Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-02-18
2002-02-05
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S034000, C530S388600
Reexamination Certificate
active
06344337
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
None.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to an immunoassay to detect identifying antigens in horses that are infected with
Sarcocystis neurona
. The immunoassay is preferably an antigen-capture-based assay that relies upon polyclonal or monoclonal antibodies against the 16 (±4) and/or 30 (±4) kDa antigens specific to
Sarcocystis neurona
to detect the presence of the 16 (±4) and/or 30 (±4) kDa antigens in equine serum or equine cerebrospinal fluid. The present invention further relates to polyclonal and monoclonal antibodies against the 16 (±4) and/or 30 (±4) kDa antigens, and DNA and clones encoding the 16 (±4) and/or 30 (±4) kDa antigens.
(2) Description of Related Art
Equine protozoal myeloencephalitis (EPM) is a neurological disease caused by the protozoan parasite
Sarcocystis neuronaa
. In recent years, EPM has caused significant health, economic, and emotional costs to horses and their owners (reviewed by McKay et al., The Compendium of Continuing Education for Practicing Veterinarians 14: 1359-1366 (1997). Opossums have been implicated as the natural reservoir of
Sarcocystis neurona
because the sexual stages of the parasite occur in the intestines of the opossum and the sporocysts are passed in the feces of the opossum. Horses accidentally eat the opossum feces containing the sporocysts when they are grazing; however, because
Sarcocystis neurona
does not appear to form mature tissue cysts in equines, equines are considered to be dead end hosts. Because opossums are ubiquitous in the United States, large numbers of horses are exposed to this parasite: approximately 50 to 60% of the horses nationwide (Blythe et al., J. Am. Vet. Med. Assoc. 210: 525-527 (1997), Saville et al., J. Am. Vet. Assoc. 210: 519-524 (1997), Bentz et al., J. Am. Vet. Med. Assoc. 210: 517-518 (1997)).
Currently, there are no in-field or horse-side diagnostic tests for determining whether a horse is currently infected with
Sarcocystis neuronaa
. A Western blot test was developed to detect antibodies to
Sarcocystis neurona
in cerebrospinal fluid of horses suspected of having EPM; however, these Western blot assays have not been reliable in predicting the presence of
Sarcocystis neurona
due to the prevalence in horses of cross-reacting antibodies to other Sarcocystis species (Granstom et al. J. Vet. Diag. Invest. 5: 88-90 (1993), Fenger et al., Vet. Parasitol. 68: 199-213 (1997), Bentz et al., ibid., Saville et al., ibid., Blythe et al., ibid.). More recently, an improved western blot diagnostic test was disclosed in U.S. application Ser. No. 09/156,954 which reliably measures the prevalence of antibodies against
Sarcocystis neurona
in horse serum or cerebrospinal fluid. The improved method measures the presence of identifying 16 (±4) and 30 (±4) kDa
Sarcocystis neurona
antigens on Western blots that have been pretreated with antibodies against bovine
Sarcocystis cruzi
which prevents binding to the western blot of antibodies that may be present in the horse serum against Sarcocystis spp. other than
Sarcocystis neuronaa
.
Currently, there are no vaccines to protect horses from the parasite, and current treatment regimens are effective in only about 50% of the horses (Martenuik et al., The Conference of Research Workers in Animal Diseases, Nov. 10-11, Chicago, Ill. (1997)). However, these studies on treatment efficacy were based on a low number of horses. The U.S. Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), National Animal Health Monitoring System (NAHMS) of the Needs Assessment Survey (NAS) has designated EPM as one of the top two infectious diseases of national importance to the horse industry. Among veterinarians and race horse owners, EPM has been ranked as the leading health care concern. In particular, 58% of the race horse owners ranked EPM as the top health care concern.
Since there are no commercially available vaccines for EPM and EPM is a significant health concern of the equine industry, considerable effort has been directed towards developing therapeutic methods for treating EPM. For example, U.S. Pat. No. 5,935,591 to Rossignol et al. describes using thiazolides as a treatment for EPM; U.S. Pat. No. 5,883,095 to Granstrom et al. describes using triazine-based anti-coccidials as a treatment for EPM; U.S. Pat. No. 5,830,893 to Russel describes using triazinediones as a treatment for EPM; U.S. Pat. No. 5,747,476 to Russel describes using a combination of pyrimethamine and a sulfonamide, preferably sulfadiazine in the absence of known therapeutic amounts of trimethoprim as a treatment for EPM; and U.S. Pat. No. 5,925,622 to Rossignol et al. describes using aryl glucuronide of 2-hydroxy-N-(5-nitro-2-thiazolyl) benzamide as a treatment for EPM.
Treatment for EPM is expensive and cumbersome because of the long duration required to achieve positive results. Because many horses cannot be successfully treated, economically and emotionally valuable animals have been lost to EPM. However, the extent of EPM's economic impact is even greater because of the large sums of money spent by horse owners for treating lame horses which have been incorrectly diagnosed with EPM, for giving prophylactic treatments that have no scientific basis, and for finding positive post-race drug test results.
EPM has been the cause of hysteria in the equine industry. The small amount of scientific data available on EPM supports a high exposure rate of horses, but there are no data available that document the rate of clinical disease resulting from exposure to the parasite. Because of this, horse owners and veterinarians assume that the rate of clinical disease is high. As a result, several alarming consequences have arisen. Horses with lameness or other neurological diseases are being misdiagnosed as having EPM. People whose livelihoods depend on horses are resorting to medicating all their horses all of the time with antimicrobials. This approach to treating EPM is very widespread in the racing industry. However, this indiscriminate use of antimicrobials has the potential of leading to resistant bacteria such as Salmonella,
E. coli
, etc. which will then enter the environment and pose a risk for humans and animals. Thus, the repercussions of EPM may extend beyond a disease that merely affects the horse industry. All of the repercussions of EPM are expensive, decrease the value realized to the U.S. equine industry, and raise the specter of a public health problem of immense proportions.
For the above reasons, there is a need for a reliable diagnostic assay that can detect those horses that are infected with
Sarcocystis neurona.
SUMMARY OF THE INVENTION
The present invention provides a method for detecting the presence of
Sarcocystis neurona
in an equine in an immunoassay, the improvement which comprises reacting a biological sample from the equine suspected of harboring the
Sarcocystis neurona
with an antibody against a
Sarcocystis neurona
antigen to form an antibody-antigen complex. In particular, the antigen is a
Sarcocystis neurona
specific antigen about 16 (±4) kDa and about 30 (±4) kDa. Optionally, the method provides a positive control consisting of the 16 (±4) and/or 30 (±4) kDa antigens or portion thereof.
In a preferred embodiment of the method a labeled antibody against the antigen or the antibody in the antibody-antigen complex is provided to the reaction for the detecting. Preferably, the label is selected from the group consisting of alkaline phosphatase, horseradish peroxidase, fluorescent compounds, luminescent compounds, colloidal gold, and magnetic particles. In a preferred embodiment, the label is biotin which is reacted with peroxidase conjugate and then detected by reaction with an appropriate color forming substrate.
Preferably, the biological sample is selected from the group consisting of serum, cerebrospinal fluid, and
Mansfield Linda S.
Murphy Alice J.
Rossano Mary G.
Vrable Ruth A.
Board of Trustees of Michigan State University
McLeod Ian C.
Park Hankyel T.
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