Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1999-07-07
2001-10-09
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C422S051000, C422S051000, C435S007100, C435S007200, C435S007310, C435S007360, C435S040510, C435S962000, C435S970000, C435S973000, C435S921000, C435S922000, C435S923000, C435S924000, C435S288300, C436S528000, C436S531000, C436S527000, C436S809000, C436S810000
Reexamination Certificate
active
06300140
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for immunoassay and reagents thereof which are suitable for detecting fungi present on the skin. Also contemplated are immunoassays for other antigens of pathogenic microorganisms.
BACKGROUND OF THE INVENTION
Physicians are constantly being confronted with situations where the differential diagnosis is between a primary dermatitis such as eczema, psoriasis, seborrheic dermatitis, versus a fungal pathogen such as dermatophytosis or candidiasis of the skin.
Currently, the preferred method of diagnosing a fungal pathogen is a potassium hydroxide wet mount (KOH), and or a fungal culture or screen. The KOH is highly technical and experience-dependent and a fungal screen or culture takes approximately 7 to 30 days for a definitive answer. The KOH wet mount has been combined with DMSO in ranges of 10 to 20 percent to enhance the breakdown of cell walls so that fungal hyphae can be more readily discerned via microscopic identification. There also has been the use of tissue dyes, such as india ink to highlight the hyphael structures for quicker identification. The problem with the above microscopic KOH (with and without DMSO or tissue dye) techniques is that they are dependent upon the individual expertise of the operator. In this scenario there could be a large margin of error both in the over identification of fungi-false positives, and in the under identification of fungi-false negatives. The rate of false positives and false negatives is purely a correlation of operator experience. For example, a board certified dermatologist should predictably have a high rate of specificity, over 90 percent, due to the rigorous training and testing specifically for diseases of the skin. However, a family physician who has limited training and limited experience in skin diseases will predictably have a higher error rate in either over or under detection of the fungi. Therefore, it would be desirable to have a test kit that would eliminate operator error, and have a predictably accurate and reproducible rate of identification of pathogenic fungi, yeasts and molds. The herein disclosed invention is directed to this end.
PRIOR ART PATENTS
Zuk in U.S. Pat. No. 4,281,061 discloses an immunoassay method in which various signals are discussed. The assay can detect an analyte in a concentration of 10
−4
to 10
−15
M. Further, the assay can be used to detect microorganisms which may be either intact or lysed.
Khanna in U.S. Pat. No. 4,582,791 discloses immunoassays in which whole cells are detected. Examples of the cells detected are virus infected cells, bacteria and fungi.
None of the prior art patents discloses the invention herein disclosed.
SUMMARY OF THE INVENTION
Treatment regimens that are available for fungal infections of the skin, hair and or nails are targeted for groups of organisms in three broad categories, i.e., dermatophtyes, candidal organisms and/or molds.
Therefore, the clinician only needs to know whether the fungus is a dermatophyte, candidal organism or a mold. It is not necessary to know the exact genus and species of the organism infecting the skin, hair or nails, but the general group category, i.e. dermatophyte—which comprises such organisms as
Trichophyton tonsurans, Microsporum audouinii, Trichophyton violaceum, Trichophyton mentagrophytes, Microsporum canis, Epidermophyton floccosum, Trichophyton concentricum, Microsporum ferrugineum, Microsporum rivalierii, Trichophyton rubrum, Trichophyton megninic and Trichophyton schoenleinii
(the majority but not all inclusive); candida—which comprises such organisms as (but not limited to)—
Candida albicans, Candida krusei, Candida stellatoidea, Candida tropicalis, Candida pseudotropicalis and Candida guillermondii;
and molds—which comprises such organisms (but not limited to)—Aspergillus, Cephalosporium,
Fusarium oxysporum
and
Scopulariopsis brevicalis.
The invention is directed to a rapid dermatophyte, candidal and mold test kit that will identify the class of organisms infecting the skin, hair and/or nails of humans and animals. This kit will employ polyclonal antibodies to the classes of organisms, not the individual organisms. The rapid identification of the class of organisms either dermatophyte, candidal or mold will immediately allow the clinician to provide the necessary treatment on the day of the patient's visit. All therapies are geared toward classes of organisms, hence, the novel kit employing polyclonal antibodies.
This form of collection would be especially rapid and useful for the entire integument and would provide sufficient antigen to react with a polyclonal antibody to the dermatophtyes. The antibody would be tagged with colored microscopic beads, which in turn could be rapidly and reproducibly identified by any operator physician or allied health professional. The colored microscopic beads attached to each specific antibody could be color coded for each class of organism that the operator is trying to identify, such as a dermatophtyes colored red, candida colored yellow and molds colored green.
It is further anticipated that polyclonal or monoclonal antibody kits could be employed to identify agricultural pathogens, i.e. molds in the field, without waiting for laboratory confirmation.
It is further anticipated that a kit could be employed for home testing in the field of allergy and allergy testing to identify potential substances implicated in allergen-induced asthma and upper respiratory disease. The invention can be directed to the field of allergy and allergy testing and treatment recommendations that would occur after triggering allergens were discovered in the patient's home or work environment.
Tinea is a common disease that affects humans as well as domestic animals. It is produced by pathogenic fungi that colonize the skin. Until now, the methods that the clinician had available for the detection of such pathogens consisted of:
a) submitting samples of scrapings obtained from the skin to a specialized laboratory for culturing,
b) direct visualization of scrapings obtained from the skin under a microscope or
c) depositing skin or nail scrapings in a flask containing fungal culture material, which change color after about 1 to 2 weeks in the presence of fungi.
These three methods pose disadvantages. For example, the culture of skin is specific and highly sensitive, but usually takes approximately one to four weeks to obtain an answer, and requires the sample be sent to a specialized laboratory. Visualization under a microscope requires the steps of harvesting skin scales; incubating them briefly with warm potassium hydroxide and the slide is then warmed and a coverslip applied. This method takes about 5 minutes, but it requires a trained professional examining the sample, since it is difficult to differentiate fungal hyphae from artifacts such as cell borders, impurities, cloth threads or hairs.
The herein disclosed invention represent novel diagnostic kits for the identification of pathogens that currently take days to weeks to diagnose. These diagnostic kits will find immediate use in their respective fields.
The inventors envision the kit for carrying out the immunoassay of this invention. would contain therein:
1) an adhesive slide,
2) a file or sandpaper, and
3) a container or containers having therein specific antibodies to the various infective agents to be tested for.
Slides with an adhesive surface to capture a skin sample for analysis under a microscope are known in the art. (Dockerty et al, Genitourinary Med. Vol. 71 (1995 pages 407-409). However, the inventors are not aware of the use of a transparent adhesive area on a transparent slide for immuno-testing a sample. The adhesive slide of this invention can be designed with an adhesive surface within a peripheral lip to obtain a sample from the skin or other surface which has to be sampled. Then an immuno-reagent or other reagent can be added to accomplish the reaction. After the immuno-reaction has taken place, the sample can be examined under a microscope to d
Bochner Bruce S.
Martin Neil F.
Robinson Howard N.
Tausk Francisco A.
Bloom Leonard
Chin Christopher L.
Leonard Bloom
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