Classifications

• • 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
• • 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

Definitions

• This invention relates to a method for diagnosing coeliac disease or gluten-sensitive enteropathy and related conditions as hereinafter defined.
• Coeliac disease is a permanent intolerance to gliadin (wheat protein) leading to intestinal villous flattening and crypt hype ⁇ lasia in susceptible subjects.
• Immune reactions to gliadin are considered to play a part in the pathogenesis of the disease, although the immunological mechanisms have not been fully elucidated.
• Antigliadin antibodies (AGA), reticulin antibodies and endomysium antibodies (EmA) are found in serum samples from patients with coeliac disease. However, AG As 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 individuals with dermatitis herpetiformis, who are also gluten sensitive.
• 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.
• coeliac disease is dependent on the histological evaluation of the small intestinal mucusal biopsy, more specifically the jejunal biopsy.
• serological assessment is an increasingly important method of screening for the condition, especially as it represents a less invasive test therefor.
• IgA EmA antibodies The measurement of IgA EmA antibodies is currently considered 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 antibodies (but requires lengthy processing (Ladinser, B. et al (1994); Gut 35 776-778).
• reticulin antibodies For the detection of reticulin antibodies rat kidney, stomach or liver tissue is used.
• Reticulin antibodies and EmA are considered by some to be 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 against reticulin and endomysium but not against gliadin 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 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.
• ELISA enzyme linked immunosorbent assay
• 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.
• 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 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.
• cell suspensions especially single cell suspensions, are easier to examine than the tissue sections typical of 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.
• immortal cell line herein is meant a cell line which can be repeatedly cultured and does not die.
• 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 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 human umbilical cell.
• Especially suitable cells for use in accordance with the invention are human umbilical vein endothelial cells (HUVECs).
• HUVECs human umbilical vein endothelial cells
• 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.
• human umbilical cells are human umbilical artery smooth muscle cells and human umbilical vein smooth muscle 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 organisation under the Catalogue No. 6209/L/l .
• the antibodies determined in accordance with the invention will typically be of the IgA type.
• IgA EmA is the predominant antibody response in coeliac disease is not fully understood. However, it is postulated to be related to antigen presentation at the gastrointestinal level.
• the cells are ruptured prior to or during contact with said body fluid.
• the body fluid can be any body fluid but is preferably serum.
• the antigen for use in immunoassays 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. Especially suitable techniques are flow cytometry and enzyme immunoassay. 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 containing a suspension of cells from an immortal cell line.
• 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 intestinal biopsy acted as controls.
• HUVEC ECV 304 No. 92091712 European Collection of Animal Cell Culture
• RPMI 1640 medium containing 10% foetal calf serum and gentamycin at 37°C in a humidified incubator with 5% CO 2 until confluent.
• the cells were harvested using trypsin EDTA and washed 2X in RPMI 1640.
• Immunofluorescence studies 30 ⁇ l aliquots of 2X10 6 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.
• PBS phosphate buffered saline
• 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 stomach was used to detect reticulin antibodies. Sections 4 micron thick were cut and mounted onto glass slides, sera were diluted and immunofluorescence carried out as described in the HUVECs and EmA studies. The sera were randomised with the normal controls and the observer was blinded as to the origin of each slide.
• Table 2 shows the correlation between IgA EmA, HUVEC and reticulin antibody responses from fifty seven sera from coeliac patients and control subjects.
• 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.
• Fresh HUVECs were harvested from culture as described in Example 1 and adjusted to 2X10 6 /ml. 50 ⁇ l aliquots of cells were washed once in PBS and reacted with 50 ⁇ l aliquots of coeliac patients' sera diluted 1/5 in PBS. The cells were incubated on ice for 30 mins. and washed I X with PBS. 20 ⁇ l 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 mark) (Becton Dickinson).
• HUVEC intracellular antigens HUVEC cells were fixed in 2% paraformaldehyde for 30 mins. The cells were adjusted to 2X10 6 /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 ⁇ l volumes to tubes containing aliquots of 2X10 6 cells. The cells were incubated on ice for 30 mins. and washed IX with SPBS. 20 ⁇ l 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.
• Each numerical value represents a different patient or control
• the mean fluorescent intensity (MFI) values ranged from 5 to 7 with the EmA negative group and from 5 to 8 with the EmA positive group.
• MFI values for the EmA negative group ranged from 19 to 42, with a mean MFI value 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 experiments were the same. However, fixing and permealisation causes the cells to give out a degree of autofluorescence which explains the difference between the MFI values for the two sets of experiments.
• A) Absorption of gliadin onto latex particles One ml of lX10 7 /ml 8 ⁇ m polystyrene beads (Polysciences, Warrington) were washed in distilled H 2 O and incubated overnight at 4°C with gliadin (Sigma) at a concentration of 1 mg/ml in 0.05 M bicarbonate buffer pH 9.6. The beads were then washed IX in PBS to remove the unbound gliadin and reincubated at 37°C for 3 hours in PBS containing bovine serum albumin (BSA) to block unoccupied sites. Any unbound BSA was removed by washing with PBS containing 0.05% Tween (PBS/Tween).
• PBS/Tween PBS/Tween
• gliadin antibodies lO ⁇ l volumes of beads (lX10 7 /ml) were added to 75 mm tubes containing 50 ⁇ l of patients' or control sera diluted 1/5 in PBS/Tween and incubated with gentle 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 ⁇ l 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 IX with 2 ml vols of PBS/Tween and resuspended in 1 ml of PBS Tween for analysis by flow cytometry. B) Abso ⁇ tion studies
• Serum samples from coeliac disease patients which tested positive for EmA, reticulin, HUVEC and gliadin antibodies were absorbed with 2X10 7 HUVEC cells previously fixed with 2% paraformaldehyde and permeabilised with SPBS.
• the abso ⁇ tion studies were carried out by initially incubating 2X10 7 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 IX in SPBS containing 10% rabbit serum and resuspended at a concentration of 2X10 7 /ml.
• Fifty ⁇ l aliquots of absorbed and non-absorbed sera diluted 1/5 in SPBS were added to 50 ⁇ l aliquots of paraformaldehyde fixed HUVEC cells at a concentration of 2X10 6 /ml. The cells were incubated at room temperature for 30 mins., washed once in SPBS and spun at 2000 RPM at 4°C for 5 mins. Fifty ⁇ l 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 IX in SPBS, resuspended in SPBS and MFI measured on a FacScan.
• Sera positive for IgA antibodies to EmA, reticulin, HUVEC and gliadin were absorbed with gliadin coated particles as follows: One ml of sera was diluted 1/5 in PBS/Tween containing 10% BSA and allowed to mix gently with 1 ml of gliadin coated polystyrene particles (lX10 7 /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 antibodies to EmA, reticulin, HUVEC and gliadin and differences noted.
• the titres for EmA pre-abso ⁇ tion were 1280, 640, 1280, 640 and 160 whereas, after abso ⁇ tion with HUVEC the titres dropped to 80, 80, 80, 40 and 10, respectively.
• the reticulin titres pre-abso ⁇ tion were 320, 160, 160 and 160 whereas, the post-abso ⁇ tion titres were 20, 10, 40 and 20, respectively.
• the gliadin IgA antibodies pre- abso ⁇ tion MFI values were 52, 59, 21, 60 and 35 whereas, the post- abso ⁇ tion MFI values were 30, 36, 12, 40, 19, respectively.
• Hut 78 (ECACC No. negative 88041901)
• EmA or reticulin antigens/antibodies in the pathogenesis of coeliac disease is unknown, as is the mechanism, whereby these antibodies are specifically produced. Since the antibodies are present in active coeliac disease and disappear on gluten withdrawal it may suggest that they are simply the result of the inflammatory process with may expose neo-embryonic antigens to the immune system.

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• 1996
  • 1996-09-13 NZ NZ319260A patent/NZ319260A/xx unknown
  • 1996-09-13 JP JP9511805A patent/JPH11511559A/ja active Pending
  • 1996-09-13 WO PCT/IE1996/000061 patent/WO1997010508A1/en not_active Application Discontinuation
  • 1996-09-13 EP EP96932788A patent/EP0850418A1/de not_active Withdrawn
  • 1996-09-13 AU AU71439/96A patent/AU707114B2/en not_active Ceased
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• 1998
  • 1998-03-10 NO NO981057A patent/NO981057L/no not_active Application Discontinuation

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