Patent Publication Number: US-2005136495-A1

Title: Method and apparatus for distinguishing Crohn&#39;s disease from ulcerative colitis and other gastrointestinal diseases by detecting the presence of fecal antibodies to Saccharomyces cerevisiae

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
      This application claims the benefit of priority to U.S. application Ser. No. 10/280,564 filed on Oct. 25, 2002 which claims the benefit of priority to U.S. Provisional Application No. 60/335,812 filed on Oct. 26, 2001, the entirety of the disclosures of which are hereby incorporated by reference. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
      Not Applicable.  
     FIELD OF THE INVENTION  
      A method and apparatus for the differentiation of Crohn&#39;s disease from other gastrointestinal illnesses, such as ulcerative colitis and irritable bowel syndrome, using the presence of fecal anti- Saccharomyces cerevisiae  antibodies (ASCA) as a marker for Crohn&#39;s disease are provided. The apparatus includes an enzyme-linked immunoassay or other immunoassay that utilizes antibodies specific to human immunoglobulins for the measurement of total endogenous ASCA in a human fecal sample. The method and apparatus may be used by healthcare providers to distinguish Crohn&#39;s disease from other gastrointestinal illnesses, such as ulcerative colitis and irritable bowel syndrome.  
     BACKGROUND OF THE INVENTION  
      It is estimated that at least one million Americans suffer from Inflammatory Bowel Disease (IBD). IBD is characterized by a chronic inflammatory response that results in histologic damage to the intestinal lining. IBD comprises two known clinical subtypes, Crohn&#39;s Disease (CD) and ulcerative colitis (UC). CD may involve the entire gastrointestinal tract and include inflammation extending into the transmural mucosa whereas UC affects solely the large bowel and includes inflammation of the innermost lining. Due to the differences between them, these two distinct diseases require a rapid differential diagnosis for optimal treatment. Conventional methods for differentiating between these clinical subtypes of IBD utilize multiple endoscopy examinations and histological analysis. These methods, however, do not permit quick differential diagnosis as each may require years for a diagnosis to be confirmed. As a result, methods are needed for the rapid differential diagnosis of CD and UC.  
      Serological methods for the differential diagnosis of CD and UC are known in the art. For example, it is known in the art to use the presence of serum anti- Saccharomyces cerevisiae  antibodies (ASCA) to diagnose CD. See Main et al., Antibody to  Saccharomyces cerevisiae  (baker&#39;s yeast) in Crohn&#39;s disease, BMJ Vol. 297 (Oct. 29, 1988); Broker et al., A Murine Monoclonal Antibody Directed Against a Yeast Cell Wall Glycoprotein Antigen of the Yeast Genus  Saccharomyces,  FEMS Microbiology Letters 118 (1994), 297-304. It is further known in the art to use the presence of serum ASCA to diagnose clinical subtypes of UC and CD in patients presenting with established diagnoses. For example, U.S. Pat. No. 5,968,741 discloses utilizing the presence of serum ASCA to diagnose a medically resistant clinical subtype of UC in patients presenting with an established diagnosis of UC. Similarly, U.S. Pat. No. 5,932,429 discloses utilizing the presence of serum ASCA to diagnose a clinical subtype of CD in patients presenting with an established diagnosis of CD.  
      Each of the above-mentioned serological methods utilizing ASCA as a marker has a number of drawbacks. For instance, each method requires an invasive procedure such as a finger prick or the like to obtain a serum sample. Further, each method utilizes only serum antibodies that are not required to cross the intestinal wall and the serum antibodies may not be accurate indicator for the proper diagnosis.  
     SUMMARY OF THE INVENTION  
      A method for testing a fecal sample, the method comprising: obtaining a fecal sample from a person; and determining the amount of anti- Saccharomyces cerevisiae  antibodies in the sample.  
      A method for testing a fecal sample, the method comprising: obtaining a fecal sample from a person; and determining the presence of anti- Saccharomyces cerevisiae  antibodies in the sample, wherein the presence of fecal anti- Saccharomyces cerevisiae  antibodies is used to aid in the differentiation of Crohn&#39;s disease from other gastrointestinal illnesses such as, ulcerative colitis and irritable bowel syndrome (IBS).  
      An assay for determining the concentration of endogenous anti- Saccharomyces cerevisiae  antibodies, the assay comprising: obtaining a human fecal sample; diluting the fecal sample; contacting the sample with extract of  Saccharomyces cerevisiae  to create a treated sample; contacting the treated sample with enzyme-linked polyclonal antibodies to create a readable sample; determining the optical density of the readable sample at 450 nm; generating a purified anti- Saccharomyces cerevisiae  antibodies standard curve; and comparing the optical density of the readable sample to the standard curve to determine the concentration of endogenous anti- Saccharomyces cerevisiae  antibodies in the fecal sample.  
      A diagnostic assay for diagnosing Crohn&#39;s disease by determining the level of endogenous anti- Saccharomyces cerevisiae  antibodies, the assay comprising: obtaining a human fecal sample; diluting the sample; contacting the sample extract  Saccharomyces cerevisiae  to create a treated sample; contacting the treated sample with enzyme-linked polyclonal antibodies to create a readable sample; adding an enzyme substrate for color development and determining the optical density of the readable sample at 450 nm to determine whether the readable sample contains an elevated level of endogenous anti- Saccharomyces cerevisiae  antibodies as compared to a reference value for healthy control subjects.  
      A kit for diagnosing Crohn&#39;s disease by testing a fecal sample from a person to be diagnosed, the kit comprising: one or more microassay plates, each the plate containing extract  Saccharomyces cerevisiae;  enzyme-linked polyclonal antibody to human anti- Saccharomyces cerevisiae  antibodies; and enzyme substrate for color development.  
      Additional aspects of invention, together with the advantages and novel features appurtenant thereto, will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned from the practice of the invention. The objects and advantages of the invention may be realized and attained by means, instrumentalities and combinations particularly pointed out in the appended claims. 
    
    
     BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS  
       FIG. 1  is a graphical representation of a standard curve of purified anti- Saccharomyces cerevisiae  antibodies.  
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      A method and apparatus for the differentiation of Crohn&#39;s disease from other gastrointestinal illnesses, such as ulcerative colitis and irritable bowel syndrome, using the presence of fecal anti- Saccharomyces cerevisiae  antibodies (ASCA) as a marker for Crohn&#39;s disease are provided. The apparatus includes an enzyme-linked immunoassay or other immunoassay that utilizes antibodies specific to human immunoglobulins for the measurement of total endogenous ASCA in a human fecal sample. The method and apparatus may be used by healthcare providers to distinguish Crohn&#39;s disease from other gastrointestinal illnesses, such as ulcerative colitis and irritable bowel syndrome. The particular embodiments described herein are intended in all respects to be illustrative rather than restrictive. Alternative embodiments will become apparent to those skilled in the art to which the present embodiment of the invention pertains without departing from its scope.  
      The present embodiment of the invention provides immunoassays, including, but not limited to, enzyme-linked immunoassays (ELISAs), that utilize antibodies specific to human ASCA for the measurement of total endogenous ASCA in fecal samples, including feces and mucosal secretions. The assay of the present invention may include, but is not limited to, the following steps: 1) obtaining a fecal sample from a person to be diagnosed, 2) diluting the sample, 3) contacting the sample with extract of  Saccharomyces cerevisiae  to create a treated sample, and 4) contacting the treated sample with enzyme-linked polyclonal antibodies to create a readable sample. Further, the optical density of the readable sample at 450 nm may be determined. The optical density of the readable sample then may be compared to a standard curve generated using purified anti- Saccharomyces cerevisiae  standard curve to determine the concentration of the endogenous anti- Saccharomyces cerevisiae  antibodies in the fecal sample. The present embodiment of the invention further provides a kit usable in such immunoassays to aid physicians and other clinical personnel in diagnosing Crohn&#39;s disease.  
      It will be understood and appreciated by those of skill in the art that a immunoassay such as a lateral flow dipstick that utilizes both monoclonal and polyclonal antibodies to total endogenous ASCA also may be used to diagnose Crohn&#39;s disease. Such is contemplated to be within the scope hereof.  
      A limited number of cases of ulcerative colitis and IBS may test positive for ASCA. Therefore, it is possible that a diagnosis of Crohn&#39;s disease cannot be established solely on the basis of a positive result with the assay of the present embodiment of the invention. However, a positive result with the assay of the present embodiment of the invention will permit the substantial preclusion of a diagnosis of a other gastrointestinal illness, such as IBS or ulcerative colitis.  
      The immunoassay of the embodiment of the present embodiment of the invention may be used as an in vitro diagnostic aid for detecting elevated levels of ASCA as a detection marker for Crohn&#39;s disease. The immunoassay of the present embodiment of the invention provides a test that is easy to use, simple to read, and accurate for distinguishing Crohn&#39;s disease from IBS or ulcerative colitis.  
      The following examples are intended in all respects to be illustrative rather than restrictive.  
     EXAMPLE 1  
      In this example using an ELISA method, a fecal sample was obtained and serially diluted 20 fold. 100 μl of the diluted sample was added to a test well of a microassay plate coated with extract of  Saccharomyces cerevisiae  and purified mannan. The sample then was incubated at 37° C. to allow antibodies to  Saccharomyces cerevisiae  to bind to the extract of  Saccharomyces cerevisiae.  Following incubation, anti-human Ig polyclonal antibodies coupled to horseradish peroxidase enzyme (conjugate) were added to the test well and allowed to bind to captured ASCA. Unbound conjugate then was washed from the well and one component substrate (tetra-methyl-benzidene and hydrogen peroxide) was added for color development. Following the substrate incubation, 0.1M sulfuric acid was added to quench the reaction and the optical density (OD) was obtained spectrophotometrically at 450 nm using a single wavelength spectrophotometer.  
      The method described above was used in a clinical study to test a total of 86 IBD patients (55.8% males and 44.2% females). The approximate 1 to 1 ratio of males to females was similar to the ratio observed in IBD patient populations. The IBS patient group ranged in age from 19 to 78 years and was 9% male and 91% female. This ratio of males to females (1:10) reflects the increased incidence for IBS in females as seen in patient populations. The healthy control (HC) patient group ranged in age from 20 to 79 years old and was 33.3% male and 66.6% female. A summary of the patient population in the clinical study is shown in Table 1.  
               TABLE 1                          Summary of patient population.                             Summary of Clinical Histories   Total           (N = 120)   Subjects                                         Total number of IBD patients   86           No. Males   48           No. Females   38           Total number of patients with Crohn&#39;s Disease   49           No. Males   26           No. Females   23           Total number of patients with ulcerative colitis   37           No. Males   22           No. Females   15           Total number of patients with irritable bowel syndrome   22           No. Males   2           No. Females   20           Total number of healthy controls   12           No. Males   4           No. Females   8                      
 
      In the clinical study, there were 37 ulcerative colitis patients, 49 Crohn&#39;s disease patients, 22 irritable bowel patients, and 12 healthy controls. Fecal samples were collected from each enrolled subject and stored at −70° C. until tested. The optical densities for each sample were determined using the method described above. Results were reported as positive for fecal ASCA if an optical density of greater than or equal to 0.200 was observed. Results were reported as negative for fecal ASCA if an optical density of less than or equal to 0.199 was observed. Other clinical data, such as stool consistency, was also determined. Table 2, below, contains the clinical data and test results for healthy patients that participated in this clinical study. Table 3, below, contains the clinical data and test results for patients with ulcerative colitis patients that participated in this clinical study. Table 4, below, contains the clinical data and test results for patients with Crohn&#39;s disease that participated in this study. Table 5, below, contains the clinical data and test results for patients with irritable bowel syndrome that participated in this study.  
               TABLE 2                          Clinical data and test results for healthy controls                                                     Previous of                           Age   chronic GI   Stool       Donor ID   Sex   Range   illness   Consistency   Optical Density   Fecal ASCA               HC1   F   40-49   NO   Solid   0.098   NEGATIVE       HC2   F   40-49   NO   Solid   0.089   NEGATIVE       HC3   M   70-79   NO   Solid   0.095   NEGATIVE       HC4   F   60-69   NO   Solid   0.085   NEGATIVE       HC5   M   70-79   NO   Solid   0.083   NEGATIVE       HC6   F   70-79   NO   Solid   0.076   NEGATIVE       HC7   F   50-59   NO   Solid   0.124   NEGATIVE       HC8   F   40-49   NO   Solid   0.095   NEGATIVE       HC9   F   50-49   NO   Solid   0.111   NEGATIVE       HC10   F   40-49   NO   Solid   0.111   NEGATIVE       HC11   M   50-60   NO   Solid   0.070   NEGATIVE       HC12   M   50-60   NO   Solid   0.054   NEGATIVE                  
 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                   
               
               
                 Clinical data and test results for ulcerative colitis patients 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Stool 
                 Disease 
                   
                   
               
               
                 Patient ID 
                 Sex 
                 Age 
                 Disease 
                 Consistency 
                 Activity 
                 Optical Density 
                 Fecal ASCA 
               
               
                   
               
               
                 UC1 
                 F 
                 46 
                 UC 
                 Liquid 
                 ACTIVE 
                 0.184 
                 NEGATIVE 
               
               
                 UC2 
                 M 
                 39 
                 UC 
                 Liquid 
                 ACTIVE 
                 0.378 
                 POSITIVE 
               
               
                 UC3 
                 F 
                 30 
                 UC 
                 Semi-Solid 
                 ACTIVE 
                 0.193 
                 NEGATIVE 
               
               
                 UC4 
                 F 
                 31 
                 UC 
                 Semi-Solid 
                 INACTIVE 
                 0.319 
                 POSITIVE 
               
               
                 UC5 
                 F 
                 30 
                 UC 
                 Semi-Solid 
                 ACTIVE 
                 0.114 
                 NEGATIVE 
               
               
                 UC6 
                 M 
                 61 
                 UC 
                 Semi-Solid 
                 INACTIVE 
                 0.115 
                 NEGATIVE 
               
               
                 UC7 
                 F 
                 68 
                 UC 
                 Liquid 
                 INACTIVE 
                 0.091 
                 NEGATIVE 
               
               
                 UC8 
                 F 
                 45 
                 UC 
                 Liquid 
                 ACTIVE 
                 0.356 
                 POSITIVE 
               
               
                 UC9 
                 F 
                 21 
                 UC 
                 Semi-Solid 
                 ACTIVE 
                 0.082 
                 NEGATIVE 
               
               
                 UC10 
                 F 
                 27 
                 UC 
                 Liquid 
                 ACTIVE 
                 0.161 
                 NEGATIVE 
               
               
                 UC11 
                 F 
                 24 
                 UC 
                 Solid 
                 INACTIVE 
                 0.104 
                 NEGATIVE 
               
               
                 UC12 
                 F 
                 74 
                 UC 
                 Semi-Solid 
                 INACTIVE 
                 0.091 
                 NEGATIVE 
               
               
                 UC13 
                 M 
                 69 
                 UC 
                 Semi-Solid 
                 ACTIVE 
                 0.070 
                 NEGATIVE 
               
               
                 UC14 
                 M 
                 19 
                 UC 
                 Solid 
                 INACTIVE 
                 0.088 
                 NEGATIVE 
               
               
                 UC15 
                 M 
                 62 
                 UC 
                 Solid 
                 INACTIVE 
                 0.054 
                 NEGATIVE 
               
               
                 UC16 
                 F 
                 70 
                 UC 
                 Solid 
                 INACTIVE 
                 0.056 
                 NEGATIVE 
               
               
                 UC17 
                 M 
                 23 
                 UC 
                 Liquid 
                 ACTIVE 
                 0.573 
                 POSITIVE 
               
               
                 UC18 
                 F 
                 52 
                 UC 
                 Solid 
                 ACTIVE 
                 0.073 
                 NEGATIVE 
               
               
                 UC19 
                 M 
                 60 
                 UC 
                 Solid 
                 INACTIVE 
                 0.062 
                 NEGATIVE 
               
               
                 UC20 
                 F 
                 52 
                 UC 
                 Liquid 
                 ACTIVE 
                 0.089 
                 NEGATIVE 
               
               
                 UC21 
                 M 
                 31 
                 UC 
                 Solid 
                 INACTIVE 
                 0.064 
                 NEGATIVE 
               
               
                 UC22 
                 M 
                 44 
                 UC 
                 Semi-Solid 
                 INACTIVE 
                 0.143 
                 NEGATIVE 
               
               
                 UC23 
                 F 
                 30 
                 UC 
                 Liquid 
                 ACTIVE 
                 0.110 
                 NEGATIVE 
               
               
                 UC24 
                 M 
                 48 
                 UC 
                 Semi-Solid 
                 INACTIVE 
                 0.096 
                 NEGATIVE 
               
               
                 UC25 
                 F 
                 37 
                 UC 
                 Liquid 
                 ACTIVE 
                 0.282 
                 POSITIVE 
               
               
                 UC26 
                 F 
                 32 
                 UC 
                 Solid 
                 ACTIVE 
                 0.107 
                 NEGATIVE 
               
               
                 UC27 
                 F 
                 46 
                 UC 
                 Liquid 
                 ACTIVE 
                 0.199 
                 NEGATIVE 
               
               
                 UC28 
                 M 
                 49 
                 UC 
                 Semi-Solid 
                 INACTIVE 
                 0.161 
                 NEGATIVE 
               
               
                 UC29 
                 F 
                 42 
                 UC 
                 Solid 
                 INACTIVE 
                 0.080 
                 NEGATIVE 
               
               
                 UC30 
                 F 
                 41 
                 UC 
                 Semi-Solid 
                 INACTIVE 
                 0.087 
                 NEGATIVE 
               
               
                 UC31 
                 F 
                 43 
                 UC 
                 Solid 
                 INACTIVE 
                 0.070 
                 NEGATIVE 
               
               
                 UC32 
                 M 
                 30 
                 UC 
                 Solid 
                 ACTIVE 
                 0.103 
                 NEGATIVE 
               
               
                 UC33 
                 F 
                 43 
                 UC 
                 Solid 
                 INACTIVE 
                 0.092 
                 NEGATIVE 
               
               
                 UC34 
                 F 
                 33 
                 UC 
                 Semi-Solid 
                 INACTIVE 
                 0.075 
                 NEGATIVE 
               
               
                 UC35 
                 M 
                 58 
                 UC 
                 Semi-Solid 
                 ACTIVE 
                 0.121 
                 NEGATIVE 
               
               
                 UC36 
                 F 
                 32 
                 UC 
                 Semi-Solid 
                 ACTIVE 
                 0.083 
                 NEGATIVE 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                   
               
               
                 Clinical Data and test results for Crohn&#39;s disease patients. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Stool 
                 Disease 
                   
                   
               
               
                 Patient ID 
                 Sex 
                 Age 
                 Disease 
                 Consistency 
                 Activity 
                 Optical Density 
                 FECAL ASCA 
               
               
                   
               
               
                 CD1 
                 M 
                 26 
                 CD 
                 Liquid 
                 INACTIVE 
                 1.900 
                 POSITIVE 
               
               
                 CD2 
                 M 
                 60 
                 CD 
                 Liquid 
                 ACTIVE 
                 2.849 
                 POSITIVE 
               
               
                 CD3 
                 F 
                 66 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.282 
                 POSITIVE 
               
               
                 CD4 
                 F 
                 74 
                 CD 
                 Semi-Solid 
                 INACTIVE 
                 0.091 
                 NEGATIVE 
               
               
                 CD5 
                 F 
                 25 
                 CD 
                 Solid 
                 INACTIVE 
                 0.162 
                 NEGATIVE 
               
               
                 CD6 
                 F 
                 66 
                 CD 
                 Semi-Solid 
                 INACTIVE 
                 1.240 
                 POSITIVE 
               
               
                 CD7 
                 M 
                 39 
                 CD 
                 No Data 
                 ACTIVE 
                 1.150 
                 POSITIVE 
               
               
                 CD8 
                 F 
                 46 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.160 
                 NEGATIVE 
               
               
                 CD9 
                 F 
                 46 
                 CD 
                 Semi-Solid 
                 INACTIVE 
                 0.074 
                 NEGATIVE 
               
               
                 CD10 
                 F 
                 56 
                 CD 
                 Solid 
                 ACTIVE 
                 0.406 
                 POSITIVE 
               
               
                 CD11 
                 M 
                 56 
                 CD 
                 Solid 
                 ACTIVE 
                 0.168 
                 NEGATIVE 
               
               
                 CD12 
                 F 
                 56 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.732 
                 POSITIVE 
               
               
                 CD13 
                 M 
                 21 
                 CD 
                 Solid 
                 ACTIVE 
                 1.369 
                 POSITIVE 
               
               
                 CD14 
                 M 
                 52 
                 CD 
                 Semi-Solid 
                 INACTIVE 
                 0.136 
                 NEGATIVE 
               
               
                 CD15 
                 M 
                 63 
                 CD 
                 Solid 
                 INACTIVE 
                 0.134 
                 NEGATIVE 
               
               
                 CD16 
                 M 
                 34 
                 CD 
                 Solid 
                 ACTIVE 
                 0.076 
                 NEGATIVE 
               
               
                 CD17 
                 F 
                 45 
                 CD 
                 Semi-Solid 
                 ACTIVE 
                 0.160 
                 NEGATIVE 
               
               
                 CD18 
                 M 
                 67 
                 CD 
                 Semi-Solid 
                 INACTIVE 
                 0.059 
                 NEGATIVE 
               
               
                 CD19 
                 F 
                 46 
                 CD 
                 No Data 
                 ACTIVE 
                 0.839 
                 POSITIVE 
               
               
                 CD20 
                 M 
                 66 
                 CD 
                 Semi-Solid 
                 INACTIVE 
                 0.084 
                 NEGATIVE 
               
               
                 CD21 
                 M 
                 63 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.780 
                 POSITIVE 
               
               
                 CD21 
                 M 
                 51 
                 CD 
                 Semi-Solid 
                 ACTIVE 
                 3.000 
                 POSITIVE 
               
               
                 CD22 
                 M 
                 34 
                 CD 
                 Semi-Solid 
                 ACTIVE 
                 1.447 
                 POSITIVE 
               
               
                 CD23 
                 M 
                 21 
                 CD 
                 Solid 
                 ACTIVE 
                 2.757 
                 POSITIVE 
               
               
                 CD24 
                 F 
                 78 
                 CD 
                 Semi-Solid 
                 INACTIVE 
                 0.092 
                 NEGATIVE 
               
               
                 CD25 
                 F 
                 27 
                 CD 
                 Semi-Solid 
                 ACTIVE 
                 0.979 
                 POSITIVE 
               
               
                 CD26 
                 M 
                 40 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.373 
                 POSITIVE 
               
               
                 CD27 
                 M 
                 51 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.978 
                 POSITIVE 
               
               
                 CD28 
                 M 
                 42 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.089 
                 NEGATIVE 
               
               
                 CD29 
                 F 
                 31 
                 CD 
                 Solid 
                 INACTIVE 
                 0.075 
                 NEGATIVE 
               
               
                 CD30 
                 F 
                 59 
                 CD 
                 Solid 
                 ACTIVE 
                 0.088 
                 NEGATIVE 
               
               
                 CD31 
                 M 
                 35 
                 CD 
                 Semi-Solid 
                 ACTIVE 
                 1.487 
                 POSITIVE 
               
               
                 CD32 
                 M 
                 37 
                 CD 
                 Semi-Solid 
                 INACTIVE 
                 1.257 
                 POSITIVE 
               
               
                 CD33 
                 F 
                 77 
                 CD 
                 Solid 
                 INACTIVE 
                 0.093 
                 NEGATIVE 
               
               
                 CD34 
                 F 
                 40 
                 CD 
                 No Data 
                 ACTIVE 
                 1.762 
                 POSITIVE 
               
               
                 CD35 
                 F 
                 38 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.098 
                 NEGATIVE 
               
               
                 CD36 
                 M 
                 51 
                 CD 
                 Liquid 
                 ACTIVE 
                 2.326 
                 POSITIVE 
               
               
                 CD37 
                 M 
                 38 
                 CD 
                 Semi-Solid 
                 ACTIVE 
                 0.091 
                 NEGATIVE 
               
               
                 CD38 
                 M 
                 37 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.372 
                 POSITIVE 
               
               
                 CD39 
                 M 
                 59 
                 CD 
                 Semi-Solid 
                 ACTIVE 
                 0.224 
                 POSITIVE 
               
               
                 CD40 
                 F 
                 41 
                 CD 
                 Solid 
                 ACTIVE 
                 0.503 
                 POSITIVE 
               
               
                 CD41 
                 M 
                 41 
                 CD 
                 Solid 
                 ACTIVE 
                 0.117 
                 NEGATIVE 
               
               
                 CD42 
                 M 
                 48 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.115 
                 NEGATIVE 
               
               
                 CD43 
                 F 
                 40 
                 CD 
                 Solid 
                 INACTIVE 
                 0.638 
                 POSITIVE 
               
               
                 CD44 
                 F 
                 72 
                 CD 
                 Solid 
                 ACTIVE 
                 0.087 
                 NEGATIVE 
               
               
                 CD45 
                 F 
                 32 
                 CD 
                 Liquid 
                 INACTIVE 
                 0.911 
                 POSITIVE 
               
               
                 CD46 
                 F 
                 24 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.341 
                 POSITIVE 
               
               
                 CD47 
                 M 
                 23 
                 CD 
                 Solid 
                 INACTIVE 
                 0.088 
                 NEGATIVE 
               
               
                 CD48 
                 F 
                 34 
                 CD 
                 Liquid 
                 ACTIVE 
                 0.599 
                 POSITIVE 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                   
               
               
                 Clinical data and test results for irritable bowel syndrome patients 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Stool 
                 Disease 
                   
                   
               
               
                 Patient ID 
                 Sex 
                 Age 
                 Disease 
                 consistency 
                 Activity 
                 Optical Density 
                 Fecal ASCA 
               
               
                   
               
               
                 IBS1 
                 F 
                 56 
                 IBS 
                 Semi-Solid 
                 ACTIVE 
                 0.132 
                 NEGATIVE 
               
               
                 IBS2 
                 F 
                 48 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.103 
                 NEGATIVE 
               
               
                 IBS3 
                 F 
                 30 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.073 
                 NEGATIVE 
               
               
                 IBS4 
                 F 
                 31 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.074 
                 NEGATIVE 
               
               
                 IBS5 
                 F 
                 72 
                 IBS 
                 Semi-Solid 
                 ACTIVE 
                 0.079 
                 NEGATIVE 
               
               
                 IBS6 
                 F 
                 47 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.088 
                 NEGATIVE 
               
               
                 IBS7 
                 F 
                 19 
                 IBS 
                 Semi-Solid 
                 ACTIVE 
                 0.105 
                 NEGATIVE 
               
               
                 IBS8 
                 F 
                 58 
                 IBS 
                 Semi-Solid 
                 ACTIVE 
                 0.107 
                 NEGATIVE 
               
               
                 IBS9 
                 F 
                 40 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.065 
                 NEGATIVE 
               
               
                 IBS10 
                 F 
                 33 
                 IBS 
                 Semi-Solid 
                 ACTIVE 
                 0.065 
                 NEGATIVE 
               
               
                 IBS11 
                 F 
                 78 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.071 
                 NEGATIVE 
               
               
                 IBS12 
                 F 
                 74 
                 IBS 
                 Semi-Solid 
                 ACTIVE 
                 0.063 
                 NEGATIVE 
               
               
                 IBS13 
                 F 
                 50 
                 IBS 
                 Semi-Solid 
                 ACTIVE 
                 0.052 
                 NEGATIVE 
               
               
                 IBS14 
                 F 
                 39 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.079 
                 NEGATIVE 
               
               
                 IBS15 
                 F 
                 54 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.080 
                 NEGATIVE 
               
               
                 IBS16 
                 M 
                 49 
                 IBS 
                 Semi-Solid 
                 ACTIVE 
                 0.238 
                 POSITIVE 
               
               
                 IBS17 
                 M 
                 53 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.123 
                 NEGATIVE 
               
               
                 IBS18 
                 F 
                 34 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.091 
                 NEGATIVE 
               
               
                 IBS19 
                 F 
                 43 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.075 
                 NEGATIVE 
               
               
                 IBS20 
                 F 
                 35 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.075 
                 NEGATIVE 
               
               
                 IBS21 
                 F 
                 51 
                 IBS 
                 Semi-Solid 
                 ACTIVE 
                 0.081 
                 NEGATIVE 
               
               
                 IBS22 
                 F 
                 40 
                 IBS 
                 Solid 
                 ACTIVE 
                 0.083 
                 NEGATIVE 
               
               
                   
               
            
           
         
       
     
      There were a total of 49 patients with Crohn&#39;s disease and 37 with ulcerative colitis. In the Crohn&#39;s disease group, a total of 55.1% patients were positive for fecal ASCA. In the ulcerative colitis group, 13.5% were positive. Of the 22 IBS patients, a single patient (4.6%) was positive for fecal ASCA. All 12 healthy controls were negative. A summary of positive results for fecal ASCA is shown in Table 6.  
               TABLE 6                          Summary of positive results for Crohn&#39;s disease, ulcerative colitis,       active IBS, and healthy controls                             Total       Fecal ASCA   Fecal ASCA       Assessments N = 120   Total   Positive   Negative               Total IBD (Crohn&#39;s disease   86   37.2% (32)    62.8% (54)       and ulcerative colitis)       Total Crohn&#39;s Disease   49   55.1% (27)    44.9% (22)       Total Ulcerative Colitis   37   13.5% (5)    86.5% (32)       Total Active IBS   22    4.6% (1)    96.4% (21)       Total Healthy Controls   12   0   100.0% (12)                  
 
      When distinguishing Crohn&#39;s disease from ulcerative colitis, fecal ASCA exhibited a sensitivity of 55.1% and specificity of 86.5%. The predictive positive and negative values were 84.4% and 59.3%, respectively, and the correlation was 68.6% as shown in Table 7.  
               TABLE 7                       Statistical evaluation using the presence of fecal ASCA to distinguish       Crohn&#39;s disease from ulcerative colitis                                                    N = 86   Crohn&#39;s disease   Ulcerative colitis                       Fecal ASCA positive   27    5           Fecal ASCA negative   22   32                                         Sensitivity   55.1%           Specificity   86.5%           Predictive Positive Value   84.4%           Predictive Negative Value   59.3%           Correlation   68.6%                      
 
      When distinguishing Crohn&#39;s disease from ulcerative colitis, irritable bowel syndrome and healthy controls, fecal ASCA exhibited a sensitivity of 55% and a specificity of 91.6%. The predictive positive and negative values were 82% and 75%, respectively, and the correlation was 77% as shown below in Table 8.  
               TABLE 8                       Statistical evaluation using fecal ASCA to distinguish Crohn&#39;s disease       from ulcerative colitis, irritable bowel syndrome/healthy controls                                            N = 120   Crohn&#39;s disease   UC/IBS/Healthy Controls               Fecal ASCA positive   27    6       Fecal ASCA negative   22   65                                     Sensitivity   55.1%           Specificity   91.6%           Predictive Positive Value   81.8%           Predictive Negative Value   74.7%           Correlation   76.7%                      
 
      The mean optical densities for each group were obtained and differences were tested for statistical significance using a two-tailed t-test giving a p-value result. Of the 33 patients that tested positive for fecal ASCA, there were 27 CD, 5 UC, and 1 IBS. Sensitivity, specificity and overall correlation were 55.1%, 91.5% and 76.7%, respectively. ASCA-positive CD showed a higher mean±SD A450 of 1.183±0.794 as compared to 0.382±0.113 for UC and the single A450 of 0.0.091±0.0.038 for IBS. There was a significant difference between CD and all other subject groups. A summary of the statistical analysis is listed in Table 9.  
               TABLE 9                          Summary of the Mean and P values of Optical Densities for Fecal ASCA                                     Mean       Optical               Optical   Standard   Density       Test Group   Density   Deviation   Range   P Value               CD   1.183   0.794   0.341-3.000   CD vs UC, IBS, HC                       P &lt; 0.005       UC   0.382   0.113   0.382-0.113   CD vs UC                       P &lt; 0.05       IBS   0.091   0.038   0.052-0.238   CD vs IBS                       P &lt; 0.005       HC   0.091   0.019   0.054-0.124   CD vs HC                       P &lt; 0.005                  
 
     EXAMPLE 2  
      In this example, the sensitivity of the fecal ASCA test was determined using serial two fold dilutions of highly purified ASCA antibodies. For the analysis, standard curves were generated using the kit dilutent. The test was consistently positive at a concentration of 0.62 μg/mL as determined by a cutoff absorbency value of ≧0.200. Individual results are shown below in Table 10. The standard curves are shown in  FIG. 1 .  
               TABLE 10                          Standard curves generated using purified ASCA antibodies                                 Purified                       ASCA       Antibodies       (μg/mL)   Test 1   Test 2   Mean   Std Dev                                                 5.00   1.702   1.856   1.779   0.108           2.50   1.117   1.099   1.108   0.012           1.25   0.634   0.624   0.629   0.007           0.62   0.303   0.329   0.316   0.018           0.31   0.191   0.164   0.177   0.019           0.16   0.115   0.113   0.114   0.001           0.08   0.090   0.077   0.083   0.009           0.04   0.063   0.065   0.064   0.001                      
 
     EXAMPLE 3  
      In this example, tests were conducted to determine what type of immunoglobulins (antibodies) were present in a fecal sample and in serum. The immunglobulin typing was done for human IgA, human IgA sec , human IgD, human IgM, and human IgG. The immunoglobulin typing was done on a fecal sample from 6 Crohn&#39;s disease patients and 2 ulcerative colitis and on a serum control sample using pre-absorbed Ig-type specific conjugates. The serum control sample was obtained from a patient with a confirmed allergy to  Saccharomyces cerevisiae.    
      Of the Crohn&#39;s disease patients, 5 patients exhibited a response to IgA and IgA sec , 4 patients exhibited a response to IgM and a single patient exhibited a response to IgG. Of the 2 ulcerative colitis patients, a single patient reacted with the Ig conjugate. The serum control only exhibited a response to individual immunoglobulins IgM and IgG. A response to IgA and IgA sec  occurred the fecal samples but not in the control serum sample. A summary of results are shown in Table 11.  
               TABLE 11                          A Summary of Immunoglobulin Typing of ASCA in a Human Fecal sample and       a Serum Control                                             Patient       IgA   IgA sec     IgD   IgM   IgG   Ig       Number   Disease   Conjugate   Conjugate   Conjugate   Conjugate   Conjugate   Conjugate               1   Crohn&#39;s   +   +   −   +   +   +           Disease       2   Crohn&#39;s   +   +   −   +   −   +           Disease       3   Crohn&#39;s   −   −   −   −   −   −           Disease       4   Crohn&#39;s   +   +   NO   +   −   +           Disease           DATA       5   Crohn&#39;s   +   +   NO   −   −   +           Disease           DATA       6   Crohn&#39;s   +   +   NO   +   −   +           Disease           DATA       7   Ulcerative   −   −   −   −   −   −           Colitis       8   Ulcerative   −   −   −   −   −   +           Colitis       Serum   Yeast   −   −   −   +   +   +       Control   Allergy                  
 
      In summary, the present embodiment of the invention provides a method and apparatus for the differentiation of Crohn&#39;s disease from other gastrointestinal illnesses, such as ulcerative colitis and irritable bowel syndrome, using the presence of fecal anti- Saccharomyces cerevisiae  antibodies (ASCA) as a marker for Crohn&#39;s disease. The apparatus includes an enzyme-linked immunoassay or other immunoassay that utilizes antibodies specific to human immunoglobins for the measurement of total endogenous ASCA in a human fecal sample. The method and apparatus may be used by healthcare providers to distinguish Crohn&#39;s disease from other gastrointestinal illnesses, such as ulcerative colitis and irritable bowel syndrome. The present embodiment of the invention has been described in relation to particular embodiments which are intended in all respects to be illustrative rather than restrictive. Alternative embodiments will become apparent to those skilled in the art to which the present embodiment of the invention pertains without departing from its scope.  
      From the foregoing, it will be seen that this embodiment of the invention is one well adapted to attain all the ends and objects hereinabove set forth together with other advantages which are obvious and which are inherent to the method.  
      It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.