Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B

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Abstract

Background

Tuberculosis is the leading cause of death due to bacterial infections worldwide, mainly caused by Mycobacterium tuberculosis. The antigen 85 complex comprises a set of major secreted proteins of M. tuberculosis, which are potential biomarkers for diagnostic.

Results

In this work, the first human single chain fragment variable (scFv) antibodies specific for the tuberculosis biomarker 85 B were selected by phage display from naïve antibody gene libraries (HAL7/8). Produced as scFv-Fc in mammalian cells, these antibodies were further characterized and analysed for specificity and applicability in different tuberculosis antigen detection assays. Sandwich detection of recombinant 85 B was successful in enzyme linked immunosorbent assay (ELISA), lateral flow immunoassay and immunoblot. Whereas detection of M. tuberculosis cell extracts and culture filtrates was only possible in direct ELISA and immunoblot assays. It was found that the conformation of 85 B, depending on sample treatment, influenced antigen detection.

Conclusions

Recombinant antibodies, selected by phage display, may be applicable for 85 B detection in various assays. These antibodies are candidates for the development of future point of care tuberculosis diagnostic kits. Using 85 B as a biomarker, the antigen conformation influenced by sample treatment is important.

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Most cited references 57

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Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.

Susanna G Von Essen, H Schägger (1987)

A discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system for the separation of proteins in the range from 1 to 100 kDa is described. Tricine, used as the trailing ion, allows a resolution of small proteins at lower acrylamide concentrations than in glycine-SDS-PAGE systems. A superior resolution of proteins, especially in the range between 5 and 20 kDa, is achieved without the necessity to use urea. Proteins above 30 kDa are already destacked within the sample gel. Thus a smooth passage of these proteins from sample to separating gel is warranted and overloading effects are reduced. This is of special importance when large amounts of protein are to be loaded onto preparative gels. The omission of glycine and urea prevents disturbances which might occur in the course of subsequent amino acid sequencing.

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Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project.

C. Dye, S. Scheele, P. DOLIN … (1999)

To estimate the risk and prevalence of Mycobacterium tuberculosis (MTB) infection and tuberculosis (TB) incidence, prevalence, and mortality, including disease attributable to human immunodeficiency virus (HIV), for 212 countries in 1997. A panel of 86 TB experts and epidemiologists from more than 40 countries was chosen by the World Health Organization (WHO), with final agreement being reached between country experts and WHO staff. Incidence of TB and mortality in each country was determined by (1) case notification to the WHO, (2) annual risk of infection data from tuberculin surveys, and (3) data on prevalence of smear-positive pulmonary disease from prevalence surveys. Estimates derived from relatively poor data were strongly influenced by panel member opinion. Objective estimates were derived from high-quality data collected recently by approved procedures. Agreement was reached by (1) participants reviewing methods and data and making provisional estimates in closed workshops held at WHO's 6 regional offices, (2) principal authors refining estimates using standard methods and all available data, and (3) country experts reviewing and adjusting these estimates and reaching final agreement with WHO staff. In 1997, new cases of TB totaled an estimated 7.96 million (range, 6.3 million-11.1 million), including 3.52 million (2.8 million-4.9 million) cases (44%) of infectious pulmonary disease (smear-positive), and there were 16.2 million (12.1 million-22.5 million) existing cases of disease. An estimated 1.87 million (1.4 million-2.8 million) people died of TB and the global case fatality rate was 23% but exceeded 50% in some African countries with high HIV rates. Global prevalence of MTB infection was 32% (1.86 billion people). Eighty percent of all incident TB cases were found in 22 countries, with more than half the cases occurring in 5 Southeast Asian countries. Nine of 10 countries with the highest incidence rates per capita were in Africa. Prevalence of MTB/HIV coinfection worldwide was 0.18% and 640000 incident TB cases (8%) had HIV infection. The global burden of tuberculosis remains enormous, mainly because of poor control in Southeast Asia, sub-Saharan Africa, and eastern Europe, and because of high rates of M tuberculosis and HIV coinfection in some African countries.

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Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis.

J Belisle, V Vissa, T Sievert … (1997)

The dominant exported proteins and protective antigens of Mycobacterium tuberculosis are a triad of related gene products called the antigen 85 (Ag85) complex. Each has also been implicated in disease pathogenesis through its fibronectin-binding capacities. A carboxylesterase domain was found within the amino acid sequences of Ag85A, B, and C, and each protein acted as a mycolyltransferase involved in the final stages of mycobacterial cell wall assembly, as shown by direct enzyme assay and site-directed mutagenesis. Furthermore, the use of an antagonist (6-azido-6-deoxy-alpha, alpha'-trehalose) of this activity demonstrates that these proteins are essential and potential targets for new antimycobacterial drugs.

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Author and article information

Contributors

Manon Fuchs

Susanne Kämpfer

Saskia Helmsing

Ralf Spallek

Wulf Oehlmann

Wiebke Prilop

Ronald Frank

Stefan Dübel

Mahavir Singh

Michael Hust

Journal

Journal ID (nlm-ta): BMC Biotechnol

Journal ID (iso-abbrev): BMC Biotechnol

Title: BMC Biotechnology

Publisher: BioMed Central

ISSN (Electronic): 1472-6750

Publication date Collection: 2014

Publication date (Electronic): 17 July 2014

Volume: 14

Page: 68

Affiliations

[1 ]Technische Universität Braunschweig, Institut für Biochemie und Biotechnologie, Spielmannstr.7, 38106 Braunschweig, Germany

[2 ]Lionex GmbH, Salzdahlumer Str. 196, 38126 Braunschweig, Germany

[3 ]Helmholtz Zentrum für Infektionsforschung, Abteilung Chemische Biologie, Inhoffenstr. 7, 38124 Braunschweig, Germany

[4 ]Leibniz-Institut für Molekulare Pharmakologie, Robert-Roessle-Str. 10, 13125 Berlin, Germany

Article

Publisher ID: 1472-6750-14-68

DOI: 10.1186/1472-6750-14-68

PMC ID: 4119940

PubMed ID: 25033887

SO-VID: c4b26b8d-7f44-4106-826c-24350ebafc2d

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B

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Abstract

Background

Results

Conclusions

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UCL: Covid-19 Research

Most cited references 57

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.

Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project.

Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis.

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