Method for measuring FceRII/CD23 IgE receptor in human feces as an indicator of a TH2 predominant immune response involved in gastrointestinal illnesses

Abstract

A method and apparatus for measuring FcεRII/CD23 IgE receptor in human feces or other biological specimens as an indicator of a TH2 immune response indicative of gastrointestinal illnesses such as ulcerative colitis and food allergy is described. The apparatus consists of either a qualitative or quantitative enzyme-linked immunoassay or other immunoassay that utilizes antibodies specific to human FcεRII/CD23 IgE receptor for the measurement of endogenous FcεRII/CD23 IgE receptor in human feces. The method and apparatus can be used by healthcare providers as an aid for determining gastrointestinal illnesses that are predominantly a TH2 predominant immune response such as ulcerative colitis, large bowel Crohn's disease (IC-like Crohn's disease), allergic colitis (food allergy) and parasitic infections.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

The FcεRII/CD23 IgE receptor is a membrane protein expressed by a wide range of cells including B lymphocytes, T lymphocytes, monocytes and eosinophils. This low-affinity IgE receptor binds the IgE immunoglobulin and signals the release of histamine as part of the TH2 immune response and regulates the production of IgE. The FcεRII/CD23 IgE receptor is proteolytically cleaved from the membrane surface, releasing a stable soluble 25 kD fragment. The soluble FcεRII/CD23 acts as a cytokine to stimulate the production of IL-5 for the recruitment of eosinophils and IL-6 for the differentiation of B lymphocytes. In in vitro experiments, soluble FcεRII/CD23 has been shown to interact with P2 integrin CD11b to stimulate proinflammatory TNF-α production by monocytes (macrophages). The FcεRII/CD23 glycoprotein is expressed at higher levels in activated eosinophils, increasing the levels of FcεRII/CD23 during cell infiltration in diseased tissue.

Currently, there are serum-based immunoassays for the detection of soluble serum FcεRII/CD23 IgE receptor as an indicator of rheumatoid arthritis and chronic lymphocytic leukemia (Bindazyme™ sCD23, The Binding Site, U.K.). However, this method does not determine the presence of elevated fecal FcεRII/CD23 IgE receptor as a specific marker for a TH2 immune response indicating gastrointestinal illnesses such as allergic colitis (food allergy), ulcerative colitis and large bowel Crohn's disease, and parasitic infections. There remains a need for a method utilizing antibodies specific for FcεRII/CD23 IgE receptor for measuring elevated fecal FcεRII/CD23 IgE receptor as an indicator of allergic colitis and other eosinophilic gastrointestinal illnesses such as food allergy that involve a TH2 immune response.

SUMMARY OF THE INVENTION

The present invention relates to methods for aiding in the determination of a TH2 immune response by determining the presence of elevated FcεRII/CD23 IgE receptor in human feces as a marker for TH2 predominant gastrointestinal illnesses. In addition, the presence of elevated fecal FcεRII/CD23 IgE receptor can be used to determine gastrointestinal illnesses such as allergic colitis, ulcerative colitis, large bowel Crohn's disease (UC-like Crohn's disease) and parasitic infections that involve a TH2 immune response. The measurement of elevated fecal FcεRII/CD23 offers a diagnostic aid for the determination of TH2 predominant gastrointestinal illnesses for optimizing medical treatment.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph generated using optical density values in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The fecal and serum FcεRII/CD23 IgE receptor specific immunoassays can be used to determine a gastrointestinal illnesses with a TH2 predominant immune response such as in the case of inflammatory bowel disease and allergic colitis for determining the optimal treatment by measuring the presence of elevated fecal FcεRII/CD23 IgE receptor (human feces and mucosal secretions). The detection of elevated fecal FcεRII/CD23 IgE receptor can also be useful for indicating other TH2 predominant gastrointestinal illnesses such as allergic colitis in cases of food allergy and other eosinophilic gastrointestinal illnesses.

A qualitative immunoassay such as a lateral flow and flow-through tests that utilize antibodies to protein fragments of human FcεRII/CD23 IgE receptor for detecting elevated FcεRII/CD23 IgE receptor in human feces, saliva or mucosal secretions can indicate the absence or presence of a TH2 immune response for predominant diseases such as allergic colitis, ulcerative colitis, large bowel Crohn's disease and parasitic infections.

In the qualitative assay, human feces is diluted and 100 μl of diluted specimen to an individual well of a microassay plate coated with antibodies, proteins or polypeptide fragments specific to FcεRII/CD23 IgE receptor. If endogenous fecal FcεRII/CD23 IgE receptor is present, it will bind to the capture antibody, proteins or polypeptide fragments during an incubation step for 1 to 2 hours. Following incubation, anti-human specific antibodies conjugated to an enzyme, anti-IgG-Horse-radish peroxidase (HRP) or streptavidin-HRP Conjugate is added and allowed to bind with antibodies to captured fecal FcεRII/CD23 IgE receptor. The test wells are incubated to allow binding of the conjugate. Unbound conjugate is then washed from the well and substrate (tetramethylbenzidene/peroxide) is added for color development. Following the substrate incubation, low pH stop solution (sulfuric acid) is added to stop the reaction and the optical density (OD) is obtained spectrophotometrically at 450 nm.

In our clinical study, we screened 49 healthy subjects (no known acute infection or chronic intestinal disorders) to determine the baseline readings for fecal FcεRII/CD23. A total of 8 (16.3%) of the 49 subjects had detectable FcεRII/CD23, indicating a TH2 immune response. Measurable FcεRII/CD23 was then evaluated in 79 pediatric subjects with inflammatory bowel disease (IBD) and 6 irritable bowel syndrome (IBS) patients. Of the 79 IBD patients, a total of 25 subjects had detectable levels of fecal FcεRII/CD23 (8 Crohn's disease and 17 ulcerative colitis). There was a 2:1 ratio of UC to CD, indicating a higher frequency of a TH2 immune response in UC than in CD. A single subject with IBS had a detectable level of fecal FcεRII/CD23. A summary of results is shown below in Table 1 and subject data are shown in Tables 2 through 4.

TABLE 1
Detection of FcεRII/CD23 in Fecal Specimens from subjects with
Crohn's disease, ulcerative colitis, IBS and in healthy persons.
Total		Fecal FcεRII/CD23	Fecal FcεRII/CD23
Assessments N = 134	Total	Positive	Negative
Total IBD (Crohn's	79	31.7% (25)	68.3% (54)
disease and
ulcerative colitis)
Total Crohn's Disease	38	21.1% (8)	78.9% (30)
Total Ulcerative	41	41.5% (17)	58.5% (24)
Colitis
Total Active IBS	6	16.7% (1)	83.3% (5)
Healthy Persons	49	16.3% (8)	83.7% (41)

TABLE 2
FcεRII/CD23 results for healthy subjects
			FcεRII/CD23
		Presence of	Test
Subject		intestinal	Absorbance
ID	Disease	inflammation	value (OD450 nm)	Interpretation
H1	Healthy	None	0.111	NEGATIVE
H2	Healthy	None	0.115	NEGATIVE
H3	Healthy	None	0.144	NEGATIVE
H4	Healthy	None	0.095	NEGATIVE
H5	Healthy	None	0.113	NEGATIVE
H6	Healthy	None	0.241	POSITIVE
H7	Healthy	None	0.130	NEGATIVE
H8	Healthy	None	0.182	POSITIVE
H9	Healthy	None	0.113	NEGATIVE
H10	Healthy	None	0.103	NEGATIVE
H11	Healthy	None	0.128	NEGATIVE
H12	Healthy	None	0.119	NEGATIVE
H13	Healthy	None	0.099	NEGATIVE
H14	Healthy	None	0.099	NEGATIVE
H15	Healthy	None	0.119	NEGATIVE
H16	Healthy	None	0.112	NEGATIVE
H17	Healthy	None	0.104	NEGATIVE
H18	Healthy	None	0.107	NEGATIVE
H19	Healthy	None	0.092	NEGATIVE
H20	Healthy	None	0.096	NEGATIVE
H21	Healthy	None	0.095	NEGATIVE
H22	Healthy	None	0.101	NEGATIVE
H23	Healthy	None	0.096	NEGATIVE
H24	Healthy	None	0.124	NEGATIVE
H25	Healthy	None	0.078	NEGATIVE
H26	Healthy	None	0.102	NEGATIVE
H27	Healthy	None	0.114	NEGATIVE
H28	Healthy	None	0.141	NEGATIVE
H29	Healthy	None	0.097	NEGATIVE
H30	Healthy	None	0.107	NEGATIVE
H31	Healthy	None	0.091	NEGATIVE
H32	Healthy	None	0.095	NEGATIVE
H33	Healthy	None	0.091	NEGATIVE
H34	Healthy	None	0.225	POSITIVE
H35	Healthy	None	0.115	NEGATIVE
H36	Healthy	None	0.222	POSITIVE
H37	Healthy	None	0.227	POSITIVE
H38	Healthy	None	0.147	NEGATIVE
H39	Healthy	None	0.181	POSITIVE
H40	Healthy	None	0.188	POSITIVE
H41	Healthy	None	0.103	NEGATIVE
H42	Healthy	None	0.096	NEGATIVE
H43	Healthy	None	0.137	NEGATIVE
H44	Healthy	None	0.129	NEGATIVE
H45	Healthy	None	0.167	POSITIVE
H46	Healthy	None	0.096	NEGATIVE
H47	Healthy	None	0.111	NEGATIVE
H48	Healthy	None	0.124	NEGATIVE
H49	Healthy	None	0.126	NEGATIVE
Cutoff = ≧0.157

TABLE 3
FcεRII/CD23 results for IBD subjects
			FcεRII/CD23
			Test
Subject		Disease	Absorbance
ID	Disease	Activity	value (OD450 nm)	Interpretation
IBD1	CD	ACTIVE	0.123	NEGATIVE
IBD2	CD	ACTIVE	0.138	NEGATIVE
IBD3	CD	ACTIVE	0.116	NEGATIVE
IBD4	UC	INACTIVE	0.116	NEGATIVE
IBD5	CD	ACTIVE	0.153	NEGATIVE
IBD6	UC	INACTIVE	0.121	NEGATIVE
IBD7	UC	ACTIVE	0.169	POSITIVE
IBD8	CD	ACTIVE	0.305	POSITIVE
IBD9	UC	INACTIVE	0.116	NEGATIVE
IBD10	CD	INACTIVE	0.123	NEGATIVE
IBD11	UC	ACTIVE	0.180	POSITIVE
IBD12	CD	ACTIVE	0.111	NEGATIVE
IBD13	UC	ACTIVE	0.111	NEGATIVE
IBD14	UC	ACTIVE	0.103	NEGATIVE
IBD15	UC	ACTIVE	0.158	POSITIVE
IBD16	UC	INACTIVE	0.157	POSITIVE
IBD17	UC	ACTIVE	0.138	NEGATIVE
IBD18	UC	ACTIVE	0.131	NEGATIVE
IBD19	CD	ACTIVE	0.107	NEGATIVE
IBD20	UC	ACTIVE	0.235	POSITIVE
IBD21	UC	ACTIVE	0.186	POSITIVE
IBD22	CD	ACTIVE	0.116	NEGATIVE
IBD23	UC	ACTIVE	0.273	POSITIVE
IBD24	CD	ACTIVE	0.098	NEGATIVE
IBD25	CD	INACTIVE	0.115	NEGATIVE
IBD26	UC	ACTIVE	0.129	NEGATIVE
IBD27	CD	INACTIVE	0.128	NEGATIVE
IBD28	CD	ACTIVE	0.150	NEGATIVE
IBD29	UC	ACTIVE	0.129	NEGATIVE
IBD30	UC	ACTIVE	0.119	NEGATIVE
IBD31	CD	ACTIVE	0.171	POSITIVE
IBD32	CD	ACTIVE	0.313	POSITIVE
IBD33	CD	ACTIVE	0.116	NEGATIVE
IBD34	CD	ACTIVE	0.152	NEGATIVE
IBD35	UC	ACTIVE	0.335	POSITIVE
IBD36	CD	ACTIVE	0.123	NEGATIVE
IBD37	UC	ACTIVE	0.129	NEGATIVE
IBD38	CD	ACTIVE	0.108	NEGATIVE
IBD39	CD	ACTIVE	0.109	NEGATIVE
IBD40	UC	ACTIVE	0.181	POSITIVE
IBD41	UC	ACTIVE	0.125	NEGATIVE
IBD42	UC	ACTIVE	0.160	POSITIVE
IBD43	CD	ACTIVE	0.116	NEGATIVE
IBD44	UC	INACTIVE	0.112	NEGATIVE
IBD45	CD	ACTIVE	0.099	NEGATIVE
IBD46	CD	ACTIVE	0.367	POSITIVE
IBD47	UC	ACTIVE	0.101	NEGATIVE
IBD48	CD	ACTIVE	0.111	NEGATIVE
IBD49	CD	ACTIVE	0.131	NEGATIVE
IBD50	CD	ACTIVE	0.173	POSITIVE
IBD51	UC	ACTIVE	0.194	POSITIVE
IBD52	UC	INACTIVE	0.233	POSITIVE
IBD53	UC	ACTIVE	0.118	NEGATIVE
IBD54	CD	ACTIVE	0.120	NEGATIVE
IBD55	CD	ACTIVE	0.137	NEGATIVE
IBD56	CD	INACTIVE	0.129	NEGATIVE
IBD57	UC	ACTIVE	0.191	POSITIVE
IBD58	UC	ACTIVE	0.281	POSITIVE
IBD59	UC	INACTIVE	0.205	POSITIVE
IBD60	CD	ACTIVE	0.191	POSITIVE
IBD61	UC	ACTIVE	0.122	NEGATIVE
IBD62	UC	INACTIVE	0.136	NEGATIVE
IBD63	UC	ACTIVE	0.347	POSITIVE
IBD64	CD	ACTIVE	0.123	NEGATIVE
IBD65	CD	INACTIVE	0.124	NEGATIVE
IBD66	UC	ACTIVE	0.126	NEGATIVE
IBD67	UC	INACTIVE	0.143	NEGATIVE
IBD68	UC	INACTIVE	0.134	NEGATIVE
IBD69	UC	ACTIVE	0.146	NEGATIVE
IBD70	CD	ACTIVE	0.186	POSITIVE
IBD71	UC	ACTIVE	0.193	POSITIVE
IBD72	CD	INACTIVE	0.113	NEGATIVE
IBD73	UC	ACTIVE	0.130	NEGATIVE
IBD74	CD	ACTIVE	0.130	NEGATIVE
IBD75	CD	ACTIVE	0.132	NEGATIVE
IBD76	UC	INACTIVE	0.137	NEGATIVE
IBD77	CD	INACTIVE	0.262	POSITIVE
IBD78	CD	ACTIVE	0.115	NEGATIVE
IBD79	UC	INACTIVE	0.145	NEGATIVE
Cutoff = ≧0.157

TABLE 4
FcεRII/CD23 results for IBS subjects
			FcεRII/CD23
			Test
Subject		Disease	Absorbance
ID	Disease	Activity	value (OD450 nm)	Interpretation
IBS1	IBS	SYMTOMATIC	0.116	NEGATIVE
IBS2	IBS	SYMTOMATIC	0.143	NEGATIVE
IBS3	IBS	SYMTOMATIC	0.115	NEGATIVE
IBS4	IBS	SYMTOMATIC	0.111	NEGATIVE
IBS5	IBS	SYMTOMATIC	0.108	NEGATIVE
IBS6	IBS	SYMTOMATIC	0.169	POSITIVE
Cutoff = ≧0.157

A standard curve was generated using the FcεRII/CD23 ELISA test and purified human FcεRII/CD23 for a quantitative assay. The curve shows linearity with an R² value of 0.99. The optical densities (OD) for each FcεRII/CD23 concentration and a graph generated using the OD values are shown below in Table 5 and FIG. 1. Using the FcεRII/CD23 standard curve, fecal specimens of healthy subjects were analyzed quantitatively for levels of fecal FcεRII/CD23. The mean±SD for fecal FcεRII/CD23 in healthy subjects was 6.8±9.5 ng/mL. The FcεRII/CD23 levels for each healthy subject are listed below in Table 6. A second test group, 3 adult subjects with UC and 3 adult subjects with CD were monitored for detectable levels of fecal FcεRII/CD23 over a range of 6 months with an average of 6 specimens (1 per month). The mean±SD for the subjects with IBD was 67.3±43.5 ng/mL. The mean level for healthy subjects was statistically different from the level determined in the subjects with IBD (Two-tailed Student T-Test; p<0.005). A majority of these subjects continued to show elevated levels for the 6-month period. The quantitative results for the IBD subjects are shown in Table 7.

TABLE 5
Standard curves generated using Purified Human FcεRII/CD23
Purified
FcεRII/CD23 Mean
(ng/mL)     OD
60          1.453
20          0.591
6.7         0.260
2.2         0.101
0.7         0.074

TABLE 6
FcεRII/CD23 levels for healthy subjects
        Fecal
Subject FcεRII/CD23
Code    (ng/mL)
001     5.6
003     7.5
007     3.6
008     5.3
011     12.8
012     3.3
014     4.7
015     10.8
016     16.0
017     9.3
020     7.2
021     0
025     14.4
026     0
033     0
035     4.5
037     0
039     6.4
042     7.0
043     16.4
045     57.9
046     4.2
047     0
049     1.6
050     31.7
052     0
055     0
056     0
064     5.0
065     6.4
066     12.5
068     6.8
070     3.2
071     0
072     5.3
073     3.6
077     0
078     5.5
080     0
081     19.0
082     5.3
083     4.1
084     0
086     0
087     3.3
088     0
090     5.0
094     6.2
096     4.4
097     3.6
099     0
112     17.9
113     20.6
114     0
120     7.8
Mean    6.8 ng/ml
Std.    9.5
Std. = Standard deviation

TABLE 7
FcεRII/CD23 levels for IBD subjects over time
			Fecal	Interpretation
Subject			FcεRII/CD23	Level ≧ 25.8* =
ID	Disease	Sample date	level (ng/mL)	Elevated
1CDF	CD	August 03	36.9	ELEVATED
		September 03	141.3	ELEVATED
		October 03	77.2	ELEVATED
		December 03	158.6	ELEVATED
		January 04	66.3	ELEVATED
1UCF	UC	August 03	16.4	BASELINE
		September 03	54.4	ELEVATED
		October 03	14.0	BASELINE
		November 03	136.9	ELEVATED
		December 03	142.6	ELEVATED
		January 04	16.4	BASELINE
2UCM	UC	August 03	34.5	ELEVATED
		September 03	123.8	ELEVATED
2UCM	UC	October 03	80.2	ELEVATED
		November 03	21.5	BASELINE
		December 03	57.4	ELEVATED
2CDF	CD	September 03	10.2	BASELINE
		October 03	22.6	BASELINE
		November 03	87.3	ELEVATED
		December 03	79.6	ELEVATED
		January 04	21.5	ELEVATED
ACRM1	CD	November 03	39.9	ELEVATED
		December 03	60.9	ELEVATED
		December 03	107.8	ELEVATED
		December 03	101.9	ELEVATED
UCM4	UC	November 03	30.7	ELEVATED
		November 03	65.4	ELEVATED
		December 03	67.2	ELEVATED
		January 04	77.8	ELEVATED
		Mean	67.3
		Std.	43.5
*Cut-off for elevated FcεRII/CD23 is the mean of healthy subjects plus 2 Std. (6.8 + 2(9.5) = 25.8).
Std. = Standard deviation

From the foregoing, it will be seen that this invention is 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.

Claims

1. A method for detecting amounts of FcεRII/CD23 IgE receptor in a fecal sample, the method comprising: obtaining a fecal sample from a human; determining the presence of FcεRII/CD23 IgE receptor in the fecal sample.

2. The method of claim 1, wherein the presence of FcεRII/CD23 IgE receptor is determined using one of an enzyme linked immunoassay, flow-through membrane test and lateral flow membrane test.

3. The method of claim 2, further comprising: determining whether the FcεRII/CD23 IgE receptor is elevated.

4. The method of claim 3, wherein if the FcεRII/CD23 IgE receptor is elevated, determining the presence of a TH2 immune response in the human.

5. The method of claim 4, wherein said TH2 immune response is indicative of allergic colitis.

6. The method of claim 4, wherein said TH2 immune response is indicative of TH2 predominant ulcerative colitis subgroup.

7. The method of claim 4, wherein said TH2 immune response is indicative of TH2 predominant Crohn's Disease subgroup.

8. The method of claim 4, wherein said TH2 immune response is indicative of a parasitic infection.

9. The method of claim 4, wherein said TH2 immune response is indicative of eosinophilic gastrointestinal illness.

10. The method of claim 1, wherein the FcεRII/CD23 IgE receptor comprises total FcεRII/CD23 IgE receptor in the fecal sample.

11. A method for detecting the presence of a TH2 response in a patient, the method comprising: obtaining a fecal sample from a patient, wherein said fecal sample comprises FcεRII/CD23 IgE receptor; contacting the fecal sample with antibodies specific to the FcεRII/CD23 IgE receptor; detecting in the sample an amount of FcεRII/CD23 IgE receptor that binds to the one of antibodies and protein fragments specific to the FcεRII/CD23 IgE receptor; and comparing the amount of bound receptor to a pre-determined cut-off value and therefrom determining the presence of a TH2 immune response in the patient.

12. The method of claim 11, wherein said TH2 immune response is indicative of allergic colitis.

13. The method of claim 11, wherein said TH2 immune response is indicative of a TH2 predominant ulcerative colitis subgroup.

14. The method of claim 11, wherein said TH2 immune response is indicative of a TH2 predominant Crohn's Disease subgroup.

15. The method of claim 11, wherein said TH2 immune response is indicative of a parasitic infection.

16. The method in claim 11, wherein the presence of fecal FcεRII/CD23 IgE receptor within a single patient is used as an aid in determining the optimal treatment and TH2 immune specific therapy such as anti-CD23 antibody infusions.

17. A assay for determining the amount of FcεRII/CD23 IgE receptor in human feces, the method comprising: obtaining a sample of one of human feces, saliva and mucosal secretions; diluting the sample; contacting the sample with antibodies or proteins specific to FcεRII/CD23 IgE receptor and allowing the FcεRII/CD23 IgE receptors in the sample to bind to the antibodies or proteins to create a bound sample; contacting the bound sample with a conjugate to create a readable sample; and determining the optical density of the readable sample at 450 nm to determine the level of FcεRII/CD23 IgE receptor.

18. The assay of claim 17, further comprising: comparing the optical density of the readable sample to a predetermined cut-off value.

19. The assay of claim 18, wherein if the optical density of the readable sample is above a cut-off value, determining the presence of a TH2 immune response in the patient.

20. A kit for diagnosing a TH2 immune response in a person by testing a fecal sample from a person to be diagnosed, the kit comprising: one or more microassay plates, each the plate containing antibodies or proteins specific to FcεRII/CD23 IgE receptor; anti-human specific antibodies conjugated to an enzyme; and enzyme substrate for color development.

21. The kit as recited in claim 21, further comprising a stop solution for quenching the reaction.