CA2231528A1 - Method for the diagnosis of coeliac disease - Google Patents
Method for the diagnosis of coeliac disease Download PDFInfo
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- CA2231528A1 CA2231528A1 CA 2231528 CA2231528A CA2231528A1 CA 2231528 A1 CA2231528 A1 CA 2231528A1 CA 2231528 CA2231528 CA 2231528 CA 2231528 A CA2231528 A CA 2231528A CA 2231528 A1 CA2231528 A1 CA 2231528A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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Abstract
A method for diagnosing coeliac disease and related conditions in a subject known or suspected of having such a condition comprises contacting a body fluid from the subject containing antibody particular to the condition with a suspension of cells from an immortal cell line having an epitopic site specific for the antibody and then determining the extent of binding of antibody to the cells. Human umbilical cells have been found to be particularly suitable for use in the method. The use of a cell line means that substantial amounts of material for the antibody estimation can be prepared by culturing the cells and storing them for future use at a very low cost.
Description
CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 Description METHOD FOR THE DIAGNOSIS OF COELIAC DISEASE
Technical Field This invention relates to a method for diagnosing coeliac disease S or gluten-sensitive enteropathy and related conditions as hereinafter defined.
Back~round Art Coeliac disease is a permanent intolerance to gli~clin (wheat protein) leading to intestinal villous flattening and crypt hyperplasia 10 in susceptible subjects. Immune reactions to gli~clin are considered to play a part in the pathogenesis of the disease, although the immunological mech~nicm~ have not been fully elucidated.
Antigliadin antibodies (AGA), reticulin antibodies and endomysium antibodies (EmA) are found in serum samples from 15 patients with coeliac disease. However, AGAs are not specific to coeliac disease and high serum AGA titres are found in individuals affected by other gastrointestinal disorders.
EmAs are closely related to reticulin antibodies and are found in most individuals with active coeliac disease and in the majority of 20 individuals with dermatitis herpetiformis, who are also gluten sensitive.
By related condition herein is meant any condition which is characterised by the presence of one or more of antigliadin, reticulin or endomysium antibodies in a body fluid. These conditions are referred to collectively hereinafter as coeliac disease.
.
The diagnosis of coeliac disease is dependent on the histological evaluation of the small intestinal mucusal biopsy, more specifically the CA 02231~28 1998-03-lO
W O 97/10508 PCT~E96/00061 jejunal biopsy. However, serological assessment is an increasingly important method of screening for the condition, especially as it represents a less invasive test therefor.
The measurement of IgA EmA antibodies is currently considered 5 to be the most important single serological test (Ferreira, M. et al (1992); Gut 33 1633-1637). The current method for IgA EmA
determination involves the use of microscope-mounted sections of monkey oesophagus.
Human umbilical cord is also used for measuring EmA
10 antibodies (but requires lengthy processing (T .~lin~er, B. et al (1994);
Gut 35 776-778).
For the detection of reticulin antibodies rat kidney, stomach or liver tissue is used.
Reticulin antibodies and EmA are considered by some to be 15 closely related (Ferreira, M. et al supra). However, the exact nature of either antigen is not known.
Foetal lung fibroblasts have been shown to secrete non-collagenous protein molecules which bind to serum IgA from patients with coeliac disease. The purified molecules removed antibodies 20 against reticulin and endomysium but not against ~ clin from serum samples from coeliac disease patients (Marttinen, A. and Maki, M.
(1993); Pediatric Research 34 No. 4 420).
The requirement for monkey or human tissue for the serological screening of individuals with coeliac disease poses ethical and moral 25 problems. Also the use of tissue samples for the detection of antibodies precludes the use of standard techniques such as enzyme linked immunosorbent assay (ELISA) and flow cytometry.
CA 02231~28 1998-03-10 D'Cruz, D.P. et al. (1991) Clin. Exp. Immunol. 85, 254-261 describes the use of human umbilical vein endothelial cells (HUVECs) as the solid phase in an ELISA assay to identify antibodies to endothelial cells in the sera of patients with systemic lupus 5 erythematosus and to investigate whether these antibodies may be a marker for disease pattern and activity with particular reference to nephritis and vasculitis.
AMEND~D SH~ET
CA 0223l~28 l998-03-lO
W O 97/10508 PCT~E96/00061 The present invention overcomes these problems by providing an in vitro method for the diagnosis of coeliac disease based on the use of cell suspensions specific for antibodies indicative of a condition of coeliac disease in a patient.
5 Disclosure of the Invention Accordingly, the invention provides a method for diagnosing coeliac disease and related conditions in a subject known or suspected of having such a condition, which comprises contacting a body fluid from said subject cont~inin~ antibody particular to said condition with 10 a suspension of cells from an immortal cell line having an epitopic site specific for said antibody and determining the extent of binding of antibody to said cells.
It will be appreciated that cell suspensions, especially single cell suspensions, are easier to examine than the tissue sections typical of 15 the known methods. The use of a cell line means that substantial amounts of material for the antibody estimation can be prepared by culturing the cells and storing them for future use at a very low cost.
By immortal cell line herein is meant a cell line which can be repeatedly cultured and does not die.
Preferably, the cells are embryonic in nature.
A common feature of the tissues that are involved in the detection of coeliac disease specific antibody is their involvement at the mucosal surfaces as indicated by the examples of oesophagus and foetal lung fibroblasts mentioned above.
A further feature that can be identified as being common to tissues that bind antibodies found in patients with coeliac disease is that they are embyronic in nature. Marttinen, A. and Maki, M.
(supra) have shown that sera from patients with coeliac disease react CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 with foetal fibroblasts. However, foetal fibroblasts undergo a limited number of passages and, accordingly, do not satisfy the criteria for an immortal cell line required as a reagent for use in a routine diagnostic assay of the type described herein.
One type of preferred cell for use in accordance with the invention is a hllm~n umbilical ce~l.
Especially suitable cells for use in accordance with the invention are hllm~n umbilical vein endothelial cells (HUVECs).
Our studies have shown that HUVECs possess antigens that react with antibodies which closely correlate with EmA.
The HUVECs can be, for example, cells from a commercially available cell line such as ECV 304 cell line, No. 92091712.
Further suitable hllm~n umbilical cells are human umbilical artery smooth muscle cells and hllm~n umbilical vein smooth muscle 1~ cells. An example of the former type of cells are those available from Technoclone, Austria under Catalogue No. 62019/L/l and an example of the latter type of cells are those available from the same org~ni.c~tion under the Catalogue No. 6209/L/l.
The antibodies determined in accordance with the invention will typically be of the IgA type. The reason why IgA EmA is the predomin~nt antibody response in coeliac disease is not fully understood. However, it is postulated to 'De related to antigen presentation at the gastrointestinal level.
However, in the case of endomysium, IgG antibodies thereto have been detected in the sera of some patients.
Suitably, the cells are ruptured prior to or during contact with said body fluid.
CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 The body fluid can be any body fluid but is preferably serum.
Because the antigen for use in immllnoassays in accordance with the invention is a cell line suspension, more especially a single cell suspension, various techniques can be used to detect bound antibody.
5 Especially suitable techniques are flow cytometry and enzyme immlmoassay. However, other techniques that can be used include a fluorescent microscopic procedure.
The invention also provides a kit for carrying out the method hereinbefore defined cont~ininp a suspension of cells from an irnmortal 10 cell line.
The invention will be further illustrated by the following Examples.
Modes for Carr~in~ Out the Invention Example 1 15 Measurement of HUVEC antibody response by fluorescent microscopy The sera used in this Example were obtained from patients attending the Gastroenterology Clinic at St. James's Hospital, Dublin.
Twenty five patients had untreated coeliac disease, 16 were on a gluten free diet and 16 who had non-specific symptoms and a normal small 20 intestinal biopsy acted as controls.
HUVEC ECV 304 No. 92091712 (European Collection of Anim~l Cell Culture) were grown in Rosemount Park Memorial ~ Institute (RPMI) 1640 medium containing 10% foetal calf serum and gentamycin at 37~C in a humidified incubator with 5% CO2 until 25 confluent. The cells were harvested using trypsin EDTA and washed 2X in RPMI 1640.
CA 02231~28 1998-03-10 W O 97/10508 PCTnE96/00061 Tmmllnofluorescence studies: 30,u1 aliquots of 2Xl06 cells/ml were allowed to air dry on poly-L-lysine (Sigma) coated glass slides at room temperature (RT). The slides were then washed for 30 mins. in 0.5 M phosphate buffered saline (PBS) pH 7.2. 30~u1 volumes of 1/5 S dilutions of a patient or control serum in PBS were layered over the cells and allowed to react for 30 mins. at RT. The slides were again washed in PBS for a further 30 mins. The cells were then stained with 50 ,ul volumes of fluorescein isothiocyanate (FITC) labelled rabbit anti hllm~n IgA or IgG (Dako) diluted 1/20 for 30 mins. and washed for 30 10 mins. in PBS. The slides were covered with a coverslip in glycerol/saline mounting media and visualised by immuno~luorescence using a Leitz Ortholux (Ortholux is a trade mark) microscope with a 50 watt mercury vapour lamp. Positivity was denoted by st~inin~ of the cell cytoplasm.
EmA and reticulin antibodies were measured using standard techniques. Briefly, EmA antibodies were measured using commercially available tissue sections of monkey oesophagus (Medica California). Patients' sera were diluted 1/5 in PBS and treated as for the HUVECs. Similarly. a composite block of rat kidney, liver and 20 stomach was used to detect reticulin antibodies. Sections ~ micron thick were cut and mounted onto glass slides, sera were diluted and immunofluorescence carried out as described in the HUVEC~s and EmA
studies. The sera were randomised with the normal controls and the observer was blinded as to the origin of each slide.
The results are shown in Table 1.
Table 1 Patient No. IgA IgA IgA Diagnosis EmA HWEC ReTIC
neg neg neg Control 2 neg neg neg Control Contd/...
CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 Table 1 contd/...
Patient No. IgA IgA IgA Diagnosis EmA Hl JVEC ReTIC
Technical Field This invention relates to a method for diagnosing coeliac disease S or gluten-sensitive enteropathy and related conditions as hereinafter defined.
Back~round Art Coeliac disease is a permanent intolerance to gli~clin (wheat protein) leading to intestinal villous flattening and crypt hyperplasia 10 in susceptible subjects. Immune reactions to gli~clin are considered to play a part in the pathogenesis of the disease, although the immunological mech~nicm~ have not been fully elucidated.
Antigliadin antibodies (AGA), reticulin antibodies and endomysium antibodies (EmA) are found in serum samples from 15 patients with coeliac disease. However, AGAs are not specific to coeliac disease and high serum AGA titres are found in individuals affected by other gastrointestinal disorders.
EmAs are closely related to reticulin antibodies and are found in most individuals with active coeliac disease and in the majority of 20 individuals with dermatitis herpetiformis, who are also gluten sensitive.
By related condition herein is meant any condition which is characterised by the presence of one or more of antigliadin, reticulin or endomysium antibodies in a body fluid. These conditions are referred to collectively hereinafter as coeliac disease.
.
The diagnosis of coeliac disease is dependent on the histological evaluation of the small intestinal mucusal biopsy, more specifically the CA 02231~28 1998-03-lO
W O 97/10508 PCT~E96/00061 jejunal biopsy. However, serological assessment is an increasingly important method of screening for the condition, especially as it represents a less invasive test therefor.
The measurement of IgA EmA antibodies is currently considered 5 to be the most important single serological test (Ferreira, M. et al (1992); Gut 33 1633-1637). The current method for IgA EmA
determination involves the use of microscope-mounted sections of monkey oesophagus.
Human umbilical cord is also used for measuring EmA
10 antibodies (but requires lengthy processing (T .~lin~er, B. et al (1994);
Gut 35 776-778).
For the detection of reticulin antibodies rat kidney, stomach or liver tissue is used.
Reticulin antibodies and EmA are considered by some to be 15 closely related (Ferreira, M. et al supra). However, the exact nature of either antigen is not known.
Foetal lung fibroblasts have been shown to secrete non-collagenous protein molecules which bind to serum IgA from patients with coeliac disease. The purified molecules removed antibodies 20 against reticulin and endomysium but not against ~ clin from serum samples from coeliac disease patients (Marttinen, A. and Maki, M.
(1993); Pediatric Research 34 No. 4 420).
The requirement for monkey or human tissue for the serological screening of individuals with coeliac disease poses ethical and moral 25 problems. Also the use of tissue samples for the detection of antibodies precludes the use of standard techniques such as enzyme linked immunosorbent assay (ELISA) and flow cytometry.
CA 02231~28 1998-03-10 D'Cruz, D.P. et al. (1991) Clin. Exp. Immunol. 85, 254-261 describes the use of human umbilical vein endothelial cells (HUVECs) as the solid phase in an ELISA assay to identify antibodies to endothelial cells in the sera of patients with systemic lupus 5 erythematosus and to investigate whether these antibodies may be a marker for disease pattern and activity with particular reference to nephritis and vasculitis.
AMEND~D SH~ET
CA 0223l~28 l998-03-lO
W O 97/10508 PCT~E96/00061 The present invention overcomes these problems by providing an in vitro method for the diagnosis of coeliac disease based on the use of cell suspensions specific for antibodies indicative of a condition of coeliac disease in a patient.
5 Disclosure of the Invention Accordingly, the invention provides a method for diagnosing coeliac disease and related conditions in a subject known or suspected of having such a condition, which comprises contacting a body fluid from said subject cont~inin~ antibody particular to said condition with 10 a suspension of cells from an immortal cell line having an epitopic site specific for said antibody and determining the extent of binding of antibody to said cells.
It will be appreciated that cell suspensions, especially single cell suspensions, are easier to examine than the tissue sections typical of 15 the known methods. The use of a cell line means that substantial amounts of material for the antibody estimation can be prepared by culturing the cells and storing them for future use at a very low cost.
By immortal cell line herein is meant a cell line which can be repeatedly cultured and does not die.
Preferably, the cells are embryonic in nature.
A common feature of the tissues that are involved in the detection of coeliac disease specific antibody is their involvement at the mucosal surfaces as indicated by the examples of oesophagus and foetal lung fibroblasts mentioned above.
A further feature that can be identified as being common to tissues that bind antibodies found in patients with coeliac disease is that they are embyronic in nature. Marttinen, A. and Maki, M.
(supra) have shown that sera from patients with coeliac disease react CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 with foetal fibroblasts. However, foetal fibroblasts undergo a limited number of passages and, accordingly, do not satisfy the criteria for an immortal cell line required as a reagent for use in a routine diagnostic assay of the type described herein.
One type of preferred cell for use in accordance with the invention is a hllm~n umbilical ce~l.
Especially suitable cells for use in accordance with the invention are hllm~n umbilical vein endothelial cells (HUVECs).
Our studies have shown that HUVECs possess antigens that react with antibodies which closely correlate with EmA.
The HUVECs can be, for example, cells from a commercially available cell line such as ECV 304 cell line, No. 92091712.
Further suitable hllm~n umbilical cells are human umbilical artery smooth muscle cells and hllm~n umbilical vein smooth muscle 1~ cells. An example of the former type of cells are those available from Technoclone, Austria under Catalogue No. 62019/L/l and an example of the latter type of cells are those available from the same org~ni.c~tion under the Catalogue No. 6209/L/l.
The antibodies determined in accordance with the invention will typically be of the IgA type. The reason why IgA EmA is the predomin~nt antibody response in coeliac disease is not fully understood. However, it is postulated to 'De related to antigen presentation at the gastrointestinal level.
However, in the case of endomysium, IgG antibodies thereto have been detected in the sera of some patients.
Suitably, the cells are ruptured prior to or during contact with said body fluid.
CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 The body fluid can be any body fluid but is preferably serum.
Because the antigen for use in immllnoassays in accordance with the invention is a cell line suspension, more especially a single cell suspension, various techniques can be used to detect bound antibody.
5 Especially suitable techniques are flow cytometry and enzyme immlmoassay. However, other techniques that can be used include a fluorescent microscopic procedure.
The invention also provides a kit for carrying out the method hereinbefore defined cont~ininp a suspension of cells from an irnmortal 10 cell line.
The invention will be further illustrated by the following Examples.
Modes for Carr~in~ Out the Invention Example 1 15 Measurement of HUVEC antibody response by fluorescent microscopy The sera used in this Example were obtained from patients attending the Gastroenterology Clinic at St. James's Hospital, Dublin.
Twenty five patients had untreated coeliac disease, 16 were on a gluten free diet and 16 who had non-specific symptoms and a normal small 20 intestinal biopsy acted as controls.
HUVEC ECV 304 No. 92091712 (European Collection of Anim~l Cell Culture) were grown in Rosemount Park Memorial ~ Institute (RPMI) 1640 medium containing 10% foetal calf serum and gentamycin at 37~C in a humidified incubator with 5% CO2 until 25 confluent. The cells were harvested using trypsin EDTA and washed 2X in RPMI 1640.
CA 02231~28 1998-03-10 W O 97/10508 PCTnE96/00061 Tmmllnofluorescence studies: 30,u1 aliquots of 2Xl06 cells/ml were allowed to air dry on poly-L-lysine (Sigma) coated glass slides at room temperature (RT). The slides were then washed for 30 mins. in 0.5 M phosphate buffered saline (PBS) pH 7.2. 30~u1 volumes of 1/5 S dilutions of a patient or control serum in PBS were layered over the cells and allowed to react for 30 mins. at RT. The slides were again washed in PBS for a further 30 mins. The cells were then stained with 50 ,ul volumes of fluorescein isothiocyanate (FITC) labelled rabbit anti hllm~n IgA or IgG (Dako) diluted 1/20 for 30 mins. and washed for 30 10 mins. in PBS. The slides were covered with a coverslip in glycerol/saline mounting media and visualised by immuno~luorescence using a Leitz Ortholux (Ortholux is a trade mark) microscope with a 50 watt mercury vapour lamp. Positivity was denoted by st~inin~ of the cell cytoplasm.
EmA and reticulin antibodies were measured using standard techniques. Briefly, EmA antibodies were measured using commercially available tissue sections of monkey oesophagus (Medica California). Patients' sera were diluted 1/5 in PBS and treated as for the HUVECs. Similarly. a composite block of rat kidney, liver and 20 stomach was used to detect reticulin antibodies. Sections ~ micron thick were cut and mounted onto glass slides, sera were diluted and immunofluorescence carried out as described in the HUVEC~s and EmA
studies. The sera were randomised with the normal controls and the observer was blinded as to the origin of each slide.
The results are shown in Table 1.
Table 1 Patient No. IgA IgA IgA Diagnosis EmA HWEC ReTIC
neg neg neg Control 2 neg neg neg Control Contd/...
CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 Table 1 contd/...
Patient No. IgA IgA IgA Diagnosis EmA Hl JVEC ReTIC
3 neg neg neg Control 4 neg neg neg Control S neg neg neg Control 6 neg neg neg Control 7 neg neg neg Control 8 neg neg neg Control 9 neg neg neg Control neg neg neg Control 11 neg neg neg Control 12 neg neg neg Control 13 neg neg neg Control 14 neg neg neg Control neg neg neg Control 16 neg neg neg Control 17 neg neg neg TCD
18 neg neg neg TCD
19 neg neg neg TCD
neg neg neg TCD
21 neg neg neg TCD
22 neg neg neg TCD
23 neg neg neg TCD
24 neg neg neg TCD
neg neg neg TCD
c 26 neg neg neg TCD
Contd/....
CA 02231~28 1998-03-lO
W O 97/10508 PCT~E96/00061 Table 1 contd/...
Patient No. IgA IgA IgA Diagnosis EmA HUVEC ReTIC
27 w pos pos wpos TCD
28 w pos pos neg TCD
29 w pos wpos neg TCD
wpos wpos neg TCD
31 pos wpos wpos TCD
32 pos pos pos TCD
33 w pos pos neg UTCD
34 w pos pos wpos UTCD
wpos wpos wpos UTCD
36 pos pos pos UTCD
37 pos pos wpos UTCD
38 pos w pos neg UTCD
39 pos pos pos UTCD
pos pos neg UTCD
41 pos pos w pos UTCD
42 pos pos w pos UTCD
43 pos pos pos UTCD
44 pos pos pos UTCD
pos pos pos UTCD
46 pos pos wpos UTCD
47 w pos w pos wpos UTCD
48 pos pos w pos UTCD
49 pos pos w pos UTCD
pos pos UTCD
Contd/....
CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 Table 1 contd/...
Patient No. IgA IgA IgA Diagnosis EmA HUVEC ReTIC
51 pos pos pos UTCD
52 pos pos pos UTCD
53 pos pos pos UTCD
54 pos pos pos UTCD
SS pos pos pos UTCD
56 pos pos pos UTCD
57 pos pos w pos UTCD
Key: w pos = weakly positive CD = Coeliac disease S TCD = Treated coeliac disease UTCD = Untreated coeliac disease In Table 1, the comparisons between the EmA, reticulin and HUVEC IgA antibody responses in patients and controls are shown.
Fifty seven sera were tested, 16 were from normal controls, 16 were from treated coeliac patients (TCD) and 25 were from untreated coeliac disease (UTCD) patients the results show that there is a strong correlation between the presence of all three antibodies, although the association of EmA and HUVEC antibodies is the strongest. Thirty one sera were positive for IgA EmA antibodies and of these the same 31 were also positive for HUVEC IgA antibodies. Of the 31 sera positive for EmA and HUVEC IgA antibodies, 24 of these were also positive for IgA reticulin antibodies and 7 sera were negativet 26 sera were negative for EmA, HUVEC and reticulin IgA antibodies.
A sllmm~ry of the results is given in Table 2 which shows the correlation between IgA EmA, HUVEC and reticulin antibody responses from fifty seven sera from coeliac patients and control subjects.
CA 02231~28 1998-03-lO
W O 97/10508 PCT~E96/00061 Table 2 No. of EmA HUVEC Reticulin sera tested Pos Neg Pos Neg Pos Neg Of the 31 sera positive for EmA antibodies all of these were positive for HUVEC IgA antibodies and 24 of these were positive for reticulin antibodies.
Example 2 Measurement of HUVEC surface and intracellular antibody responses using flow cytometry To determine whether the HUVEC antigen reacting with the coeliac disease sera was of surface (extracellular) or intracellular origin, studies were carried out on non-permeabilised cells (surface) and permeabilised cells (intracellular) using flow cytometry. Ten EmA
positive and 10 EmA negative sera were used.
Surface staining: Fresh HUVECs were harvested from culture as described in Example 1 and adjusted to 2X106/rnl. 50 ,ul aliquots of cells were washed once in PBS and reacted with 5O~L1 aliquots of coeliac patients' sera diluted 1/5 in PBS. The cells were incubated on ice for 30 mins. and washed lX with PBS. 20,u1 aliquots of FITC
conjugated rabbit anti human IgA diluted 1/20 were then added to the tubes and allowed to react for 30 mins. The cells were washed once in PBS and read on a FacScan flow cytometer (FacScan is a trade P
mark) (Becton Dickinson). U937 and HL60 cells which are non-foetal cell lines, were used as control cells and treated in a similar manner.
-CA 02231~28 1998-03-10 WO 97/10508 PCTnE96/00061 HUVEC intracellular anti~ens: HUVEC cells were fixed in 2%
paraformaldehyde for 30 mins. The cells were adjusted to 2X106/ml in 0.05% Saponin/PBS (SPBS) and given two further washes in SPBS.
Patient and control sera were diluted 1/5 in SPBS and added in 50 ~1 S volumes to tubes cont7Jinin~ aliquots of 2Xl06 cells. The cells were incubated on ice for 30 mins. and washed lX with SPBS. 20~1 aliquots of FITC conjugated rabbit anti human IgA diluted 1/20 were then added to the tubes and allowed to react for 30 mins. The cells were washed once in SPBS and read on a FacScan flow cytometer.
The results are shown in Table 3.
Table 3 Surface and intracellular stainin~ of HUVEC antigens usin~ I~A
antibodies from EmA positive CD sera and EmA negative normal controls Surface Surface/Intracellular EmA neg* EmA pos* EmA ne~* EmA pos*
6 6 ' 31 95 15 ~k Each numerical value represents a different patient or control It will be observed that no difference was seen between the EmA positive and EmA negative sera when non-permealised HUVECs were used. The mean fluorescent intensity (MFI) values ranged from 5 to 7 with the EmA negative group and from 5 to 8 CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 with the EmA positive group. VVhen the cells were fixed with 2%
paraformaldehyde and permealised with 0.1% saponin there was a marked difference between the two groups. The MFI values for the ErnA negative group ranged from 19 to 42, with a mean MFI value S of 28.2, whereas the MFI values for the EmA positive group ranged from 40 to 192 with a mean MFI value of 109.5. The instrument settings for the two experirnents were the sarne. However, fixing and permç~ ion causes the cells to give out a degree of autofluorescence which explains the difference between the MFI
10 values for the two sets of experiments.
~xample 3 Measurement of ~liadin antibody responses by flow cytometry A) Absorption of ~ din onto latex particles: One ml of lXl07/ml 8~1m polystyrene beads (Polysciences, Warrington) were 15 washed in distilled H20 and incubated overnight at 4~C with gIiadin (Sigma) at a concentration of 1 mg/rnl in 0.05 M bicarbonate buffer pH 9.6. The beads were then washed lX in PBS to remove the unbound gliadin and reincubated at 37~C for 3 hours in PBS
cont~ining bovine serum albumin (BSA) to block unoccupied sites.
20 Any unbound BSA was removed by washing with PBS Cont~inin~
0.05% Tween (PBS/Tween).
Measurement of ~liadin antibodies: 10,ul volumes of beads (lX107/ml) were added to 75 rnm tubes cont~inin~ 50 ,ul of patients' or control sera diluted 1/5 in PB~/Tween and incubated with gentle 25 mixing for 30 mins. at room temperature. The beads were washed 2X
in PBS/Tween to remove unbound sera by centrifuging at 2000 RPM
for 5 mins. 20,u1 aliquots of a 1/20 dilution of FITC conjugated rabbit anti IgA was then added to each tube and incubated with gentle mixing for 30 mins. at room temperature. The beads were again washed lX
30 with 2 ml vols of PBS~Tween and resuspended in 1 ml of PBS/Tween for analysis by flow cytometry.
CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 B) Absorption studies Serum sarnples from coeliac disease patients which tested positive for EmA, reticulin, HUVEC and gliadin antibodies were absorbed with 2X107 HUVEC cells previously fixed with 2%
18 neg neg neg TCD
19 neg neg neg TCD
neg neg neg TCD
21 neg neg neg TCD
22 neg neg neg TCD
23 neg neg neg TCD
24 neg neg neg TCD
neg neg neg TCD
c 26 neg neg neg TCD
Contd/....
CA 02231~28 1998-03-lO
W O 97/10508 PCT~E96/00061 Table 1 contd/...
Patient No. IgA IgA IgA Diagnosis EmA HUVEC ReTIC
27 w pos pos wpos TCD
28 w pos pos neg TCD
29 w pos wpos neg TCD
wpos wpos neg TCD
31 pos wpos wpos TCD
32 pos pos pos TCD
33 w pos pos neg UTCD
34 w pos pos wpos UTCD
wpos wpos wpos UTCD
36 pos pos pos UTCD
37 pos pos wpos UTCD
38 pos w pos neg UTCD
39 pos pos pos UTCD
pos pos neg UTCD
41 pos pos w pos UTCD
42 pos pos w pos UTCD
43 pos pos pos UTCD
44 pos pos pos UTCD
pos pos pos UTCD
46 pos pos wpos UTCD
47 w pos w pos wpos UTCD
48 pos pos w pos UTCD
49 pos pos w pos UTCD
pos pos UTCD
Contd/....
CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 Table 1 contd/...
Patient No. IgA IgA IgA Diagnosis EmA HUVEC ReTIC
51 pos pos pos UTCD
52 pos pos pos UTCD
53 pos pos pos UTCD
54 pos pos pos UTCD
SS pos pos pos UTCD
56 pos pos pos UTCD
57 pos pos w pos UTCD
Key: w pos = weakly positive CD = Coeliac disease S TCD = Treated coeliac disease UTCD = Untreated coeliac disease In Table 1, the comparisons between the EmA, reticulin and HUVEC IgA antibody responses in patients and controls are shown.
Fifty seven sera were tested, 16 were from normal controls, 16 were from treated coeliac patients (TCD) and 25 were from untreated coeliac disease (UTCD) patients the results show that there is a strong correlation between the presence of all three antibodies, although the association of EmA and HUVEC antibodies is the strongest. Thirty one sera were positive for IgA EmA antibodies and of these the same 31 were also positive for HUVEC IgA antibodies. Of the 31 sera positive for EmA and HUVEC IgA antibodies, 24 of these were also positive for IgA reticulin antibodies and 7 sera were negativet 26 sera were negative for EmA, HUVEC and reticulin IgA antibodies.
A sllmm~ry of the results is given in Table 2 which shows the correlation between IgA EmA, HUVEC and reticulin antibody responses from fifty seven sera from coeliac patients and control subjects.
CA 02231~28 1998-03-lO
W O 97/10508 PCT~E96/00061 Table 2 No. of EmA HUVEC Reticulin sera tested Pos Neg Pos Neg Pos Neg Of the 31 sera positive for EmA antibodies all of these were positive for HUVEC IgA antibodies and 24 of these were positive for reticulin antibodies.
Example 2 Measurement of HUVEC surface and intracellular antibody responses using flow cytometry To determine whether the HUVEC antigen reacting with the coeliac disease sera was of surface (extracellular) or intracellular origin, studies were carried out on non-permeabilised cells (surface) and permeabilised cells (intracellular) using flow cytometry. Ten EmA
positive and 10 EmA negative sera were used.
Surface staining: Fresh HUVECs were harvested from culture as described in Example 1 and adjusted to 2X106/rnl. 50 ,ul aliquots of cells were washed once in PBS and reacted with 5O~L1 aliquots of coeliac patients' sera diluted 1/5 in PBS. The cells were incubated on ice for 30 mins. and washed lX with PBS. 20,u1 aliquots of FITC
conjugated rabbit anti human IgA diluted 1/20 were then added to the tubes and allowed to react for 30 mins. The cells were washed once in PBS and read on a FacScan flow cytometer (FacScan is a trade P
mark) (Becton Dickinson). U937 and HL60 cells which are non-foetal cell lines, were used as control cells and treated in a similar manner.
-CA 02231~28 1998-03-10 WO 97/10508 PCTnE96/00061 HUVEC intracellular anti~ens: HUVEC cells were fixed in 2%
paraformaldehyde for 30 mins. The cells were adjusted to 2X106/ml in 0.05% Saponin/PBS (SPBS) and given two further washes in SPBS.
Patient and control sera were diluted 1/5 in SPBS and added in 50 ~1 S volumes to tubes cont7Jinin~ aliquots of 2Xl06 cells. The cells were incubated on ice for 30 mins. and washed lX with SPBS. 20~1 aliquots of FITC conjugated rabbit anti human IgA diluted 1/20 were then added to the tubes and allowed to react for 30 mins. The cells were washed once in SPBS and read on a FacScan flow cytometer.
The results are shown in Table 3.
Table 3 Surface and intracellular stainin~ of HUVEC antigens usin~ I~A
antibodies from EmA positive CD sera and EmA negative normal controls Surface Surface/Intracellular EmA neg* EmA pos* EmA ne~* EmA pos*
6 6 ' 31 95 15 ~k Each numerical value represents a different patient or control It will be observed that no difference was seen between the EmA positive and EmA negative sera when non-permealised HUVECs were used. The mean fluorescent intensity (MFI) values ranged from 5 to 7 with the EmA negative group and from 5 to 8 CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 with the EmA positive group. VVhen the cells were fixed with 2%
paraformaldehyde and permealised with 0.1% saponin there was a marked difference between the two groups. The MFI values for the ErnA negative group ranged from 19 to 42, with a mean MFI value S of 28.2, whereas the MFI values for the EmA positive group ranged from 40 to 192 with a mean MFI value of 109.5. The instrument settings for the two experirnents were the sarne. However, fixing and permç~ ion causes the cells to give out a degree of autofluorescence which explains the difference between the MFI
10 values for the two sets of experiments.
~xample 3 Measurement of ~liadin antibody responses by flow cytometry A) Absorption of ~ din onto latex particles: One ml of lXl07/ml 8~1m polystyrene beads (Polysciences, Warrington) were 15 washed in distilled H20 and incubated overnight at 4~C with gIiadin (Sigma) at a concentration of 1 mg/rnl in 0.05 M bicarbonate buffer pH 9.6. The beads were then washed lX in PBS to remove the unbound gliadin and reincubated at 37~C for 3 hours in PBS
cont~ining bovine serum albumin (BSA) to block unoccupied sites.
20 Any unbound BSA was removed by washing with PBS Cont~inin~
0.05% Tween (PBS/Tween).
Measurement of ~liadin antibodies: 10,ul volumes of beads (lX107/ml) were added to 75 rnm tubes cont~inin~ 50 ,ul of patients' or control sera diluted 1/5 in PB~/Tween and incubated with gentle 25 mixing for 30 mins. at room temperature. The beads were washed 2X
in PBS/Tween to remove unbound sera by centrifuging at 2000 RPM
for 5 mins. 20,u1 aliquots of a 1/20 dilution of FITC conjugated rabbit anti IgA was then added to each tube and incubated with gentle mixing for 30 mins. at room temperature. The beads were again washed lX
30 with 2 ml vols of PBS~Tween and resuspended in 1 ml of PBS/Tween for analysis by flow cytometry.
CA 02231~28 1998-03-10 W O 97/10508 PCT~E96/00061 B) Absorption studies Serum sarnples from coeliac disease patients which tested positive for EmA, reticulin, HUVEC and gliadin antibodies were absorbed with 2X107 HUVEC cells previously fixed with 2%
5 paraformaidehyde and permeabiiised with SPBS. lhe absorption studies were carried out by initially incubating 2Xl07 HUVEC
permeabilised cells for 1 hour at room temperature in 10 ml of 10%
rabbit serum in SPBS to block non-specific binding. The cells were washed lX in SPBS cont~inin~ 10% rabbit serum and resuspended at a concentration of 2Xl07/ml. One ml of a 1/5 dilution of SPBS of the coeliac disease serum was then mixed with 1 ml of 2Xl07 cells and incubated with gentle mixing at 4~C overnight. The following day the cells were spun in a refrigerated centrifuge at 2000 RPM for 10 mins and the absorbed serum removed. For comparison, another sample 15 of the patients' serum was treated identically with the exception that no HUVEC cells were added. IgG and IgA measurements were made of the absorbed and non-absorbed sera and the concentration of IgA
antibody adjusted so that the levels of antibodies were the same in both specimens. Doubling dilutions from 1/5 to 1/640 of the 20 absorbed and non-absorbed serum were made in SPBS. EmA, reticulin, HUVEC and gliadin antibody levels were measured in the absorbed and non-absorbed sera and differences noted.
C) Absorption studies on HUVEC and polystyrene particles usin~ flow cytometry Fifty ,ul aliquots of absorbed and non-absorbed sera diluted 1/5 in SPBS were added to 50,u1 aliquots of paraformaldehyde fixed HUVEC cells at a concentration of 2X106/ml. The cells were incubated ~ at room temperature for 30 mins., washed once in SPBS and spun at 2000 RPM at 4~C for S mins. Fifty ,ul of FITC anti IgA diluted 1/20 in SPBS were then added to each tube and allowed to react at room temperature for a further 30 mins. The cells were washed lX in SPBS, resuspended in SPBS and MFI measured on a FacScan.
CA 02231~28 1998-03-lO
W O 97/10508 PCT~E96/00061 D) Studies carried out usin~ polystyrene beads coated with ~liadin: Briefly, 50,ul aliquots of absorbed and non-absorbed sera diluted 1/5 in PBS/Tween were added to 50,ul aliquots of lX107/ml polystyrene beads previously coated with ~ lin. The beads were 5 incubated at room temperature for 30 mins., washed once in PBS/Tween and spun at 2000 RPM at 4~C for S mins. Fifty ,ul of FITC anti IgA diluted 1/20 in PBS/Tween were then added to the eac tube and allowed to react at room temperature for a ~urther 30 mins.
The beads were washed lX in PBS/Tween, resuspended in PBS/Tween 10 and the MFI measured on a FacScan.
E) Absorption studies with gliadin Sera positive for IgA antibodies to EmA, reticulin, HUVEC
and gli~-lin were absorbed with gliadin coated particles as follows:
One ml of sera was diluted 1/5 in PBS/Tween cont~ining 10% BSA
15 and allowed to mix gently with 1 ml of gliadin coated polystyrene particles ( 1 X 1 07/ml) overnight at 4~C. Similar volumes of sera from the same patients were treated identically with the exception that they were absorbed with polystyrene coated particles coated with 1% BSA.
Both absorbed and non-absorbed sera were then tested for IgA
20 antibodies to EmA, reticulin, HUVEC and gliadin and differences noted.
The results are shown in Tables 4 and ~.
W O 97/10508 PCT~E96/00061 Table 4 Measurement of I~A antibody responses to ~ lin, HUVEC and EmA
before and after absorption with HUVEC and ~ lin SUBSTRATE
Absorbed with: Exp No Gliadin MFI* EmA Titre HUVEC MFI*
Not absorbed 112 640 441 Gliadin 1 38 640 443 Not absorbed 90 640 473 Gliadin 2 35 640 480 *MFI = Median Fluorescence Intensity CA 02231528 1998-03-lO
WO 97/10508 PCT~E96/00061 Table 5 Measurement of I~A antibody responses to ~ 1in, HUVEC~ EmA and reticulin before and after absorption with HUVEC
Substrate Exp Gliadin* HWEC* EMA Titre Reticulin Titre No Pre- Post- Pre- Post- Pre- Post- Pre- Post-Absorb Absorb Absorb Absorb Absorb Absorb Absorb Absorb 52 30 (42) 583 61(90) 1280 80(94) 32020(94) 2 59 36(39) 425 101 (76) 640 80(88) 16010(94) 3 21 12(43) 70 12(83) 1280 80(94) 16040(75) 4 60 40(33) 99 15(85) 640 40(88) 16020(88) 19(46) 218 22(90) 160 10(94) ND ND
* = M[ean Fluorescent Intensity 5 ( ) = % Changes ND = Not Done It will be observed from Table 4 that the antigens in the HUVEC did not cross react with ~ lin The results show that while 10 absorption with gliadin markedly reduced the IgA response to the wheat protein from MFI values of 112 and 90 pre-absorption to MFI
values of 38 and 35 post-absorption, respectively, the IgA responses for HUVEC were unaffected with MFI values of 441 and 473 pre-absorption and MFI values of 443 and 480 post-absorption.
15 Similarly, while absorption with HUVEC reduced the HUVEC IgA
MFI values from 441, 473 to MFI values of 75 and 110, respectively, they only marginally reduced the IgA gliadin responses from pre-absorption MFI values of 112, 90 to post-absorption MFI values of 90 and 70, respectively.
CA 0223l~28 l998-03-lO
WO 97/10508 PCT~E96/00061 ~ Table 5, the results of absorption studies using different patients sera are shown. These show that in five separate experiments absorption with HUVEC subst~nti~lly reduced both the EmA and reticulin titres whereas the IgA gli~clin antibody responses were only 5 slightly reduced.
The titres for EmA pre-absorption were 1280, 640, 1280, 640 and 160 whereas, after absorption with HUVEC the titres dropped to 80, 80, 80, 40 and 10, respectively. The reticulin titres pre-absorption were 320, 160, 160 and 160 whereas, the post-absorption titres were 20, 10, 40 and 20, respectively. The gli~clin IgA antibodies pre-absorption MFI values were 52, 59, 21, 60 and 35 whereas, the post-absorption MFI values were 30, 36, 12, 40, 19, respectively. These data show that the level of reduction in the EmA and reticulin titres were similar in each absorption experiment and varied from 4 to 16 15 fold, whereas the maximum reduction in the gliadin antibody responses were less than two fold.
Example 4 An experiment was carried out to determine the suitability of various cell types for use in the method according to the invention for 20 the detection of EmA. The results are shown in Table 6.
W O 97/10508 PCT~E96/00061 Table 6 CELL TYPE RESPONSE
HUVEC +++
Human umbilical arteryl l +
smooth muscle cells Human umbilical vein ~ I +
smooth muscle cells IMR 90 +++
U937 (ECACC No. negative 85011440) Hl 60 (ECACC No. negative 85011431) Hut 78 (ECACC No. negative 88041901) LoVo (ECACC No. +/-87060101) It will be noted from the above Examples that identical positive and negative correlations were obtained when serum antibodies were measured using monkey oesophagus or HUVEC. Sera that were positive for EmA and HUVEC antibodies did not always show reticulin positivity which may reflect the lack of sensitivity of the tissue used rather than that ErnA and reticulin antibodies are different.
The HUVEC antibodies were found by flow cytometry not to react with the surface of the cells but only when the celIs were permeabilised with saponin which suggests that the target antigens are intracellular. D
AbsoIption studies using permealised HUVEC gave simil~r findings to those described by Marttinen, A. and Maki, M. (supra) using foetal lung fibroblasts in that, whereas they showed that IgA
15 antibodies to proteins secreted by these cells could bind to both CA 02231~28 1998-03-10 WO 97/10508 PCT~E96/00061 endomysium and reticulin, we showed that absorption with HUVEC
subst~nti~lly removed reticulin and EmA and only partially reduced gliadin antibody responses. In contrast gliadin coated particles did not remove HUVEC EmA antibody reactivity. These findings suggest 5 that HUVECs contain both reticulin and EmA antigens or that these antigens are one and the same.
The role of EmA or reticulin antigens/antibodies in the pathogenesis of coeliac disease is unknown, as is the mech~ni.cm, whereby these antibodies are specifically produced. Since the 10 antibodies are present in active coeliac disease and disappear on gluten withdrawal it may suggest that they are simply the result of the infl~mm~tory process with may expose neo-embryonic antigens to the immllne system.
permeabilised cells for 1 hour at room temperature in 10 ml of 10%
rabbit serum in SPBS to block non-specific binding. The cells were washed lX in SPBS cont~inin~ 10% rabbit serum and resuspended at a concentration of 2Xl07/ml. One ml of a 1/5 dilution of SPBS of the coeliac disease serum was then mixed with 1 ml of 2Xl07 cells and incubated with gentle mixing at 4~C overnight. The following day the cells were spun in a refrigerated centrifuge at 2000 RPM for 10 mins and the absorbed serum removed. For comparison, another sample 15 of the patients' serum was treated identically with the exception that no HUVEC cells were added. IgG and IgA measurements were made of the absorbed and non-absorbed sera and the concentration of IgA
antibody adjusted so that the levels of antibodies were the same in both specimens. Doubling dilutions from 1/5 to 1/640 of the 20 absorbed and non-absorbed serum were made in SPBS. EmA, reticulin, HUVEC and gliadin antibody levels were measured in the absorbed and non-absorbed sera and differences noted.
C) Absorption studies on HUVEC and polystyrene particles usin~ flow cytometry Fifty ,ul aliquots of absorbed and non-absorbed sera diluted 1/5 in SPBS were added to 50,u1 aliquots of paraformaldehyde fixed HUVEC cells at a concentration of 2X106/ml. The cells were incubated ~ at room temperature for 30 mins., washed once in SPBS and spun at 2000 RPM at 4~C for S mins. Fifty ,ul of FITC anti IgA diluted 1/20 in SPBS were then added to each tube and allowed to react at room temperature for a further 30 mins. The cells were washed lX in SPBS, resuspended in SPBS and MFI measured on a FacScan.
CA 02231~28 1998-03-lO
W O 97/10508 PCT~E96/00061 D) Studies carried out usin~ polystyrene beads coated with ~liadin: Briefly, 50,ul aliquots of absorbed and non-absorbed sera diluted 1/5 in PBS/Tween were added to 50,ul aliquots of lX107/ml polystyrene beads previously coated with ~ lin. The beads were 5 incubated at room temperature for 30 mins., washed once in PBS/Tween and spun at 2000 RPM at 4~C for S mins. Fifty ,ul of FITC anti IgA diluted 1/20 in PBS/Tween were then added to the eac tube and allowed to react at room temperature for a ~urther 30 mins.
The beads were washed lX in PBS/Tween, resuspended in PBS/Tween 10 and the MFI measured on a FacScan.
E) Absorption studies with gliadin Sera positive for IgA antibodies to EmA, reticulin, HUVEC
and gli~-lin were absorbed with gliadin coated particles as follows:
One ml of sera was diluted 1/5 in PBS/Tween cont~ining 10% BSA
15 and allowed to mix gently with 1 ml of gliadin coated polystyrene particles ( 1 X 1 07/ml) overnight at 4~C. Similar volumes of sera from the same patients were treated identically with the exception that they were absorbed with polystyrene coated particles coated with 1% BSA.
Both absorbed and non-absorbed sera were then tested for IgA
20 antibodies to EmA, reticulin, HUVEC and gliadin and differences noted.
The results are shown in Tables 4 and ~.
W O 97/10508 PCT~E96/00061 Table 4 Measurement of I~A antibody responses to ~ lin, HUVEC and EmA
before and after absorption with HUVEC and ~ lin SUBSTRATE
Absorbed with: Exp No Gliadin MFI* EmA Titre HUVEC MFI*
Not absorbed 112 640 441 Gliadin 1 38 640 443 Not absorbed 90 640 473 Gliadin 2 35 640 480 *MFI = Median Fluorescence Intensity CA 02231528 1998-03-lO
WO 97/10508 PCT~E96/00061 Table 5 Measurement of I~A antibody responses to ~ 1in, HUVEC~ EmA and reticulin before and after absorption with HUVEC
Substrate Exp Gliadin* HWEC* EMA Titre Reticulin Titre No Pre- Post- Pre- Post- Pre- Post- Pre- Post-Absorb Absorb Absorb Absorb Absorb Absorb Absorb Absorb 52 30 (42) 583 61(90) 1280 80(94) 32020(94) 2 59 36(39) 425 101 (76) 640 80(88) 16010(94) 3 21 12(43) 70 12(83) 1280 80(94) 16040(75) 4 60 40(33) 99 15(85) 640 40(88) 16020(88) 19(46) 218 22(90) 160 10(94) ND ND
* = M[ean Fluorescent Intensity 5 ( ) = % Changes ND = Not Done It will be observed from Table 4 that the antigens in the HUVEC did not cross react with ~ lin The results show that while 10 absorption with gliadin markedly reduced the IgA response to the wheat protein from MFI values of 112 and 90 pre-absorption to MFI
values of 38 and 35 post-absorption, respectively, the IgA responses for HUVEC were unaffected with MFI values of 441 and 473 pre-absorption and MFI values of 443 and 480 post-absorption.
15 Similarly, while absorption with HUVEC reduced the HUVEC IgA
MFI values from 441, 473 to MFI values of 75 and 110, respectively, they only marginally reduced the IgA gliadin responses from pre-absorption MFI values of 112, 90 to post-absorption MFI values of 90 and 70, respectively.
CA 0223l~28 l998-03-lO
WO 97/10508 PCT~E96/00061 ~ Table 5, the results of absorption studies using different patients sera are shown. These show that in five separate experiments absorption with HUVEC subst~nti~lly reduced both the EmA and reticulin titres whereas the IgA gli~clin antibody responses were only 5 slightly reduced.
The titres for EmA pre-absorption were 1280, 640, 1280, 640 and 160 whereas, after absorption with HUVEC the titres dropped to 80, 80, 80, 40 and 10, respectively. The reticulin titres pre-absorption were 320, 160, 160 and 160 whereas, the post-absorption titres were 20, 10, 40 and 20, respectively. The gli~clin IgA antibodies pre-absorption MFI values were 52, 59, 21, 60 and 35 whereas, the post-absorption MFI values were 30, 36, 12, 40, 19, respectively. These data show that the level of reduction in the EmA and reticulin titres were similar in each absorption experiment and varied from 4 to 16 15 fold, whereas the maximum reduction in the gliadin antibody responses were less than two fold.
Example 4 An experiment was carried out to determine the suitability of various cell types for use in the method according to the invention for 20 the detection of EmA. The results are shown in Table 6.
W O 97/10508 PCT~E96/00061 Table 6 CELL TYPE RESPONSE
HUVEC +++
Human umbilical arteryl l +
smooth muscle cells Human umbilical vein ~ I +
smooth muscle cells IMR 90 +++
U937 (ECACC No. negative 85011440) Hl 60 (ECACC No. negative 85011431) Hut 78 (ECACC No. negative 88041901) LoVo (ECACC No. +/-87060101) It will be noted from the above Examples that identical positive and negative correlations were obtained when serum antibodies were measured using monkey oesophagus or HUVEC. Sera that were positive for EmA and HUVEC antibodies did not always show reticulin positivity which may reflect the lack of sensitivity of the tissue used rather than that ErnA and reticulin antibodies are different.
The HUVEC antibodies were found by flow cytometry not to react with the surface of the cells but only when the celIs were permeabilised with saponin which suggests that the target antigens are intracellular. D
AbsoIption studies using permealised HUVEC gave simil~r findings to those described by Marttinen, A. and Maki, M. (supra) using foetal lung fibroblasts in that, whereas they showed that IgA
15 antibodies to proteins secreted by these cells could bind to both CA 02231~28 1998-03-10 WO 97/10508 PCT~E96/00061 endomysium and reticulin, we showed that absorption with HUVEC
subst~nti~lly removed reticulin and EmA and only partially reduced gliadin antibody responses. In contrast gliadin coated particles did not remove HUVEC EmA antibody reactivity. These findings suggest 5 that HUVECs contain both reticulin and EmA antigens or that these antigens are one and the same.
The role of EmA or reticulin antigens/antibodies in the pathogenesis of coeliac disease is unknown, as is the mech~ni.cm, whereby these antibodies are specifically produced. Since the 10 antibodies are present in active coeliac disease and disappear on gluten withdrawal it may suggest that they are simply the result of the infl~mm~tory process with may expose neo-embryonic antigens to the immllne system.
Claims (13)
1. A method for diagnosing coeliac disease and related conditions in a subject known or suspected of having such a condition, which comprises contacting a body fluid from said subject containing antibody particular to said condition with a suspension of cells from an immortal cell line having an epitopic site specific for said antibody and determining the extent of binding of antibody to said cells.
2. A method according to Claim 1, wherein the cells are embryonic in nature.
3. A method according to Claim 1, wherein the cells are human umbilical cells.
4. A method according to Claim 3, wherein the cells are human umbilical vein endothelial cells (HUVECs).
5. A method according to Claim 4, wherein the HUVECs are cells of ECV 304 cell line, No. 92091712.
6. A method according to Claim 3, wherein the cells are human umbilical artery smooth muscle cells or human umbilical vein smooth muscle cells.
7. A method according to any preceding claim, wherein the antibodies are of the IgA type.
8. A method according to any preceding claim, wherein the cells are ruptured prior to or during contact with said body fluid.
9. A method according to any preceding claim, wherein the body fluid is serum.
10. A method according to any preceding claim, wherein bound antibody is estimated by flow cytometry.
11. A method according to any one of Claims 1-9, wherein bound antibody is estimated by an enzyme immunoassay procedure.
12. A kit for carrying out a method according to any one of Claims 1-11 containing a suspension of cells from an immortal cell line.
13. A method for diagnosing coeliac disease and related conditions in a subject known or suspected of having such a condition, substantially as hereinbefore described and exemplified.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IE950725 | 1995-09-15 | ||
| IE950725 | 1995-09-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2231528A1 true CA2231528A1 (en) | 1997-03-20 |
Family
ID=11040894
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2231528 Abandoned CA2231528A1 (en) | 1995-09-15 | 1996-09-13 | Method for the diagnosis of coeliac disease |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0850418A1 (en) |
| JP (1) | JPH11511559A (en) |
| AU (1) | AU707114B2 (en) |
| CA (1) | CA2231528A1 (en) |
| NO (1) | NO981057L (en) |
| NZ (1) | NZ319260A (en) |
| WO (1) | WO1997010508A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6703208B1 (en) | 1999-10-20 | 2004-03-09 | Immco Diagnostics | Immunological assay for detection of antibodies in celiac disease |
| ITRM20020144A1 (en) * | 2002-03-15 | 2003-09-15 | Dipartimento Di Scienze Biomed | SEROLOGICAL METHOD FOR DETECTING THE ATROPHY OF INTESTINAL VILLAS IN PATIENTS WITH CELIAC DISEASE. |
| ES2217931B1 (en) * | 2002-07-01 | 2006-02-16 | Universidad De Barcelona | METHOD OF DIAGNOSIS OF THE EVOLUTION OF THE INTESTINAL ABSORTIVE MASS IN AN INDIVIDUAL. |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL155894B1 (en) * | 1987-12-23 | 1992-01-31 | Przed Zagraniczne W Polsce Pla | Test for assessing gluten-dependent enteropathies |
-
1996
- 1996-09-13 JP JP9511805A patent/JPH11511559A/en active Pending
- 1996-09-13 NZ NZ319260A patent/NZ319260A/en unknown
- 1996-09-13 EP EP96932788A patent/EP0850418A1/en not_active Withdrawn
- 1996-09-13 CA CA 2231528 patent/CA2231528A1/en not_active Abandoned
- 1996-09-13 AU AU71439/96A patent/AU707114B2/en not_active Ceased
- 1996-09-13 WO PCT/IE1996/000061 patent/WO1997010508A1/en not_active Application Discontinuation
-
1998
- 1998-03-10 NO NO981057A patent/NO981057L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| NO981057D0 (en) | 1998-03-10 |
| AU707114B2 (en) | 1999-07-01 |
| EP0850418A1 (en) | 1998-07-01 |
| JPH11511559A (en) | 1999-10-05 |
| WO1997010508A1 (en) | 1997-03-20 |
| NO981057L (en) | 1998-03-10 |
| NZ319260A (en) | 1999-08-30 |
| AU7143996A (en) | 1997-04-01 |
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