71. Pre-warm the centrifuge rotor before use. It may be necessary
to reduce centrifuge deceleration to prevent the biphase from
being disrupted by a sudden stop.
72. Be sure that the centrifuge tubes are suitable for use with
phenol (the recommendation here is Teflon).
73. Any ammonium sulfate percentage may be used depending on
your needs; however, many M. tuberculosis CFPs are differentiated for purification by beginning with a 40% ammonium
sulfate cut of CFP followed by a 50% ammonium sulfate cut
and subsequent 70% ammonium sulfate cut, hence why this
example is used.
74. The solution may begin to become cloudy as protein precipitates out of solution, but it should be easy to differentiate
between this and undissolved ammonium sulfate because the
ammonium sulfate will appear as crystals that settle to the
bottom of the container.
75. A shaker or rocker may also be used if a stir plate is unavailable
for incubation.
76. There should be little to no protein left in the 70% supernatant;
the supernatant is generally discarded once it is confirmed that
the protocol was successfully implemented.
Acknowledgments
This work was supported by ATCC contract 2010-0516-0005
(a subcontract of NIH-NIAID Contract HHSN272201000027c)
and ATCC contract 2016-0550-0002 (a subcontract of
NIH-NIAID contract HHSN272201600013c. Original work is
credited to NIH-NIAID Contract AI-75320.
References
1. Lalvani A, Pareek M (2010) A 100 year update
on diagnosis of tuberculosis infection. Br Med
Bull 93:69–84. https://doi.org/10.1093/
bmb/ldp039
2. Auguste P, Tsertsvadze A, Pink J et al (2016)
Accurate diagnosis of latent tuberculosis in
children, people who are immunocompromised or at risk from immunosuppression and
recent arrivals from countries with a high incidence of tuberculosis: systematic review and
economic evaluation. Health Technol Assess
20. https://doi.org/10.3310/hta20380
3. Targowski T, Chelstowska S, Plusa T (2014)
IGRA as a predictive factor of silent pulmonary
changes in individuals following exposure to
tuberculosis. Lung 192:869–874. https://doi.
org/10.1007/s00408-014-9637-y
4. Pai M, Behr M (2016) Latent Mycobacterium
tuberculosis infection and interferon-gamma
release assays. Microbiol Spectr 4. https://doi.
org/10.1128/microbiolspec.tbtb2-00232016
5. Pai M, Denkinger CM, Kik SV et al (2014)
Gamma interferon release assays for detection
of Mycobacterium tuberculosis infection. Clin
Microbiol Rev 27:3–20. https://doi.org/10.
1128/CMR.00034-13
6. Salgame P, Geadas C, Collins L et al (2015)
Latent tuberculosis infection – revisiting and
revising concepts. Tuberculosis 95:373–384
106
Megan Lucas et al.
to reduce centrifuge deceleration to prevent the biphase from
being disrupted by a sudden stop.
72. Be sure that the centrifuge tubes are suitable for use with
phenol (the recommendation here is Teflon).
73. Any ammonium sulfate percentage may be used depending on
your needs; however, many M. tuberculosis CFPs are differentiated for purification by beginning with a 40% ammonium
sulfate cut of CFP followed by a 50% ammonium sulfate cut
and subsequent 70% ammonium sulfate cut, hence why this
example is used.
74. The solution may begin to become cloudy as protein precipitates out of solution, but it should be easy to differentiate
between this and undissolved ammonium sulfate because the
ammonium sulfate will appear as crystals that settle to the
bottom of the container.
75. A shaker or rocker may also be used if a stir plate is unavailable
for incubation.
76. There should be little to no protein left in the 70% supernatant;
the supernatant is generally discarded once it is confirmed that
the protocol was successfully implemented.
Acknowledgments
This work was supported by ATCC contract 2010-0516-0005
(a subcontract of NIH-NIAID Contract HHSN272201000027c)
and ATCC contract 2016-0550-0002 (a subcontract of
NIH-NIAID contract HHSN272201600013c. Original work is
credited to NIH-NIAID Contract AI-75320.
References
1. Lalvani A, Pareek M (2010) A 100 year update
on diagnosis of tuberculosis infection. Br Med
Bull 93:69–84. https://doi.org/10.1093/
bmb/ldp039
2. Auguste P, Tsertsvadze A, Pink J et al (2016)
Accurate diagnosis of latent tuberculosis in
children, people who are immunocompromised or at risk from immunosuppression and
recent arrivals from countries with a high incidence of tuberculosis: systematic review and
economic evaluation. Health Technol Assess
20. https://doi.org/10.3310/hta20380
3. Targowski T, Chelstowska S, Plusa T (2014)
IGRA as a predictive factor of silent pulmonary
changes in individuals following exposure to
tuberculosis. Lung 192:869–874. https://doi.
org/10.1007/s00408-014-9637-y
4. Pai M, Behr M (2016) Latent Mycobacterium
tuberculosis infection and interferon-gamma
release assays. Microbiol Spectr 4. https://doi.
org/10.1128/microbiolspec.tbtb2-00232016
5. Pai M, Denkinger CM, Kik SV et al (2014)
Gamma interferon release assays for detection
of Mycobacterium tuberculosis infection. Clin
Microbiol Rev 27:3–20. https://doi.org/10.
1128/CMR.00034-13
6. Salgame P, Geadas C, Collins L et al (2015)
Latent tuberculosis infection – revisiting and
revising concepts. Tuberculosis 95:373–384
106
Megan Lucas et al.