Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-04-15
2004-04-13
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007310, C435S007920, C435S007930, C436S514000, C436S536000
Reexamination Certificate
active
06720152
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention generally relates to methods of detection of a fungus and fungal infections through the use of protein antigens specific for the fungus. This invention also relates to methods for identifying such antigens, to antibodies raised against these antigens, to the use of such antigens and/or antibodies in assay methods for the determination of the presence of the fungus or diseases caused by the fungus in humans, other animals, plants, food, feed, and inorganic materials such as soil or air. The invention also relates to assay kits suitable for carrying out such diagnostic methods. In a preferred embodiment, the invention particularly relates to the fungus
Histoplasma capsulatum
(hereinafter
H. capsulatum
) and the infections caused by this fungus, histoplasmosis.
Rapid, positive detection of fungi has long been difficult because antibodies which are made to fungi are generally nonspecific. That is, these antibodies often cross-react with other fungi. This problem is thought to be due to the abundance of highly immunogenic carbohydrate antigenic determinants which are present, for example, in the fungal cell wall. The cross-reactive nature of fungal antigens is exemplified by previously known approaches to develop diagnostic methods to identify infections of
H. capsulatum. H. capsulatum
is a pathogenic dimorphic fungus that grows as multicellular mycelia in nature and as unicellular budding yeasts in humans and animals. Inhalation of airborne propagules results in a morphological transformation to the yeast form which causes pulmonary infection and occasional progressive disease, particularly in immunosuppressed patients. Histoplasmosis is highly endemic in the Ohio and Mississippi valleys in the United States, and it is also widely distributed in Latin America, southern Europe, Asia, Australia and Africa.
The diagnosis of histoplasmosis in humans is often suggested by results of a careful clinical evaluation and radiologic studies, but laboratory tests are necessary to confirm the diagnosis. Isolation of the organism from blood or tissue provides a definitive diagnosis. Serological tests are also important diagnostic tools for histoplasmosis. The most widely available tests are the immunodiffusion assay, which detects antibodies to heat-sensitive glycoproteins called H and M antigens, and the more sensitive complement fixation test, which is traditionally performed with yeast and mycelial antigens. More sensitive antibody assays such as radio-immunoassay and enzyme immunoassay have been used to detect IgM and IgG antibodies to crude fungal extracts. See, generally, references 39, 40, and 41.
Attempts to develop antibody serology tests for diagnosis of histoplasmosis have been hampered by poor specificity caused by immunologic cross-reactivity between various fungal species. The problem of cross-reactivity to other fungi may be worsened in the diagnosis of histoplasmosis by the fact that
H. capsulatum
is taxonomically closely related to two other pathogens,
Coccidioides immitis
, and
Blastomyces dermatitidis
. See Kwon-Chung,
Science
177:368-369 (1972), and McGinnis et al.,
Mycotaxon
8:157-164 (1979). These fungi, which may be present along with
H. capsulatum
, cause coccidiomycosis and blastomycosis, diseases with etiologies similar to histoplasmosis. While
H. capsulatum, C. immitis
, and
B. dermatitidis
are known as imperfect fungi due to their rare or nonexistent sexual stage, studies have shown that
H. capsulatum
and
B. dermatitidis
are in the same telomorph genus, Ajellomyces, and those two fungi may be in the same taxonomic family as
C. immitis
, the Gymnoascaceae, order Onygenales, of the Ascomycetes. Another fungal pathogen, Candida sp., is also an ascomycete. See generally, Kwan-Chung et al.,
Medical Mycology
, Lea and Febiger, Philadelphia (1992), which is incorporated by reference.
In view of the cross-reactivity and poor specificity common with fungal antibodies, there is a need for improved methods for identifying antigens which are specific to a target fungus. Such antigens are useful in diagnosis of diseases caused by the fungus and in determining the presence or absence of the fungus.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to develop methods suitable for identifying protein antigens specific to a target fungus and, particularly, to
H. capsulatum.
It is also an object to develop assays and assay reagents having improved specificity for identifying target fungal antigens and antibodies, and for the diagnosis of diseases caused by a target fungus and, particularly, by
H. capsulatum.
Therefore, the present invention is directed to a method for identifying a protein antigen of a target fungus. A cDNA gene expression library is obtained for the target fungus, and the library is expressed to form an array of target-fungus proteins. Antisera to the target fungus and to a nontarget fungus are also obtained, each of which comprises antibodies to the target fungus and nontarget fungus, respectively. The nontarget fungus has at least one antigenic determinant (e.g. a protein determinant or glycoprotein determinant) in common (i.e., shared with) the target fungus. A protein antigen specific to the target fungus is then identified by identifying a target-fungus protein which is bound by the antibodies to the target fungus, but which is not substantially bound by antibodies to the nontarget fungus. That is, while antibodies to the target fungus are immunoreactive with the identified protein antigen, antibodies to the nontarget fungus are not substantially immunoreactive with the identified protein antigen.
The invention is also directed to substantially purified or isolated antibodies or antibody fragments. In one embodiment, the antibody or antibody fragment is immunoreactive with a protein antigen identified according to the aforementioned method. In another embodiment, the antibody or antibody fragment is immunoreactive with an antigen of
H. capsulatum
, but which is not substantially immunoreactive with antigens of each of
Coccidioides immitis, Blastomyces dermatitidis
or Candida sp. In a further embodiment, antibody or antibody fragment is immunoreactive with a protein antigen having the amino acid sequence set forth in SEQ ID NO: 3 or with a portion thereof that is specific to
H. capsulatum.
The invention is directed, moreover, to a method for determining the presence or absence of a target-fungus antibody in a vertebrate such as a mammal. In one embodiment, the method comprises obtaining an antibody-containing sample from the vertebrate, contacting the sample with a target-fungus protein antigen, and determining whether an antibody in the sample immunoreacts with the target-fungus protein antigen. In an alternative embodiment directed to determining the presence or absence of antibodies to
H. capsulatum
in a mammal, the method comprises obtaining an antibody-containing sample from the mammal, contacting the sample with a protein antigen of
H. capsulatum
which is bound by antibodies to
H. capsulatum
but which is not substantially bound by antibodies to each of
Coccidioides immitis, Blastomyces dermatitidis
or Candida sp., and determining whether an antibody in the sample immunoreacts with the protein antigen of
H. capsulatum
. In an additional embodiment for determining antibodies to
H. capsulatum
, the method comprises obtaining an antibody-containing sample from the mammal, contacting the sample with a protein antigen having an amino acid sequence as set forth in SEQ ID NO: 3, and determining whether an antibody in the sample immunoreacts with the protein antigen.
The invention is further directed to a method for determining the presence or absence of a target-fungus protein antigen in a sample. The method generally comprises obtaining a sample to be tested for the presence or absence of the target-fungus protein antigen, contacting the sample with an antibody or antibody fragment which is immunoreactive with a target-fungus protein antigen, and determin
Chandrashekar Ramaswamy
Weil Gary J.
Barnes-Jewish Hospital
Duffy Patricia A.
Sonnenschein Nath & Rosenthal
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