Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2002-11-13
2004-02-10
Devi, S. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007200, C435S180000, C436S533000, C436S528000, C436S518000
Reexamination Certificate
active
06689571
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a particle agglutination assay for
Pythium insidiosum
antibodies in a serum sample. In particular, the present invention uses antigens from
Pythium insidiosum
with the latex particles, wherein the particles are agglutinated in the presence of the serum antibody or antibodies. The result is the rapid detection of
Pythium insidiosum
antibodies in a sample serum, usually in five (5) minutes or less.
(2) Description of Related Art
Infections caused by fungal and parafungal organisms are occurring with increasing frequency in patients with debilitating illnesses such as leukemia and AIDS, as well as those undergoing immunosuppressive therapy. Within this group of organisms are the traditional pathogenic fungi and a long list of newly recognized emerging opportunistic fungal and parafungal organisms. Among the emerging pathogens is the oomycete
Pythium insidiosum
, a fungal-like organism in the Kingdom Kromista, Phylum Pseudofungi.
Pythium insidiosum
is not only physiologically distinct from members of the Kingdom Fungi, but also phylogenetically. This may explain why anti-fungal drugs do not have any effect on pythiosis.
Pythiosis insidiosi
particularly occurs in humans and lower animals in the tropical, subtropical, and temperate areas of the world (Cock, W. A. W., et al., J. Clin. Microbiol. 25:344-349 (1987)). The disease was described in the beginning of the 20
th
century in equines of tropical and subtropical countries including India and Indonesia as well as the USA. Soon, however, it was evident that the disease not only affected equines but other mammalian species as well. In lower animals infections of the cutaneous tissues, lymphatic vessels, intestines, lungs, and bones have been found. In humans, a deadly arteritis infection, subcutaneous invasion and keratitis occurs.
Thus,
Pythium insidiosum
is a protist that causes infection in mammals, most often horses and dogs, but sometimes humans. It enters the body through wounds or the GI tract from contaminated plants, soil or water. It causes granulomatous lesions to form wherever it gains a foothold in the body, and can spread to other sites via the lymphatic system. It is 100% fatal if not treated. Early diagnosis greatly increases the chances for survival of the host, as treatment becomes less effective the longer the infection progresses.
The currently available drugs used to treat fungal infections have had little or no effect on
Pythium insidiosum
. Reports of treatment with either amphotericin B or surgery, commonly used to treat this disease in both humans and lower animals, have indicated that 60% of the patients died of their infections. In cases of arterial invasion in humans, amphotericin B did not eliminate the infection (Rinaldi, M. G., et al., Mycology Observer 9:7 (1989); and Thianprasit, M., Trop Dermathol 4:1-4 (1990)), whereas in surgery the main problem has been to determine how much of the infected tissues has to be removed. Thus, relapses are common in surgically treated patients, who must also endure the pain and distress that such an invasive traumatic procedure inflicts on them.
My U.S. Pat. Nos. 5,948,413 and 6,287,573, which are incorporated herein by reference, describes very effective vaccines of
Pythiosis insidiosi
proteins for the treatment of Pythiosis. The problem has been to identify the disease at an early enough stage so that the vaccine can be maximally effective.
Various immunodiffusion (ID) and enzyme linked immunoabsorbant assays (ELISA) have been developed to detect
Pythiosis insidiosum
but they are more expensive and less reliable because of cross-reactivity of the antibodies (Grooters, A.M., et al., J. Vet. Intern. Med. 16:142-146 (2002); Imwidthaya, P., et al., Mycopathologia 106:109-112 (1989); Mendoza, L., et al., Clin. Diagnost. Lab. Immunol. 4:715-718 (1997); and Mendoza, L., et al., J. Clin. Microbiol. 23:813-816 (1986)). The main problem has been that these tests have to be performed by qualified laboratories and professionals. All of the currently available diagnostic tests require specialized knowledge and equipment, and there are only two laboratories in the U.S. that are equipped to perform them. A clinician who suspects pythiosis must send a serum or tissue sample to one of these two laboratories to be tested, which delay the beginning of treatment.
Latex agglutination assay methods and kits for testing for mammalian animal and human fungal pathogens are well known. One reference dealing with such methods is Serodiagnosis of Mycotic Diseases, American Lecture Series. Thomas, Springfield, Ill., USA pp 131-139 (1977) by Palmer, D. F., et al. There has been no suggestion of the use of this type of assay for
Pythiosis insidiosi.
OBJECTS
It is therefore an object of the present invention to provide an agglutination assay and test kit for
Pythium insidiosum
antibodies in serum sample.
It is further an object of the present invention to provide an assay and test kit which is rapid and reliable so as to be performable in the field or at the bedside of the patient.
These and other objects will become increasingly apparent by reference to the following description and drawings.
SUMMARY OF THE INVENTION
The present invention relates to a particle agglutination assay kit for detecting
Pythium insidiosum
antibodies in a serum sample which comprises:
(a) at least one
Pythium insidiosum
antigen which is unique to
Pythium insidiosum
and complexes with
Pythium insidiosum
antibodies which can be present in a serum sample;
(b) particles which complex with the
Pythium insidiosum
antigen;
(c) a negative control serum which is free of
Pythium insidiosum
antibodies;
(d) a positive control serum which contains
Pythium insidiosum
antibodies; and
(e) at least one device for separately mixing the antigen and the particles with (1) the serum sample; (2) the positive control serum, and (3) the negative control serum, wherein if the serum sample contains the
Pythium insidiosum
antibodies which complex with the antigen, the particles are agglutinated and are compared to the positive and negative control serums. Preferably there are multiple of the antigens which are expressed from cells of the
Pythium insidiosum
in a culture medium and then isolated and purified from the culture medium.
In particular the present invention relates to a method for assaying for
Pythium insidiosum
antibodies in a serum sample which comprises:
(a) providing:
(1) at least one
Pythium insidiosum
antigen which is unique to
Pythium insidiosum
and complexes with
Pythium insidiosum
antibodies which can be present in a serum sample;
(2) particles which complex with the
Pythium insidiosum
antigen;
(3) a negative control serum which is free of
Pythium insidiosum
antibodies;
(4) a positive control serum which contains
Pythium insidiosum
antibodies; and
(5) at least one device for separately mixing the antigen and the particles with (1) the serum sample; (2) the positive control serum, and (3) the negative control serum, wherein in the assay if the serum sample contains the
Pythium insidiosum
antibodies which complex with the antigen, the particles are agglutinated and are compared to the positive and negative control serums; and
(b) testing the serum sample, negative and positive controls with the particles and the antigen to determine if there is agglutination of the latex particles by the sample serum. The method can be used for the testing on human serum or animal serum. Preferably the testing is of horse serum. Preferably the particles are latex. The incorporated prior art describes numerous types of particles which can be used.
REFERENCES:
patent: 4829012 (1989-05-01), Cambiaso et al.
patent: 5290517 (1994-03-01), Samuels et al.
patent: 5534441 (1996-07-01), Miyazaki et al.
patent: 5652149 (1997-07-01), Mileaf et al.
patent: 5795719 (1998-08-01), Richard et al.
patent: 5948413 (1999-09-01), Mendoza
patent: 6287573 (2001-09-01), Mendoza
Rinaldi, M.G., et al., Mycology Observer 9:7 (19
Board of Trustees of Michigan State University
Devi S.
McLeod Ian C.
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