Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1997-02-24
2003-07-29
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S005000, C435S007700, C435S007220, C435S007920, C435S863000, C435S975000, C435S007100, C435S007900
Reexamination Certificate
active
06599691
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a rapid immunoassay kit and method for semi-quantitatively detecting antibodies in human saliva to antigens of disease-related microorganisms, e.g., antibodies to
Mycobacterium tuberculosis
. This invention also encompasses an alternative embodiment that permits quantitative, though less rapid, detection of antibodies in saliva by adapting the methodology of the semi-quantitative immunoassay to an enzyme linked immunosorbant assay (ELISA). Within this invention, this alternative embodiment is referred to as the quantitative immunoassay, or similar, to distinguish it from the rapid, semi-quantitative immunoassay.
2. Description of the Prior Art
Though not substantially related to the invention described herein, there have been several efforts of peripheral interest. Ebersole has described a SEROLOGICAL METHOD FOR THE IDENTIFICATION OF MICROORGANISMS in U.S. Pat. No. 4,458,014 for the identification of diseases of the mouth. Chen et al. have described in U.S. Pat. No. 4,866,167 a DETECTION OF HUMAN ORAL CELLS BY NUCLEIC ACID HYBRIDIZATION to detect oral bacterial species. The methods of both Ebersole and Chen et al. are technically complex, time consuming, not rapid and are not based on detecting antibodies in saliva to antigens of disease-related microorganisms.
Olson et al. have described an IMMUNOLOGICAL COLOR CHANGE TEST INVOLVING TWO DIFFERENTLY COLORED REAGENT SPOTS in U.S. Pat. No. 4,639,419. Their patent describes a substantially different methodology than that described herein. This test is an agglutination reaction directed toward identifying antigenic material wherein a colored substrate and colored reagent combine, in positive reactions, to give the appearance of a third color.
Higerd and Goust have described an IMMUNOSUPPRESSIVE EXTRACELLULAR PRODUCT FROM ORAL BACTERIA in U.S. Pat. No. 4,268,434. Their patent relates to a method of producing an extracellular immunosuppressive bacterial material from various bacteria to suppress the natural immunity in patients where this outcome is desired, e.g., organ transplant patients. This procedure has substantially different objectives and methodology than the invention described herein.
Antibodies are naturally produced biomolecules which react specifically with usually foreign biomolecules called antigens. Disease-related microbial infections, e.g.,
Mycobacterium tuberculosis
which causes tuberculosis, are usually characterized by the production of antibodies to the specific antigens of disease-related microorganisms. Antibodies are also produced with other diseases and afflictions, e.g., autoimmune diseases where there is an often destructive antibody response to the host—not necessarily related to a microbial antigen. In the case of autoimmune diseases, the host usually supplies the antigens of disease-related microorganisms. Within this invention, the term “disease-related antigens” includes microbial antigens and other substances capable of possessing antigenic properties and which are associated with specific diseases, conditions and disorders, including infectious diseases and autoimmune diseases. Antibodies are expressed in saliva; their detection in saliva is fundamental and unique to this invention.
This invention, as an example, can determine individuals actively or previously infected with
Mycobacterium tuberculosis
and thus aid in the diagnosis of tuberculosis.
Mycobacterium tuberculosis
causes tuberculosis (1-4) and this is widely acknowledged throughout the medical community. There are several screening tests for tuberculosis. The Mantoux test uses tuberculin purified protein derivative (PPD) which is injected intracutaneously (e.g., Tubersol®, Connaught Laboratories Limited, Willowdale, Ontario, Canada) (1). A delayed hypersensitivity reaction develops in individuals having previous infection with
Mycobacterium tuberculosis
. The injection site is normally read within 48 to 72 hours after intracutaneous injection of the antigen; a palpable induration measuring 10 mm in diameter or more is considered a positive reaction. This procedure is accepted as an aid in the diagnosis of tuberculosis infection.
The Heaf test uses a multiple puncture disk which introduces needles through concentrated Old Tuberculin applied to the skin (1). The tine test uses tuberculin adhering to metal tines; inoculation is accomplished by simple pressure into the skin (1). The Heaf and tine tests are acceptable for screening but should be confirmed by the Mantoux test (1). Antigenic material can also be applied by scratch, i.e., Pirquet's test (2). Similar to the Mantoux test, these tests generally require 48 to 72 hours after inoculation before results can be determined. The Bacillus of Caimette and Guerin (BCG) is a live, attenuated strain of
Mycobacterium bovis
which has been used with varying success as a vaccine against tuberculosis in countries where the prevalence of tuberculosis is high (5).
Mycobacterium bovis
is not normally found in humans, but since it shares antigens present in
Mycobacterium tuberculosis
, it can serve as an antigen source to detect host antibodies to
Mycobacterium tuberculosis
. BCG causes tuberculin conversion to positive; it has also been used to stimulate the immune system against a variety of medical conditions. In this invention, both PPD and BCG can serve as suitable antigens to detect host antibodies to desired mycobacteria.
Other antigens have been described. Maes has described A60-ANTIGEN FROM MYCOBACTERIA AND USE THEREOF AS TUBERCULIN VACCINE in U.S. Pat. No. 4,965,192. This patent describes the A60-antigen as effective for detecting prior exposure of an individual to mycobacterial infections through the use of a cutaneous test. This patent is similar to other inoculation tests mentioned earlier except that a new antigen is used and 24 to 48 hours are required to observe the responses at the test site.
Mycobacterium tuberculosis
whole cells (inactivated), lipoarabinomannan of
Mycobacterium tuberculosis
(6-8) and other mycobacterial derivatives can serve as antigen sources to detect host antibodies to mycobacteria in this invention.
To continue with the
Mycobacterium tuberculosis
example of this invention, a major advantage is that tuberculosis screening can be done rapidly—in approximately 5 minutes—in one visit and in a non-invasive manner. The advantages of this invention are significant when compared with earlier tests that are invasive, take 48 to 72 hours to obtain results and require two visits of the subject, e.g., the Mantoux and other tuberculosis screening tests. These earlier tests are considered too slow and are invasive. Similar limitations apply to other medical screening and diagnostic tests that are not rapid, invasive (e.g., require blood or serum samples) or involve culturing or other complicated and expensive laboratory procedures. The premise of this use of the assay is that individuals infected with
Mycobacterium tuberculosis
develop antibodies to this bacterial species which are present in their saliva and which react with mycobacterial antigens. The antibodies are then labeled and color development detected and read visually after addition of an appropriate enzymatic substrate, if required. Color development signifies positive individuals and permits semi-quantitative assessment of antibody levels. Active or previous infection with
Mycobacterium tuberculosis
is, therefore, determined. This assay aids in the diagnosis of tuberculosis and is rapid, non-invasive, uncomplicated and inexpensive. Less rapid but quantitative simultaneous assessment of multiple (or single) saliva samples is accomplished by adapting the semi-quantitative assay to an ELISA.
What is needed is a rapid, simple, non-invasive assay to semi-quantitatively detect antibodies in saliva to antigens of disease-related microorganisms, e.g., antibodies to
Mycobacterium tuberculosis
that react with mycobacterial antigens. This assay uses human saliva, is non-invasive, can be developed and read in less tha
Ralls Stephen Alden
Simonson Lloyd Grant
Graser Jennifer E.
Hemby, Jr. Joseph K.
Spevack A. David
The United States of America as represented by the Secretary of
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