, a major constraint in the use of attenuated mycobac-
terial vaccines such as BCG is that vaccination of humans or cattle
interferes with detection of tuberculosis by tuberculin skin test.
The development of tests, which can distinguish between infec-
tion with M. tuberculosis or M. bovis and vaccination with BCG,
could greatly assist in the diagnosis of early infection as well as to
enhance the use of tuberculosis vaccines on a wider scale.
The tuberculin skin test (Mantoux test) is used in humans and
animals to distinguish between infected and uninfected individ-
The test was originally introduced in the 1890s by Robert
Koch, who used a boiled crude preparation of M. tuberculosis
cultures as the antigen, referred to as old tuberculin. However, this
was replaced in the early 1940s by Florence Seibert who introduced
and standardized a puriﬁed protein derivative of tuberculin (PPD).
The tuberculin skin test cannot distinguish between an M. tuber-
culosis infection and BCG vaccination or exposure to environmental
mycobacteria. These cross-reactions are generally attributed to the
presence in PPD of antigens shared by other Mycobacterium
PPD is prepared by heat sterilization of 6-week-old M.
tuberculosis, M. bovis or Mycobacterium avium grown in broth
medium, followed by ﬁlter sterilization and protein concentration
using ultraﬁltration or ammonium sulphate precipitation.
Considering the widespread use of this immunological reagent, it is
surprising that little is known about the active components of
Some proteins that are probably present in PPD prepara-
tions have been tested as diagnostic reagents. The recombinant
proteins MPB59, MPB64, MPB70 and ESAT-6 were evaluated in
a differential diagnostic test, but only ESAT-6 was shown to be
E-mail address: firstname.lastname@example.org (J. McFadden).
These authors contributed equally to this work.
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1472-9792/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
Tuberculosis 89 (2009) 423–430