University of Pretoria

Adcock Ingram Limited

Jan Adrianus Verschoor

Carl Baumeister

Anne Lenaerts

Elzbieta Johannsen

Walter Allen Shaw

Sandra Noel Bye

Mervyn Beukes

Sandra Van Wyngaardt

Dismore Gilbert Ramathudi Siko

Immobilising isolated mycolic acid antigens of tuberculous mycobacterial origin or a synthetic analogue thereof on a screen-printed electrode by binding of the antigens to a self-assembled monolayer comprising a thiolated hydrophobic substance to produce immobilized mycolic acids antigens in the form of a mycolic acid antigen-containing self-assembled monolayer coating on a surface of the electrode.

Method of diagnosing tuberculosis

Mycolic acid antigen containing liposomes, and an electrode immobilised antigens of tuberculous mycobacterial origin are produced. A diagnostic sample from a human suspected of having tuberculosis is diluted and divided into first and second samples. A control sample comprising the first sample and a redox probe is exposed to the liposomes. A test sample comprising the second sample and a redox probe is exposed to liposomes not containing mycolic acid antigens. The control sample is contacted with the immobilised antigens to allow antibodies therein to bind to the antigens. The test sample is contacted with the immobilised antigens to allow any antibodies therein to bind to the antigens. The degree of binding in the samples is compared by electrochemical impedance spectroscopy. Any observed lesser binding by the control sample is an indicator of antibodies to the antigens in the diagnostic sample, indicating tuberculosis in the human.

A liposomal composition comprising a sterol-modified lipid and a purified mycobacterial lipid cell wall component or analog or derivative thereof is described. The composition is useful as a lipid antigen-presenting vehicle for the detection of lipid antigen specific biomarker antibodies in antibody containing biological samples in the diagnosis of active tuberculosis. The purified lipid cell wall component is typically a purified mycolic acid or a mixture of mycolic acids from athat produces mycolic acids. The sterol-modified lipid is typically a phospholipid.

Liposomal composition comprising a sterol-modified lipid and a purified mycobacterial lipid cell wall component and its use in the diagnosis of tuberculosis

The present invention relates to a method of detecting an active tuberculosis infection in a serum sample from an individual. More specifically, the disclosed method uses a single chain variable fragment from a monoclonal immunoglobulin specific for mycolic acids of mycobacterial origin to detect surrogate markers, the presence of which indicate the individual has an active tuberculosis infection. Markers useful for practicing the disclosed method can be serum mycolic acid antigen, serum anti-mycolic acid antibodies or both. In a preferred embodiment, the method is practiced using a lateral flow assay format.

Method of detecting surrogate markers in a serum sample

A method of detecting a surrogate marker for active tuberculosis involves obtaining first, second and third samples from a subject suspected of having active tuberculosis, diluting the first sample and exposing part of it to an immobilized mycolic acid antigen in a test vessel and part of it to an immobilized mycolic antigen in a control vessel. The second sample is exposed to mycolic acid antigen-containing liposomes and the third sample is exposed to liposomes not containing mycolic acid antigens. The second sample is added to the test vessel and the third to the control vessel and binding of antibodies to the mycolic acid and antigen in both the test and control vessel is detected. The degree of binding between the test and control vessels is compared and lesser binding in the test vessel is an indicator of the presence of antibodies to the mycolic acid antigen.

Method for detecting mycobacterial infection

A composition including a purified lipid cell-wall component or analog or derivative thereof and a suitable pharmaceutical carrier, medium, excipient or adjuvant is described. The composition is useful in prophylactic and therapeutic methods of treating a microbial infection in a subject, typically a mycobacterial infection such as tuberculosis, and immune disorders, inflammatory conditions and allergies in a subject, typically autoimmune diseases. It is also useful in diagnostic methods. The purified lipid cell-wall component is typically a purified mycolic acid or a mixture of purified mycolic acids from a bacterium which produces mycolic acids. The bacterium is fromor

Composition comprising a carrier and a purified mycobacterial lipid cell-wall component and its use in the treatment and diagnosis of disease

The invention relates to a diagnostic test for the detection and identification of Mycobacterium species in biological specimens of human and animal origin. The test is based on the immunological detection of one or more antigens originating from Mycobacterium. To enable the detection of an antibody-antigen reaction, the antibodies specific for these antigens can be labelled with an enzyme or fluorescent dye or attached to latex particles or any other suitable label. The diagnostic test may be in a form of ELISA and may or may not require concentration of the Mycobacterium antigens prior to the actual test. The invention comprises: the selection and procurement of a suitable mycobacterial species and strain, the isolation and purification of the antigens, preparation of the necessary conjugates for the immunization of the experimental animals using various carrier molecules, the development of an assay for monitoring the antibody production, caracterization of the antibodies specific for the Mycobacterium antigen/antigens and the development of a diagnostic assay/kit.

Method for detecting the presence of a mycobacterium species and a kit and antibodies for use therein

A composition comprising a purified lipid cell-wall component or analog or derivative thereof and a suitable pharmaceutical carrier, medium, excipient or adjuvant is described. The composition is useful in prophylactic and therapeutic methods of treating a microbial infection in a subject, typically a mycobacterial infection such as tuberculosis, and immune disorders, inflammatory conditions and allergies in a subject, typically autoimmune diseases. It is also useful in diagnostic methods. The purified lipid cell-wall component is typically a purified mycolic acid or a mixture of purified mycolic acids from a bacterium which produces mycolic acids. The bacterium is from Mycobacterium, Corynebacterium, Nocardia or Rhodococcus.

Composition comprising a carrier and a purified mycobacterial lipid cell-wall component and its use in the prevention, treatment and diagnosis of disease

A method of purifying mycolic acids, salts thereof or derivatives thereof. The method involves a) providing a mixture including the mycolic acids, salts thereof or derivatives thereof and contaminants; b) dissolving the mixture in a bi-phasic solvent to form a dissolved mixture including the mycolic acids, salts thereof or derivatives thereof and contaminants; c) purifying the mycolic acids, salts thereof or derivatives thereof by subjecting the dissolved mixture to countercurrent distribution separation involving a sufficient number of cycles to separate the mycolic acids, salts thereof or derivatives thereof from the contaminants; and d) removing the separated, purified mycolic acids, salts thereof or derivatives thereof from the bi-phasic solvent. Residual impurities may be extracted from the removed mycolic acids, salts thereof or derivatives thereof with acetone.

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Pretoria, South Africa

Jan Adrianus Verschoor