Inflammatory bowel disease first step assay system

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...

Reexamination Certificate

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C435S007240, C435S007950, C435S007310, C436S506000, C436S513000

Reexamination Certificate

active

06218129

ABSTRACT:

BACKGROUND OF THE INVENTION
Field of the Invention
The invention relates generally to the fields of inflammatory bowel disease and immunology and more specifically to serological methods for distinguishing inflammatory bowel disease from other disorders.
BACKGROUND INFORMATION
Inflammatory bowel disease (IBD), which occurs world-wide and afflicts millions of people, is the collective term used to describe two gastrointestinal disorders of unknown etiology: Crohn's disease (CD) and ulcerative colitis (UC). IBD together with irritable bowel syndrome (IBS) will affect one-half of all Americans during their lifetime, at a cost of greater than $2.6 billion dollars for IBD and greater than $8 billion dollars for IBS. A primary determinant of these high medical costs is the difficulty of diagnosing digestive diseases. The cost of IBD and IBS is compounded by lost productivity, with persons suffering from these disorders missing at least 8 more days of work annually than the national average.
Inflammatory bowel disease has many symptoms in common with irritable bowel syndrome, including abdominal pain, chronic diarrhea, weight loss and cramping, making definitive diagnosis extremely difficult. Of the 5 million people suspected of suffering from IBD in the U.S., only 1 million are diagnosed as such. The difficulty in differentially diagnosing IBD and IBS hampers early and effective treatment of these diseases. Thus, there is a need for rapid and sensitive testing methods for definitively distinguishing IBD from IBS.
Progress has been made in precisely diagnosing, in many cases, Crohn's disease and ulcerative colitis. However, current methods for diagnosing an individual as having Crohn's disease or ulcerative colitis, while highly specific, are relatively costly, requiring labor intensive immunofluorescence assays and careful analysis of cell staining patterns. Although these costly assays are easily justified for those individuals previously diagnosed with or strongly suggested to have IBD, a less expensive but highly sensitive alternative would be advantageous for first determining if an individual has inflammatory bowel disease at all. Such a highly sensitive primary screening assay would provide physicians with an inexpensive means for rapidly distinguishing individuals with IBD from those having IBS, thereby facilitating earlier and more appropriate therapeutic intervention and minimizing uncertainty for patients and their families. If desired, such a primary screening assay could be combined with a subsequent, highly specific assay for determining if an individual diagnosed with IBD has Crohn's disease or ulcerative colitis.
Unfortunately, such a highly sensitive and inexpensive primary screening assay for distinguishing IBD from other digestive diseases presenting with similar symptoms is currently not available. Thus, there is a need for a method of rapidly diagnosing inflammatory bowel disease at a very early stage of disease progression. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides a highly sensitive method of diagnosing inflammatory bowel disease (IBD) in an individual. The method includes the steps of isolating a sample from the individual; determining by non-histological means whether the sample is positive for anti-neutrophil cytoplasmic antibodies (ANCA); determining whether the sample is positive for anti-
Saccharomyces cerevisiae
immunoglobulin A (ASCA-IgA); determining whether the sample is positive for anti-
Saccharomyces cerevisiae
immunoglobulin G (ASCA-IgG); and diagnosing the individual as having IBD when the sample is positive for ANCA, ASCA-IgA or ASCA-IgG, and diagnosing the individual as not having IBD when the sample is negative for ANCA, ASCA-IgA and ASCA-IgG, provided that the method does not include histological analysis of neutrophils. In a method of the invention, ANCA, ASCA-IgA or ASCA-IgG positivity can be conveniently determined, for example, using an immunoassay.
The present invention further provides a highly sensitive method of diagnosing IBD in an individual. This method of the invention includes the steps of isolating a sample from the individual; determining by non-histological means whether the sample has an ANCA level above an ANCA cut-off value (X); determining whether the sample has an ASCA-IgA level above an ASCA-IgA cut-off value (Y); determining whether the sample has an ASCA-IgG level above an ASCA-IgG cut-off value (Z); and diagnosing the individual as having IBD when the ANCA level is above X, the ASCA-IgA level is above Y, or the ASCA-IgG level is above Z, and diagnosing the individual as not having IBD when the ANCA level is below X, the ASCA-IgA level is below Y, and the ASCA-IgG value is below Z, where X, Y, and Z are independently selected to achieve optimized sensitivity, specificity, negative predictive value, positive predictive value or overall agreement, provided that the method does not include histological analysis of neutrophils.
In a highly sensitive method of diagnosing IBD provided by the present invention, X, Y and Z can be independently selected such that, for example, the sensitivity of diagnosing an individual with IBD is at least about 70%, and can be selected such that, additionally, the specificity of diagnosing an individual with IBD is 30-60%. In addition, X, Y and Z can be independently selected such that the sensitivity of diagnosing an individual with IBD is at least about 70%, the specificity of diagnosing an individual with IBD is 30-60%, and the negative predictive value in a population having an IBD disease prevalence of about 15% is at least about 90% and can be, for example, at least about 95%.
Furthermore, X, Y and Z can be independently selected such that the sensitivity of diagnosing an individual with IBD is at least about 90%, and can be selected such that, additionally, the specificity of diagnosing an individual with IBD is 20-60%. If desired, X, Y and Z can be independently selected such that the sensitivity of diagnosing an individual with IBD is at least about 90%, the specificity of diagnosing an individual with IBD is 20-60%, and the negative redictive value in a population having an IBD disease revalence of about 15% is at least about 90%. The negative predictive value can be, for example, at least about 95%. In addition, X, Y and Z can be independently selected such that, for example, the sensitivity of diagnosing an individual with IBD is about 90%, the specificity is about 37%, and the negative predictive value in a population having an IBD disease prevalence of about 15% is at least about 95%. In one embodiment, X can be selected to be 0.7 multiplied by two standard deviations above the background value of ANCA-negative UC sera, Y can be selected to be 12 ELISA units, and Z can be selected to be 60 ELISA units.
In a method of the invention for diagnosing inflammatory bowel disease, the ANCA, ASCA-IgA and ASCA-IgG levels can be determined using, for example, a serum sample or saliva sample. ANCA levels can be determined using an antigen specific for ANCA such as fixed neutrophils, and ASCA-IgA and ASCA-IgG levels can be determined using an antigen specific for ASCA such as yeast cell wall phosphopeptidomannan (PPM), which can be prepared, for example, from strain ATCC #38926.
The invention additionally provides a highly efficient method of analyzing multiple samples for IBD by first assaying all samples for the presence or absence of ANCA; next assaying only ANCA-negative samples for the presence or absence of ASCA-IgA; and next assaying only ANCA-negative and ASCA-IgA-negative samples for the presence or absence of ASCA-IgG, where the presence of pANCA, ASCA-IgA or ASCA-IgG in a sample is indicative of IBD and where the absence of ANCA, ASCA-IgA and ASCA-IgG is indicative of the absence of IBD. In such a method of the invention, the presence of ANCA, ASCA-IgA and ASCA-IgG can be conveniently determined, for example, using an immunoassay.


REFERENCES:
Barnes et al.,

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