Abstract:
A method and apparatus for the differentiation of ulcerative colitis from Crohn&#39;s disease and other gastrointestinal illnesses using the presence of anti-neutrophil cytoplasmic antibodies (ANCA) as a marker of ulcerative colitis is described. The apparatus consists of either a qualitative enzyme-linked immunoassay or other immunoassay that utilizes antibodies specific to human immunoglobulins for the measurement of total endogenous ANCA in a human sample. The method and apparatus can be used by healthcare providers to distinguish ulcerative colitis from Crohn&#39;s disease and other gastrointestinal illnesses.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims the benefit of priority to U.S. Provisional Application No. 60/408,809, filed Sep. 5, 2002. 
     
    
     
       STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
         [0002]    Not applicable.  
         BACKGROUND OF THE INVENTION  
         [0003]    This invention relates to non-invasive methods for differentiating clinical subtypes of Inflammatory Bowel Disease, namely Crohn&#39;s disease (CD) and ulcerative colitis (UC). More specifically, this invention relates to a method and apparatus for aiding in the differentiation of Crohn&#39;s disease from ulcerative colitis by determining the presence of anti-neutrophil cytoplasmic antibodies (ANCA), wherein the presence of ANCA is indicative of ulcerative colitis. In addition, the presence of fecal ANCA may be used to differentiate ulcerative colitis from other gastrointestinal illnesses such as Irritable Bowel Syndrome.  
           [0004]    An estimated 1 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. Crohn&#39;s disease may involve the entire gastrointestinal tract and include inflammation extending into the transmural mucosa, whereas ulcerative colitis affects solely the large bowel and includes inflammation of the innermost lining. These two distinct diseases require a rapid differential diagnosis for optimal treatment. Conventional methods utilizing multiple endoscopy examinations and histological analysis may take years to confirm a diagnosis. U.S. Pat. No. 6,218,120 discloses a method of determining the presence of serum ANCA as a marker to diagnose IBD. However, it does not disclose a method for diagnosing ulcerative colitis in a patient diagnosed with IBD. Further, the method does not disclose testing human feces for the presence of ANCA.  
           [0005]    Accordingly, there remains a need in the diagnostic industry for a non-invasive method of differentially diagnosing ulcerative colitis from Crohn&#39;s disease or other gastrointestinal illnesses.  
         SUMMARY OF THE INVENTION  
         [0006]    Accordingly, in one of its aspects, the present invention provides non-invasive methods for differentiating between diagnoses of ulcerative colitis and Crohn&#39;s disease.  
           [0007]    In another of its aspects, the present invention provides methods for differentiating between ulcerative colitis and Crohn&#39;s disease wherein the presence of fecal ANCA is used as a marker for ulcerative colitis.  
           [0008]    In a further aspect, the present invention provides immunoassays, e.g., and enzyme-linked immunoassays, that utilize antibodies specific to human immunoglobulins for the measurement of total endogenous ANCA in human feces.  
           [0009]    In yet another of its aspects, the present invention provides methods differentially diagnosing ulcerative colitis from other gastrointestinal illnesses such as Irritable Bowel Syndrome (IBS). In still another of its aspects, the present invention provides methods for diagnosing ulcerative colitis wherein the presence of ANCA is used as a marker for ulcerative colitis.  
           [0010]    According to the present invention, the foregoing and other aspects are achieved by a non-invasive method for aiding in the differentiation of ulcerative colitis from Crohn&#39;s disease in a patient presenting with IBD. In the method of the present invention, fecal ANCA are used as a marker and the presence of ANCA indicates a differential diagnosis of ulcerative colitis. This rapid diagnosis may then be used by healthcare professionals to prescribe proper treatment.  
           [0011]    Aspects of the present invention are further achieved by immunoassays that utilize antibodies specific to human immunoglobulins for the measurement of total endogenous ANCA in human feces.  
           [0012]    Additional aspects of the 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, instrumentality&#39;s and combinations particularly pointed out in the appended claims. 
       
    
    
     BRIEF DESCRIPTION OF THE VIEW OF THE DRAWING  
       [0013]    [0013]FIG. 1 is a graphical representation of a standard curve of anti-neutrophil cytoplasmic antibodies in accordance with an embodiment of the present invention. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0014]    The present invention is directed to non-invasive methods for differentiating between ulcerative colitis and Crohn&#39;s disease using the presence of fecal ANCA as an indicator of ulcerative colitis. The present invention also is directed to a method for differentiating between ulcerative colitis and other gastrointestinal illnesses such as IBS. The present invention is further directed to immunoassays that utilize antibodies specific to human immunoglobulins for the measurement of total endogenous ANCA in human feces. 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 invention pertains without departing from its scope.  
         [0015]    ANCA specific immunoassays may be used to differentiate ulcerative colitis and indeterminate colitis from Crohn&#39;s disease by measurement of the presence of total endogenous ANCA. In addition to fecal matter, a sample of whole blood, serum, plasma or other bodily fluid or tissue may be tested for ANCA to diagnose ulcerative colitis. This differential diagnosis may then be used by healthcare professionals for determining optimal treatment. A qualitative immunoassay, such as a later flow dipstick that utilizes both monoclonal and polyclonal antibodies to endogenous human ANCA to indicate the presence of ulcerative colitis.  
         [0016]    In the qualitative immunoassay, the fecal or bodily sample is diluted 10 fold and added to a well containing immobilized neutrophilic antigens. If endogenous fecal ANCA is present, it will bind to the neutrophilic antigens during an incubation step at 37° C. Following the incubation, polyvalent antibodies to human immunoglobulin coupled to an enzyme, such as a horseradish peroxidase enzyme, (conjugate) is added and allowed to bind to captured ANCA. Unbound conjugate is then washed from the well and one component substrate (e.g., tetramethylbenzidene and hydrogen peroxide) is added for color development. Following the substrate incubation, 0.1M sulfuric acid is added to stop the reaction and the optical density (OD) is obtained spectrophotometrically at 450 nm.  
         [0017]    In a clinical study, a total of 98 IBD patients were enrolled and comprised 51% males and 49% females with an age range of 0 to 69 years. The approximate 1 to 1 ratio is similar to the ratio observed in IBD patient populations. The IBS patient group had an age range of 5 to 39 years with 57% males and 43% females. The healthy controls were 55% male and 45% female and comprised the age range of 20 to 79 years. Individual numbers for each age group are shown in Table 1.  
                                           TABLE 1                           Summary of patient population.                Summary of Clinical Histories   Total           (N = 116)   Subjects                            Total number of IBD patients   98           No. Males   50           No. Females   48           Total number of patients with Crohn&#39;s Disease   47           No. Males   26           No. Females   21           Total number of patients with ulcerative colitis   51           No. Males   24           No. Females   27           Total number of patients with irritable bowel syndrome   7           No. Males   4           No. Females   3           Total number of healthy persons   11           No. Males   6           No. Females   5                      
 
         [0018]    There were 51 ulcerative colitis (UC) patients, 47 Crohn&#39;s disease (CD) patients, 7 irritable bowel patients (IBS), and 11 healthy (H) adults recruited for the study. Fecal specimens were collected from each enrolled patient and stored at −70° C. until tested. Specimen consistency ranged from solid to liquid. The level of fecal ANCA was determined using the qualitative ANCA ELISA as previously described. Disease activity was defined using elevated fecal lactoferrin as an indicator of intestinal inflammation. A dilution of 1:10 was used in the qualitative ELISA test and results were reported as positive (absorbance values &gt;0.140) or negative (absorbance values &lt;0.140). The mean optical densities, standard deviation and P values (two-tailed student T-test with unequal variance) were determined for the ANCA positive ulcerative colitis patients. Of the 26 patients that tested positive for fecal ANCA, there were four patients had Crohn&#39;s Disease, 21 had ulcerative colitis and one patient was healthy. ANCA-positive ulcerative colitis showed a mean±SD OD 450  of 0.311±0.166. The mean optical density for the ulcerative colitis patients was significantly different from IBS and healthy persons (p value&lt;0.0005). A summary of the statistical analysis is listed in Table 2.  
                                                                   TABLE 2                           Summary of the mean, standard deviation and P values       for qualitative ELSA test Optical Densities                    Mean       Optical           Group   Num-   Optical   Standard   Density       ID   ber   Density   Deviation   Range   P values                    ANCA + UC   21   0.311   0.166   0.141-0.804   UC vs CD                           p &lt; 0.5       ANCA + CD   4   0.209   0.115   0.141-0.381   UC vs CD,                           IBS, H                           p &lt; 0.0005       IBS   7   0.078   0.027   0.047-0.121   UC vs CD,                           IBS                           p &lt; 0.005       Healthy   11   0.071   0.041   0.039-0.104   UC vs IBS,                           H p &lt; 0.0005                  
 
         [0019]    In the group of patients with IBD, there were 47 with Crohn&#39;s disease and 51 with ulcerative colitis. In the ulcerative colitis group, 41% were positive. In the Crohn&#39;s disease group, a total of 9% patients were positive using the qualitative ELISA test. Of the 11 healthy persons, 1 was positive and all 7 IBS patients were negative by the qualitative ELISA test. A summary of positive results for the qualitative ELISA test are shown in Table 3 and individual results are listed in Table 4 and Table 5.  
                                                   TABLE 3                           Summary of positive results for Crohn&#39;s disease, ulcerative       colitis, and IBS            Total       Fecal ANCA   Fecal ANCA       Assessments N = 116   Total   Positive   Negative                    Total IBD (Crohn&#39;s disease   98   26% (25)   75% (73)       and ulcerative colitis)       Total Crohn&#39;s Disease   47    9% (4)   91% (43)       Total Ulcerative Colitis   51   41% (21)   59% (30)       Total IBS   7   0   7       Total Healthy Persons   11    9% (1)   91% (10)                  
 
         [0020]    When distinguishing ulcerative colitis from Crohn&#39;s disease, the qualitative ELISA test exhibited a sensitivity of 41% and specificity of 92%. The predictive positive and negative values were 84% and 59%, respectively, and the correlation was 65% (Table 4).  
                                               TABLE 4                       Statistical evaluation using the qualitative ELISA test to       distinguish Crohn&#39;s disease from ulcerative colitis                                    N = 98   Ulcerative colitis   Crohn&#39;s disease                       ANCA positive   21    4           ANCA negative   30   43                            Sensitivity   41%           Specificity   92%           Predictive Positive Value   84%           Predictive Negative Value   59%           Correlation   65%                      
 
         [0021]    When distinguishing ulcerative colitis from irritable bowel syndrome and healthy persons, the qualitative ELISA test exhibited a sensitivity of 41% and a specificity of 92%. The predictive positive and negative values were 81% and 67%, respectively, and the correlation was 70% as shown in Table 5.  
                                               TABLE 5                       Statistical evaluation using the qualitative ELISA test to       distinguish ulcerative colitis from Crohn&#39;s disease, irritable       bowel syndrome and healthy persons                                            Crohn&#39;s disease           N = 116   Ulcerative colitis   IBS/Healthy                       ANCA positive   21    5           ANCA negative   30   60                            Sensitivity   41%           Specificity   92%           Predictive Positive Value   81%           Predictive Negative Value   67%           Correlation   70%                      
 
         [0022]    The sensitivity of the qualitative ELISA test was determined using serial two fold dilutions of human ANCA positive serum. For the analysis, standard curves were generated using the sample diluent. The test was consistently positive to a titer of 0.063 as determined by a cutoff absorbance value of ≧0.200. Individual results are shown below in Table 6 and standard curves are shown in FIG. 1.  
                                             TABLE 6                           Standard curves generated using qualitative ELISA test       (cut-offs are in bold)                Human                               ANCA           Serum   Test 1   Test 2   Test 3   Mean   Std Dev                       1.000   1.441   1.469   1.525   1.478   0.043           (Neat)           0.500   1.098   0.941   1.014   1.018   0.079           0.250   0.717   0.595   0.666   0.659   0.061           0.125   0.492   0.428   0.444   0.455   0.033           0.063     0.327     0.303   0.320   0.317   0.012           0.032   0.196     0.295       0.221       0.237     0.051           0.016   0.132   0.184   0.179   0.165   0.029           Diluent   0.067   0.093   0.109   0.090   0.021                      
 
         [0023]    Table 7, below, contains the clinical data and test results for patients with ulcerative colitis that participated in the study. Table 8, below, contains the clinical data and test results for patients with Crohn&#39;s disease that participated in the study. Table 9, below, contains the clinical data and test results for patients with irritable bowel syndrome that participated in the study. Table 10, below, contains the clinical data and test results for health patients that participated in the study.  
                                             TABLE 7                           Clinical data and ELISA results for ulcerative colitis patients.            Patient       Age       Disease   ELISA   ELISA       ID   Sex   Range   Disease   Activity   OD 450     Result               UC1   F   10-19   UC   INACTIVE   0.053   NEGATIVE       UC2   F   5-9   UC   INACTIVE   0.107   NEGATIVE       UC3   F   5-9   UC   ACTIVE   0.058   NEGATIVE       UC4   M   10-19   UC   INACTIVE   0.048   NEGATIVE       UC5   M   10-19   UC   ACTIVE   0.512   POSITIVE       UC6   F   10-19   UC   ACTIVE   0.061   NEGATIVE       UC7   M   5-9   UC   ACTIVE   0.211   POSITIVE       UC8   M   10-19   UC   ACTIVE   0.106   NEGATIVE       UC9   M   10-19   UC   INACTIVE   0.804   POSITIVE       UC10   M   10-19   UC   ACTIVE   0.091   NEGATIVE       UC11   F   10-19   UC   ACTIVE   0.169   POSITIVE       UC12   F   10-19   UC   ACTIVE   0.209   POSITIVE       UC13   F   10-19   UC   ACTIVE   0.351   POSITIVE       UC14   F   10-19   UC   ACTIVE   0.198   POSITIVE       UC15   F   5-9   UC   ACTIVE   0.098   NEGATIVE       UC16   F   5-9   UC   ACTIVE   0.050   NEGATIVE       UC17   F   10-19   UC   ACTIVE   0.091   NEGATIVE       UC18   M   10-19   UC   ACTIVE   0.603   POSITIVE       UC19   M   10-19   UC   ACTIVE   0.091   NEGATIVE       UC20   F   10-19   UC   ACTIVE   0.142   POSITIVE       UC21   M   10-19   UC   ACTIVE   0.074   NEGATIVE       UC22   F   10-19   UC   ACTIVE   0.105   NEGATIVE       UC23   M   10-19   UC   INACTIVE   0.256   POSITIVE       UC24   F   0-4   UC   ACTIVE   0.308   POSITIVE       UC25   F   5-9   UC   ACTIVE   0.072   NEGATIVE       UC26   M   10-19   UC   INACTIVE   0.237   POSITIVE       UC27   M   10-19   UC   ACTIVE   0.048   NEGATIVE       UC28   M   10-19   UC   ACTIVE   0.049   NEGATIVE       UC29   M   10-19   UC   ACTIVE   0.059   NEGATIVE       UC30   F   10-19   UC   INACTIVE   0.047   NEGATIVE       UC31   M   10-19   UC   ACTIVE   0.055   NEGATIVE       UC32   M   10-19   UC   INACTIVE   0.044   NEGATIVE       UC33   F   10-19   UC   ACTIVE   0.043   NEGATIVE       UC34   M   5-9   UC   ACTIVE   0.046   NEGATIVE       UC35   M   10-18   UC   INACTIVE   0.043   NEGATIVE       UC36   M   10-17   UC   INACTIVE   0.040   NEGATIVE       UC37   F   10-19   UC   ACTIVE   0.047   NEGATIVE       UC38   F   0-4   UC   ACTIVE   0.049   NEGATIVE       UC39   F   5-9   UC   INACTIVE   0.363   POSITIVE       UC40   F   10-19   UC   INACTIVE   0.046   NEGATIVE       UC41   M   10-19   UC   ACTIVE   0.118   NEGATIVE       UC42   F   50-59   UC   ACTIVE   0.230   POSITIVE       UC43   M   10-19   UC   ACTIVE   0.051   NEGATIVE       UC44   F   30-39   UC   ACTIVE   0.060   NEGATIVE       UC45   F   50-59   UC   ACTIVE   0.465   POSITIVE       UC46   M   50-59   UC   ACTIVE   0.274   POSITIVE       UC47   F   30-39   UC   ACTIVE   0.141   POSITIVE       UC48   M   60-69   UC   ACTIVE   0.184   POSITIVE       UC49   F   40-49   UC   ACTIVE   0.397   POSITIVE       UC50   F   40-49   UC   ACTIVE   0.337   POSITIVE       UC51   M   30-39   UC   ACTIVE   0.143   POSITIVE                  
 
         [0024]    [0024]                                             TABLE 8                           Clinical data and ELISA results for Crohn&#39;s disease patients.            Patient       Age       Disease   ELISA   ELISA       ID   Sex   Range   Disease   Activity   OD 450     Result               CD1   M   10-19   CD   ACTIVE   0.050   NEGATIVE       CD2   M   10-19   CD   ACTIVE   0.113   NEGATIVE       CD3   M   10-19   CD   ACTIVE   0.050   NEGATIVE       CD4   F   10-19   CD   ACTIVE   0.381   POSITIVE       CD5   F   10-19   CD   ACTIVE   0.058   NEGATIVE       CD6   M   10-19   CD   INACTIVE   0.068   NEGATIVE       CD7   M   10-19   CD   ACTIVE   0.066   NEGATIVE       CD8   M   5-9   CD   ACTIVE   0.059   NEGATIVE       CD9   F   10-19   CD   ACTIVE   0.059   NEGATIVE       CD10   F   10-19   CD   ACTIVE   0.065   NEGATIVE       CD11   F   10-19   CD   INACTIVE   0.055   NEGATIVE       CD12   M   10-19   CD   INACTIVE   0.071   NEGATIVE       CD13   F   10-19   CD   ACTIVE   0.065   NEGATIVE       CD14   M   10-19   CD   ACTIVE   0.098   NEGATIVE       CD15   F   10-19   CD   ACTIVE   0.099   NEGATIVE       CD16   M   10-19   CD   ACTIVE   0.166   POSITIVE       CD17   F   10-19   CD   ACTIVE   0.147   POSITIVE       CD18   M   10-19   CD   ACTIVE   0.057   NEGATIVE       CD19   F   10-19   CD   ACTIVE   0.084   NEGATIVE       CD20   M   10-19   CD   ACTIVE   0.053   NEGATIVE       CD21   F   10-19   CD   ACTIVE   0.074   NEGATIVE       CD22   M   10-19   CD   ACTIVE   0.054   NEGATIVE       CD23   M   0-5   CD   ACTIVE   0.055   NEGATIVE       CD24   M   10-19   CD   ACTIVE   0.067   NEGATIVE       CD25   M   10-19   CD   ACTIVE   0.099   NEGATIVE       CD26   M   5-9   CD   ACTIVE   0.086   NEGATIVE       CD27   F   10-19   CD   ACTIVE   0.043   NEGATIVE       CD28   F   10-19   CD   ACTIVE   0.064   NEGATIVE       CD29   M   5-9   CD   INACTIVE   0.039   NEGATIVE       CD30   M   10-19   CD   ACTIVE   0.071   NEGATIVE       CD31   F   10-15   CD   ACTIVE   0.109   NEGATIVE       CD32   M   10-19   CD   INACTIVE   0.057   NEGATIVE       CD33   M   10-19   CD   ACTIVE   0.141   POSITIVE       CD34   M   10-19   CD   INACTIVE   0.045   NEGATIVE       CD35   F   10-19   CD   ACTIVE   0.051   NEGATIVE       CD36   F   10-19   CD   ACTIVE   0.132   NEGATIVE       CD37   F   10-19   CD   INACTIVE   0.046   NEGATIVE       CD38   M   10-19   CD   ACTIVE   0.057   NEGATIVE       CD39   F   20-29   CD   INACTIVE   0.051   NEGATIVE       CD40   F   20-29   CD   ACTIVE   0.053   NEGATIVE       CD41   M   50-59   CD   ACTIVE   0.060   NEGATIVE       CD42   F   50-59   CD   ACTIVE   0.062   NEGATIVE       CD43   M   20-29   CD   ACTIVE   0.056   NEGATIVE       CD44   F   60-69   CD   ACTIVE   0.130   NEGATIVE       CD45   M   60-69   CD   ACTIVE   0.078   NEGATIVE       CD46   F   40-49   CD   ACTIVE   0.116   NEGATIVE       CD47   M   60-69   CD   ACTIVE   0.057   NEGATIVE                    
         [0025]    [0025]                                         TABLE 9                           Clinical data and ELISA results for Irritable bowel       syndrome patients.                    Age       ELISA   ELISA       Patient ID   Sex   Range   Disease   OD 450     Results               IBS1   F   10-19   IBS   0.056   NEGATIVE       IBS2   M   10-19   IBS   0.047   NEGATIVE       IBS3   M   5-9   IBS   0.099   NEGATIVE       IBS4   M   10-19   IBS   0.068   NEGATIVE       IBS5   M   10-19   IBS   0.092   NEGATIVE       IBS6   F   20-29   IBS   0.121   NEGATIVE       IBS7   F   30-39   IBS   0.064   NEGATIVE                    
         [0026]    [0026]                                         TABLE 10                           Clinical data and ELISA results for healthy persons.                        Age   ELISA   ELISA           Subject ID   Sex   Range   OD 450     Results                       D1   F   40-49   0.087   NEGATIVE           D2   M   20-29   0.078   NEGATIVE           D5   M   20-29   0.178   POSITIVE           D15   M   50-59   0.041   NEGATIVE           D17   M   50-59   0.039   NEGATIVE           D18   F   40-49   0.069   NEGATIVE           D19   F   60-69   0.050   NEGATIVE           D20   M   70-79   0.039   NEGATIVE           D21   F   70-79   0.104   NEGATIVE           D22   M   60-69   0.045   NEGATIVE           D24   F   50-59   0.054   NEGATIVE                        
         [0027]    In summary, the present invention is directed to non-invasive methods for aiding in the differentiation of ulcerative colitis from Crohn&#39;s disease by determining the presence of ANCA as a marker of ulcerative colitis. The present invention is further drawn to immunoassays, e.g., qualitative enzyme-linked immunoassays, that utilize antibodies specific to human immunoglobulins for the measurement of total endogenous ANCA in human feces. The present 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 invention pertains without departing from its scope.  
         [0028]    From the foregoing, it will be seen that this 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.  
         [0029]    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.