quantity of bulk materials free of environmental contaminants.
Such quality reagents are necessary for developing highly sensitive,
accurate, and reproducible diagnostic tests, subunit vaccines, and
therapeutics for pathogenic mycobacteria. Two traditional assays
used in concert for the discovery and analysis of mycobacterial
antigens and the host response to mycobacterium spp. are the tuberculin skin test (TST) and Interferon-Gamma Release Assays
(IGRA) [1]. While the TST (using purified protein derivative)
and the IGRA are commonly used to detect previous exposure to
or infection with M. tuberculosis complex [2–4], both lack the
ability to differentiate active and latent infections [5, 6], thus
demonstrating a need for better characterization of M. tuberculosis
proteome [7, 8]. In addition to these traditional vaccine and diagnostic pipelines [9], M. tuberculosis proteins are also included in
studies exploring their potential as pathogen-specific biomarkers
for detection of tuberculosis cases, including latent tuberculosis
infection (LTBI) and potentially during treatment response [10–
12]. M. tuberculosis proteins are shown to integrate into host exosomes, and in complex samples, such as serum, targeted mass
spectrometry assays augment detection levels of low-abundance
peptides for a more sensitive approach [13]. Alternative proteomic
screenings, such as those technologies using “affinity binding
reagents,” may offer a more rapid and personalized diagnostic
approach but rely on uncompromised structural epitopes from
native proteins [7]. Therefore, the generation of discreet, wellcharacterized native protein fractions and purified native proteins
compliments these novel innovative approaches to further our
understanding of tuberculosis pathogenesis.
Quality reagents for M. tuberculosis remain in high demand; in
addition, there is an expanding need for qualified reagents for
nontuberculous mycobacteria (NTM). Studies have demonstrated
that prior infections with NTMs can interfere with diagnosis of
M. bovis [14, 15]. This is of particular concern with regard to
livestock, where there is potential for a considerable economic
impact [16], and in select regions where endemic disease in wildlife
populations poses a risk of animal to human transmission
[14]. Annotation of several NTM genomes demonstrates high to
variable homology of protein antigen orthologs to those found in
M. tuberculosis complex organisms; isolation of native proteins from
these NTMs could provide for a more targeted and accurate diagnostic approach [15]. A rise in pulmonary and cutaneous NTM
incidences [17, 18] further poses a public health concern. Thus, the
use of established biochemical and proteomics methodologies is
required to extend studies into these understudied and important
pathogens.
In this chapter, we provide methods for inactivation of cell
pastes for safe downstream use in non-BSL3 laboratories and
some downstream processing methods to generate numerous
78
Megan Lucas et al.
Such quality reagents are necessary for developing highly sensitive,
accurate, and reproducible diagnostic tests, subunit vaccines, and
therapeutics for pathogenic mycobacteria. Two traditional assays
used in concert for the discovery and analysis of mycobacterial
antigens and the host response to mycobacterium spp. are the tuberculin skin test (TST) and Interferon-Gamma Release Assays
(IGRA) [1]. While the TST (using purified protein derivative)
and the IGRA are commonly used to detect previous exposure to
or infection with M. tuberculosis complex [2–4], both lack the
ability to differentiate active and latent infections [5, 6], thus
demonstrating a need for better characterization of M. tuberculosis
proteome [7, 8]. In addition to these traditional vaccine and diagnostic pipelines [9], M. tuberculosis proteins are also included in
studies exploring their potential as pathogen-specific biomarkers
for detection of tuberculosis cases, including latent tuberculosis
infection (LTBI) and potentially during treatment response [10–
12]. M. tuberculosis proteins are shown to integrate into host exosomes, and in complex samples, such as serum, targeted mass
spectrometry assays augment detection levels of low-abundance
peptides for a more sensitive approach [13]. Alternative proteomic
screenings, such as those technologies using “affinity binding
reagents,” may offer a more rapid and personalized diagnostic
approach but rely on uncompromised structural epitopes from
native proteins [7]. Therefore, the generation of discreet, wellcharacterized native protein fractions and purified native proteins
compliments these novel innovative approaches to further our
understanding of tuberculosis pathogenesis.
Quality reagents for M. tuberculosis remain in high demand; in
addition, there is an expanding need for qualified reagents for
nontuberculous mycobacteria (NTM). Studies have demonstrated
that prior infections with NTMs can interfere with diagnosis of
M. bovis [14, 15]. This is of particular concern with regard to
livestock, where there is potential for a considerable economic
impact [16], and in select regions where endemic disease in wildlife
populations poses a risk of animal to human transmission
[14]. Annotation of several NTM genomes demonstrates high to
variable homology of protein antigen orthologs to those found in
M. tuberculosis complex organisms; isolation of native proteins from
these NTMs could provide for a more targeted and accurate diagnostic approach [15]. A rise in pulmonary and cutaneous NTM
incidences [17, 18] further poses a public health concern. Thus, the
use of established biochemical and proteomics methodologies is
required to extend studies into these understudied and important
pathogens.
In this chapter, we provide methods for inactivation of cell
pastes for safe downstream use in non-BSL3 laboratories and
some downstream processing methods to generate numerous
78
Megan Lucas et al.