Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-07-07
2004-05-11
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S150100, C424S163100, C424S168100, C424S248100, C435S004000, C435S007100, C435S174000, C435S253100
Reexamination Certificate
active
06733983
ABSTRACT:
The present application claims the benefit of International patent application serial number PCT/NL98/00701 filed Dec. 8, 1998, which in turn is based on the European patent application serial number EP 97203851.7 filed Dec. 8, 1997.
The invention relates to a method for identifying a Mycobacterium species responsible for a mycobacterial infection in a human or animal, and to diagnostic kits for use in said method.
The genus Mycobacterium contains about 50 species. It is responsible for a number of diseases which are known collectively as mycobacterioses. The best known and widest spread of these are leprosy, caused by
M. leprae
, and tuberculosis caused by
M. tuberculosis
. Both of these diseases affect more than ten million people all over the world. Most other mycobacteria normally occur only as environmental saprophytes. However, they can also cause opportunist diseases, which happens often, but not exclusively, in organisms suffering from problems with their immune systems, such as AIDS patients or people undergoing immunosuppression. The opportunist types comprise the slow-growing species
M. avium
, and the closely related
M. intracellulare
and
M. scrofulaceum
(often together referred to as the MAIS complex),
M. kansai, M. marinum
and
M. ulcerans
, and the fast-growing species
M. chelonae
and
M. fortultum
. Although quite rare in the Western world for several decades, the occurrence of opportunist mycobacterial diseases and tuberculosis has shown a significant increase with the incidence of AIDS. Further, it has been reported that mycobacteria or antigens of mycobacteria play a role in the etiology of a plurality of other diseases, such as sarcoidosis and Crohn's disease, as well as different auto-immune diseases, such as auto-immune dermatitis, rheumatoid arthritis and diabetes. It has been suggested that this role can be attributed to a structural mimicry between epitopes of mycobacteria and those of the host organism.
The cell walls of mycobacteria are very complex and contain many different lipids, some of which have structures unique to the genus. These structures comprise mycolinic acids and esters, peptido-glycolipide, arabino-galactane and lipo-arabinomanane. The lipid-rich cell walls of a mycobacterial cell are responsible for the notable coloring properites of the mycobacteria. They also enable mycobacteria to counter an attack by the immune system of a host organism. A number of species, after being taken up into macrophages, surround themselves with a thick layer of secreted lipids.
Many of the different components of the mycobacteria interact with the immune system of a host organism. These components comprise proteins and hydrocarbon antigens, which can either be actively secreted by the mycobacteria or can form part of the cell wall or cell membrane. In addition, they may be present in the cytoplasm, for example in the cytoplasmic matrix, ribosomes and enzymes. Mycobacteria further also possess immuno-modulating components, such as immunosuppressing compounds and adjuvants. As of consequence, a single mycobacterial species can induce a large variety of immune responses in different forms having diverse specificities. This makes it very difficult to derive protein antigens which are suitable for the detection of species-specific humoral responses as a basis for a highly sensitive and specific diagnostic test for the above mentioned diseases, particularly tuberculosis. Because mycobacteria have a frequent occurrence, both human and animal body fluids contain nearly at all times anti-mycobacterial antibodies.
In the past, researchers have attempted to develop a sufficiently sensitive diagnostic test for mycobacterioses. The focus of these attempts has mostly been on finding species-specific glycolipid antigens for the detection of specific humoral immune responses, because of the problems with the specificity of protein antigens.
In the international patent application 94/14069, it has been disclosed to make use of the antibody response of an organism to immuno-dominant mycobacterial cross-reactive antigen components (further referred to as Im-CRAC) for developing a diagnostic test for mycobacterial infections. The Im-CRAC is believed to provide indirect information on the nature of the immune recognition of, and response to, a specific mycobacterial pathogen.
The method proposed in WO-A-94/14069 is based on the discovery that the clinical manifestation of mycobacterial diseases is related to the varying capability of an individual host to produce a humoral response to different mycobacterial immuno-cross-reactive antigen components (Im-CRAC). Each mycobacterial infection generates its own specific antibody response to a number of specified antigens. Analysis of the antibody-response, e.g. by immunoblotting, has demonstrated that the immuno-dominant Im-CRAC vary in accordance with the immunopathological manifestation of the mycobacterial diseases. Said analysis results in different and distinguishing band patterns of mycobacterial antigens for different individuals which are infected with different Mycobacterium species. The band pattern, which is obtained after an immunoblot, can be referred to as an Antigen Bar Code. The antigen-antibody reactions which are shown in the bar code are, when taken together, unique for a certain Mycobacterium species.
The present invention aims at providing an improved method for identifying a Mycobacterium species in a diagnostic test. Although satisfactory in most aspects, it is still desirable to have a diagnostic test which is even more sensitive than the method described in WO-A-94/14069. Furthermore, there is a need for a diagnostic test which can be used in the determination of previous vaccination, and for monitoring therapy of organisms infected with a Mycobacterium species. During therapy, there are situations where low levels of certain antibodies occur, which may disturb the accuracy and/or sensitivity of the test making use of the antibody-antigen cross-reactions, as outlined hereinabove.
SUMMARY OF THE INVENTION
It has now been found that a highly sensitive diagnostic test can be performed by contacting a sample of a body fluid with both antibodies and antigens. It has been found that, besides antibodies, several mycobacterial components are present in animal and human body fluids, of which the presence can be determined by using cross-reactions with a chosen set of antibodies in a reliable manner.
The invention therefore provides a method for identifying a Mycobacterium species comprising the steps of:
a) contacting at least one immuno-cross reactive antigen component of a mycobacterial species with a sample of a body fluid of a human or animal individual;
b) contacting at least one antibody, which is capable of reacting with a mycobacterial antigen, with said body fluid sample;
c) detecting the presence of antigen-antibody complexes, and identifying the Mycobacterium species present in said body fluid sample.
REFERENCES:
patent: 4391904 (1983-07-01), Litman et al.
patent: 5817473 (1998-10-01), Das et al.
patent: 0407605 (1991-01-01), None
patent: WO94/14069 (1994-06-01), None
patent: WO97/09429 (1997-03-01), None
Araj, G.F.., et al. “Improved Detection of Mycobacterial Antigens in Clinical Specimens by Combined Enzyme-Linked Immunosorbent Assays”, Diagn. Microbiol. Infect. Dis., vol. 17, pp. 119-127, 1993.*
Roche, P.W., et al, “Antibody Responses to the 18-kDa Protein ofMycobacterium lepraein Leprosy and Tuberculosis Patients”, International Journal of Leprosy, vol. 60, No. 2, pp. 201-207, 1992.*
Vega-Lopez et al, Recognition of mycobacerial antigens by sera from patients with leprosy. Journal of Clinical Microbiology. vol. 26, No. 12, pp. 2472-2479. 1988.*
Havlir, D. V. et al., “Human Immune Response toMycobacterium TuberculosisAntigens.”Infection and Immunity(1991) 59:665-670.
Rambukkana, A. et al., “Subcellular Distribution of Monoclonal Antibody Defined Epitopes on ImmunodominantMycobacterium TuberculosisProteins in the 30-kDa Region: Identification and Localization of 29/33-kDa Doublet
Goerdayal Soenita
Houthoff Hendrik-Jan
Kolk Arend
Kroon-Swart Saskia
Kuyper Sjoukje
Hoffmann & Baron , LLP
Kreatech Biotechnology B.V.
Swartz Rodney P
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