Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Oct;20(5):16-22.

Purification of an IgA Monoclonal Antibody Specific for the Acr Protein of Mycobacterium tuberculosis by Immunoaffinity Chromatography

Fátima Reyes  1 , Oscar Otero  1 , Frank Camacho  1 , María Elena Sarmiento  1 , Armando Acosta  1
Affiliations

Purification of an IgA Monoclonal Antibody Specific for the Acr Protein of Mycobacterium tuberculosis by Immunoaffinity Chromatography

Fátima Reyes et al. Malays J Med Sci. 2013 Oct.

Abstract

Background: A monoclonal antibody (mAb) of the IgA isotype, designated TBA61, is specific for the Acr protein of Mycobacterium tuberculosis (MTB). TBA61 has been used in studies exploring protection against tuberculosis (TB), and its efficacy has been proven using different challenge models. To purify the mouse IgA isotype, a combination of methods, such as globulin precipitation, ion exchange, and gel filtration, is usually required to achieve a satisfactory degree of purity.

Methods: To minimise the number of chromatographic steps, we proposed to employ immunoaffinity chromatography using the Acr protein of MTB as a specific ligand for this mAb. For this purpose, the HspX gene was cloned and expressed in Escherichia coli, and recombinant Acr (rAcr) was coupled to a cyanogen bromide-activated Sepharose 4B matrix, which was used to purify TBA61 mAb from ascites produced in mice in a single step.

Results: The recovery from the purification procedure was 1.46 mg per mL of ascites. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot showed a high purity. The purified mAb retained its reactivity against the Acr protein based on enzyme-linked immunosorbent assay (ELISA) and western blot.

Conclusion: The purification method used is rapid, simple, and specific and can be easily scaled up.

Keywords: Acr protein; IgA; Mycobacterium tuberculosis; affinity; chromatography; monoclonal antibody.

PubMed Disclaimer

Figures

Figure 1:
Figure 1:
Amplification of the hspX gene using specific primers. Lane 1 shows 100 bp DNA ladder. Lane 2 indicates a band of 500 bp corresponding to the hspX gene.
Figure 2:
Figure 2:
(a) Purification of rAcr protein: SDS-PAGE analysis of rAcr protein, purified by IMAC. Soluble fraction (Lane 1), flowthrough fraction (Lane 2), washing fraction (Lane 3), elution fraction (Lane 4). (b) Purification of rAcr protein: Western blot analysis of rAcr protein, purified by IMAC. Soluble fraction (Lane 1), flowthrough fraction (Lane 2), washing fraction (Lane 3), elution fraction (Lane 4).
Figure 3:
Figure 3:
(a) SDS-PAGE analysis of the purified TBA61. Ascites (Lane 1), flowthrough fraction (Lane 2) and purified TBA61 (Lane 3). (b) Western blot analysis of the purified TBA61 employing different anti heavy chain specific mouse immunoglobulins (α IgA, α IgG, α IgM). Purified TBA61 under non reducing conditions (Lane 1), purified TBA61 under reducing conditions (Lane 2).
Figure 4:
Figure 4:
(a) Reactivity of the purified TBA61 against Acr by Western blot. Purified TBA61 (Lane 1), TBG65 MAb as positive control (Lane 2), 9A11D6 MAb as negative control (Lane 3). (b) Reactivity of the purified TBA61 against Acr by indirect ELISA. TBG65 mAb as positive control, NR: 9A11D6 MAb as negative control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. World Health Organization . Geneva (CH): World Health Organization; 2013. [cited 2013]. Tuberculosis. [Internet] doi: http://www.who.int/mediacentre/factsheets/fs104/en/
    1. World Health Organization . Geneva (CH): WHO report; 2008. Global Tuberculosis Control 2008: Surveillance, Planning, Financing; pp. 1–37.
    1. Center for Disease Control and Prevention (CDC) Emergence of Mycobacterium tuberculosis with extensive resistance to secondline drugs worldwide, 2002–2004. MMWR Morb Mort Wkly Rep. 2006;55(11):301–305. - PubMed
    1. Anonymous The role of BCG vaccine in the prevention and control of tuberculosis in the United States. A joint statement by the advisory council for the elimination of tuberculosis and the advisory committee on immunization practices. MMWR Recomm Rep. 1996;45(RR-4):1–18. - PubMed
    1. Falero-Diaz G, Challacombe S, Rahman D, Mistry M, Douce G, Dougan G, et al. Transmission of IgA and IgG monoclonal antibodies to mucosal fluids following intranasal or parenteral delivery. Int Arch Allergy Immunol. 2000;122(2):143–150. - PubMed

LinkOut - more resources