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WO1998015836A1 - Assay for determining anti-endomysial antibodies - Google Patents

Assay for determining anti-endomysial antibodies Download PDF

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Publication number
WO1998015836A1
WO1998015836A1 PCT/EP1997/005420 EP9705420W WO9815836A1 WO 1998015836 A1 WO1998015836 A1 WO 1998015836A1 EP 9705420 W EP9705420 W EP 9705420W WO 9815836 A1 WO9815836 A1 WO 9815836A1
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Prior art keywords
marker
endomysial
solid phase
human
diagnostic kit
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PCT/EP1997/005420
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French (fr)
Inventor
Maurizio Vecchi
Original Assignee
Maurizio Vecchi
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Publication of WO1998015836A1 publication Critical patent/WO1998015836A1/en

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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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention relates to a new method for diagnosing celiac disease.
  • the present invention relates to a new method for assaying anti-endomysial antibodies present in the serum of patients suffering from celiac disease .
  • Celiac disease is characterized by persistent intolerance to gluten, which induces atrophy of the jejunal villi with crypt hyperplasia and poor absorption of numerous nutrient substances, with the consequent clinical signs and symptoms, such as weight loss (or lack of weight gain in children) , diarrhoea, swelling of the abdomen, anaemia caused by iron or vitamin deficiency and osteoporosis, to name but a few.
  • weight loss or lack of weight gain in children
  • diarrhoea swelling of the abdomen
  • anaemia caused by iron or vitamin deficiency and osteoporosis to name but a few.
  • Diagnosing celiac disease has always been based on testing for characteristic lesions and for their regression after a suitable period of dieting by means of jejunal biopsy.
  • serological methods which allow non- invasive screening of at -risk populations and of individuals suspected of having this disease.
  • the assaying of anti-gliadin (AGA) , anti-reticulin (ARA) , anti-jejunal and anti-endomysial (EMA) serum IgA antibodies is characterized by very high levels of sensitivity and specificity in the diagnosis of this disease.
  • EMAs are characterized by a sensitivity and specificity of virtually 100% and the assay based on these antibodies is thus held as the most accurate test.
  • a conventional technique used for determining EMAs in the serum of patients involves the use of immunofluorescence methods on monkey distal oesophagus tissue since this tissue is particularly rich in reactive antigens. After the human serum has been incubated on the monkey tissue bound to a sample holder slide, the presence of any anti-endomysial antibodies in the serum may be evaluated by using an anti -human IgA fluorescent antibody which binds to any IgA antibodies attached to the tissue. This results in a particularly intense fluorescence, which may be detected using special microscopes .
  • the problem is thus one of providing a method for determining EMA antibodies contained in the serum of individuals who are clinically suspected of having celiac disease, which overcomes all of the abovementioned drawbacks .
  • EMA anti-endomysial human serum IgA
  • stage (b) bringing the solid phase obtained in stage (a) into contact with the said sample which may contain the said anti-endomysial human serai IgA;
  • stage (c) bringing the solid phase obtained in stage (b) into contact with an aqueous phase containing an anti-human IgA antibody bound to a marker;
  • the method according to the present invention comprises the use of an enzyme as a marker bound to the anti-human IgA antibody.
  • the present invention comprises the use of the enzyme alkaline phosphatase .
  • the antigen which reacts with anti-endomysial antibodies is obtained from animal tissue. It has proved particularly advantageous to use human umbilical cord tissue as the source of antigen which reacts with anti-endomysial antibodies .
  • the anti -human IgA antibody bound to a marker is a rabbit IgG anti -human IgA antibody.
  • the present invention also relates to a diagnostic kit for carrying out the method described above, comprising, stored separately from one another:
  • the sera of known diagnosis are essential components of the diagnostic kit of the present invention, since their analysis using the method of the present invention determines the confidence interval of this test.
  • the diagnostic kit according to the invention also comprises washing buffers in aqueous phase .
  • the said marker is an enzyme and, in particular, the said enzyme is alkaline phosphatase .
  • the present invention also relates to a diagnostic kit for carrying out the method described above, comprising, stored separately from one another:
  • the diagnostic kit according to the invention also comprises washing buffers in aqueous phase .
  • the said marker is an enzyme and, in particular, the said enzyme is alkaline phosphatase .
  • the method according to the invention has many advantages over methods using immunofluorescence techniques. Firstly, the preparation of the reaction plates for the method according to the present invention requires considerably less of antigen and much less time compared with the preparation of slides for immunofluorescence .
  • a further advantage of the method according to the present invention is that the actual analysis of EMAs may be carried out much more quickly than when using immunofluorescent techniques, since the samples do not need to be analysed one by one, under a microscope by the laboratory staff, but are instead scanned by measuring equipment at much higher speeds.
  • the most important advantage of the method according to the present invention is that the analysis results are obtained by means of the quantitative reading of a physical magnitude, such as the optical density, which cannot give rise to errors caused by the subjectivity of a qualitative evaluation, as in the case of immunofluorescence .
  • It is also possible to provide a ready-to-use diagnostic kit comprising a plate (solid phase) and to supply the antigen and the anti-human IgA antibody conjugated with the enzyme separately, so as to speed up the entire procedure of the diagnostic test considerably.
  • Figure 1 is a diagram representing the average and the standard deviation of the optical density readings of sera from individuals suffering from celiac disease (CE) and from healthy individuals (controls) , which are obtained by the method according to the invention, and
  • Figure 2 is a diagram representing the distribution of the optical density readings of sera from individuals suffering from celiac disease (CE) and from healthy individuals (controls) .
  • CE celiac disease
  • human umbilical cord is cut into approximately 1 cm-long fragments which are then frozen using liquid nitrogen and stored at a temperature of -80°C. Preparation of the homogenate
  • One or more fragments of umbilical cord are thawed and washed with phosphate-buffered saline (0.15 M NaCl, pH 7.2, PBS) until the blood material contained therein has been completely removed.
  • the fragments of cord are then placed in phosphate buffer at a temperature varying between 0 and 4°C (10 mM Na 2 HP0 4 , 10 mM KH 2 P0 4 , pH 7.2) containing leupeptin, aprotinin and pepstatin, each at a concentration of 1 ⁇ g/ml .
  • the tissue is subsequently homogenized by four cycles of 30 sec each, while maintaining the material at 0-4°C.
  • the product thus obtained is then centrifuged at 1500 x g for 20 minutes. The supernatant is removed and, after evaluating its protein concentration, it is stored at -20°C until the ELISA experiments are carried out. Implementation of the ELISA method
  • the ELISA plates made of polystyrene, are coated over a period of 14 hours at 4°C using 100 ⁇ l aliquots of cord homogenate diluted in phosphate-buffered saline (0.15 M NaCl) , until a final protein concentration of 0.3 mg/ml is obtained. The plates are then washed three times with 200 ⁇ l aliquots of phosphate-buffered saline containing 0.05% Tween 20 for each well.
  • test serum diluted to 1:10 in phosphate-buffered saline containing 0.05 % Tween 20 and 2 % bovine serum albumin are subsequently incubated in each well for one hour at 37°C.
  • the plates are then washed as above and incubated at 37°C with 100 ⁇ l aliquots of rabbit IgG anti-human IgA conjugated with alkaline phosphatase, diluted to 1:500 in phosphate- buffered saline.
  • the plates are then washed again as above, after which they are incubated at room temperature with p-nitrophenyl phosphate diluted in diethanolamine solution (97 ml/1), NaN 3 (0.2 g/1) and MgCl 2 (100 ⁇ ng/1) in distilled water, pH 9.8 in darkness for 40 minutes.
  • the optical density of each well may be measured using an ELISA detector at a wavelength of 405 nm.
  • the threshold value below which the test serum is considered healthy was calculated by adding to the average of the optical density readings obtained for the control sera, two standard deviations of this average, so as to obtain a value likely to include 95 % of the values obtained in a population of normal individuals.
  • the value thus calculated was divided by the average of the readings obtained in the normal control population, thereby obtaining a numerical coefficient which may be used in each subsequent experiment in order to calculate the threshold value by using a pool of normal control sera .
  • Figure 1 illustrates the average and the standard deviation of the optical density readings for the 107 serum samples analysed.
  • Figure 2 illustrates the distribution of the optical density readings of the 107 samples.
  • the fragments of umbilical cord are put into an ice-cold buffer having the following composition: 0.35 M NaCl
  • tissue is homogenized by means of 3 cycles (30 seconds each one) ; the total volume is measured and ammonium sulphate is added to a final concentration of 0.7 M.
  • the pH of the buffer used in the process may range between 7 and 8; various buffer systems which are conventionally used in this field such as, for example, the Tris/HCl system, may also be used.
  • the human serum may be used undiluted or diluted down to 1:100.
  • anti -human IgA antibodies from other animals (mice, pigs, etc.) may be used.
  • Various measuring techniques other than the ELISA method may also be used; for example, it is possible to use RIA (radioimmunoassay) techniques in which a radioactive marker is used instead of an enzyme. In this case, the radioactivity will be measured rather than the enzymatic activity of complexes bound to the surface of the solid phase.
  • RIA radioimmunoassay
  • other enzymes such as, for example, peroxidase, may be used when applying the ELISA method, without any substantial changes being observed in the results obtained.

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  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The present invention relates to a new sandwich-type method for diagnosing celiac disease, based on the measurement of anti-endomysial human seral IgA (EMA), which may be present in the serum of an individual. This method comprises the successive stages of: a) immobilizing an antigen which reacts with anti-endomysial antibodies on a solid phase; b) bringing the solid phase obtained in a) into contact with serum which may contain anti-endomysial human seral IgA; c) bringing the solid phase obtained in b) into contact with an aqueous phase containing an anti-human IgA antibody bound to a marker; d) detecting the presence of this marker in the solid phase obtained in c). The method according to the present invention has many advantages over the methods of the prior art; firstly, the analyses take considerably less time to carry out, there is no subjective interpretation of the results by the experimenter and considerably less antigen is required.

Description

ASSAY FOR DETERMINING ANTI-ENDOMYSIAL ANTIBODIES
DESCRIPTION
The present invention relates to a new method for diagnosing celiac disease.
More precisely, the present invention relates to a new method for assaying anti-endomysial antibodies present in the serum of patients suffering from celiac disease .
Celiac disease is characterized by persistent intolerance to gluten, which induces atrophy of the jejunal villi with crypt hyperplasia and poor absorption of numerous nutrient substances, with the consequent clinical signs and symptoms, such as weight loss (or lack of weight gain in children) , diarrhoea, swelling of the abdomen, anaemia caused by iron or vitamin deficiency and osteoporosis, to name but a few. These adverse changes are reversible once gluten has been excluded from the diet.
Diagnosing celiac disease has always been based on testing for characteristic lesions and for their regression after a suitable period of dieting by means of jejunal biopsy. However, in recent years, it has become increasingly common to use serological methods which allow non- invasive screening of at -risk populations and of individuals suspected of having this disease. The assaying of anti-gliadin (AGA) , anti-reticulin (ARA) , anti-jejunal and anti-endomysial (EMA) serum IgA antibodies is characterized by very high levels of sensitivity and specificity in the diagnosis of this disease.
These assays have thus become a very commonly used means of diagnosis when celiac disease is clinically suspected and since they may be applied to a wide range of individuals, they have made it possible to reveal a very high prevalence of the disease (about 1 in 250) , even in people who were erroneously considered to be at low risk such as, for example, the population of the United States.
Among the antibodies listed above, EMAs are characterized by a sensitivity and specificity of virtually 100% and the assay based on these antibodies is thus held as the most accurate test.
A conventional technique used for determining EMAs in the serum of patients involves the use of immunofluorescence methods on monkey distal oesophagus tissue since this tissue is particularly rich in reactive antigens. After the human serum has been incubated on the monkey tissue bound to a sample holder slide, the presence of any anti-endomysial antibodies in the serum may be evaluated by using an anti -human IgA fluorescent antibody which binds to any IgA antibodies attached to the tissue. This results in a particularly intense fluorescence, which may be detected using special microscopes .
However, there are many drawbacks involved in the use of this technique. Firstly, these tissues are very expensive to use and they especially pose ecological problems since monkeys are protected species; secondly, although results obtained using this method are easy to interpret, it nevertheless remains a subjective technique, linked to the observer's personal evaluation, and anyway depends on the local concentration of endomysium present in the oesophageal tissue, which may vary from one sample to another.
Some publications, for example B. Ladinser, E. Rossipal and K. Pittschieler, Gut 1994; 35; 776-778, have disclosed that anti-endomysial reactivity may be evaluated, with sensitivity and specificity levels similar to those using the monkey distal oesophagus technique, on human umbilical cord tissue.
The use of this technique makes it possible to avoid the abovementioned drawbacks of cost and ecological unfriendliness associated with the use of monkey oesophageal tissue since human umbilical cord is widely available and is normally disposed of after birth. However, the problem associated with the application of immunofluorescence - that is the subjectivity of the evaluation and the slowness and complexity of the analysis - still remains.
The problem is thus one of providing a method for determining EMA antibodies contained in the serum of individuals who are clinically suspected of having celiac disease, which overcomes all of the abovementioned drawbacks .
This problem is solved, according to the invention, by a new sandwich-type method for measuring the anti-endomysial human serum IgA (EMA) which may be present in a sample, this method comprising the stages of:
(a) immobilizing an antigen which reacts with anti- endomysial antibodies on a solid phase;
(b) bringing the solid phase obtained in stage (a) into contact with the said sample which may contain the said anti-endomysial human serai IgA;
(c) bringing the solid phase obtained in stage (b) into contact with an aqueous phase containing an anti-human IgA antibody bound to a marker;
(d) detecting the presence of the said marker in the solid phase obtained in stage (c) .
Preferably, the method according to the present invention comprises the use of an enzyme as a marker bound to the anti-human IgA antibody. In particular, the present invention comprises the use of the enzyme alkaline phosphatase .
In a further aspect of the present invention, the antigen which reacts with anti-endomysial antibodies is obtained from animal tissue. It has proved particularly advantageous to use human umbilical cord tissue as the source of antigen which reacts with anti-endomysial antibodies .
Preferably, the anti -human IgA antibody bound to a marker is a rabbit IgG anti -human IgA antibody.
The present invention also relates to a diagnostic kit for carrying out the method described above, comprising, stored separately from one another:
(a) a plate constituting a solid phase
(b) a liquid phase containing an antigen which reacts with anti-endomysial antibodies;
(c) an anti-human IgA antibody, bound to a marker, in aqueous phase ;
(d) a number of sera from healthy individuals;
(e) a number of sera from individuals suffering from celiac disease.
The sera of known diagnosis are essential components of the diagnostic kit of the present invention, since their analysis using the method of the present invention determines the confidence interval of this test.
Preferably, the diagnostic kit according to the invention also comprises washing buffers in aqueous phase .
In one particular embodiment of the diagnostic kit according to the present invention, the said marker is an enzyme and, in particular, the said enzyme is alkaline phosphatase .
Lastly, the present invention also relates to a diagnostic kit for carrying out the method described above, comprising, stored separately from one another:
(a) a plate constituting a solid phase, having an antigen which reacts with the anti-endomysial antibodies bound thereto;
(c) an anti-human IgA antibody, bound to a marker, in aqueous phase;
(d) a number of sera from healthy individuals; (e) a number of sera from individuals suffering from celiac disease.
Preferably, the diagnostic kit according to the invention also comprises washing buffers in aqueous phase .
In one particular embodiment of the diagnostic kit according to the present invention, the said marker is an enzyme and, in particular, the said enzyme is alkaline phosphatase .
The method according to the invention has many advantages over methods using immunofluorescence techniques. Firstly, the preparation of the reaction plates for the method according to the present invention requires considerably less of antigen and much less time compared with the preparation of slides for immunofluorescence .
A further advantage of the method according to the present invention is that the actual analysis of EMAs may be carried out much more quickly than when using immunofluorescent techniques, since the samples do not need to be analysed one by one, under a microscope by the laboratory staff, but are instead scanned by measuring equipment at much higher speeds. However, the most important advantage of the method according to the present invention is that the analysis results are obtained by means of the quantitative reading of a physical magnitude, such as the optical density, which cannot give rise to errors caused by the subjectivity of a qualitative evaluation, as in the case of immunofluorescence .
It is also possible to provide a ready-to-use diagnostic kit comprising a plate (solid phase) and to supply the antigen and the anti-human IgA antibody conjugated with the enzyme separately, so as to speed up the entire procedure of the diagnostic test considerably.
A specific example of the application of the method according to the present invention will be given below, by way of non-limiting illustration, with reference to the appended figures, in which:
Figure 1 is a diagram representing the average and the standard deviation of the optical density readings of sera from individuals suffering from celiac disease (CE) and from healthy individuals (controls) , which are obtained by the method according to the invention, and
Figure 2 is a diagram representing the distribution of the optical density readings of sera from individuals suffering from celiac disease (CE) and from healthy individuals (controls) .
EXAMPLE Processing the umbilical cord
Immediately after delivery, human umbilical cord is cut into approximately 1 cm-long fragments which are then frozen using liquid nitrogen and stored at a temperature of -80°C. Preparation of the homogenate
One or more fragments of umbilical cord are thawed and washed with phosphate-buffered saline (0.15 M NaCl, pH 7.2, PBS) until the blood material contained therein has been completely removed. The fragments of cord are then placed in phosphate buffer at a temperature varying between 0 and 4°C (10 mM Na2HP04, 10 mM KH2P04, pH 7.2) containing leupeptin, aprotinin and pepstatin, each at a concentration of 1 μg/ml . The tissue is subsequently homogenized by four cycles of 30 sec each, while maintaining the material at 0-4°C. The product thus obtained is then centrifuged at 1500 x g for 20 minutes. The supernatant is removed and, after evaluating its protein concentration, it is stored at -20°C until the ELISA experiments are carried out. Implementation of the ELISA method
The ELISA plates, made of polystyrene, are coated over a period of 14 hours at 4°C using 100 μl aliquots of cord homogenate diluted in phosphate-buffered saline (0.15 M NaCl) , until a final protein concentration of 0.3 mg/ml is obtained. The plates are then washed three times with 200 μl aliquots of phosphate-buffered saline containing 0.05% Tween 20 for each well.
100 μl aliquots of test serum diluted to 1:10 in phosphate-buffered saline containing 0.05 % Tween 20 and 2 % bovine serum albumin are subsequently incubated in each well for one hour at 37°C. The plates are then washed as above and incubated at 37°C with 100 μl aliquots of rabbit IgG anti-human IgA conjugated with alkaline phosphatase, diluted to 1:500 in phosphate- buffered saline. The plates are then washed again as above, after which they are incubated at room temperature with p-nitrophenyl phosphate diluted in diethanolamine solution (97 ml/1), NaN3 (0.2 g/1) and MgCl2 (100 τng/1) in distilled water, pH 9.8 in darkness for 40 minutes. At this point, the optical density of each well may be measured using an ELISA detector at a wavelength of 405 nm.
The threshold value below which the test serum is considered healthy was calculated by adding to the average of the optical density readings obtained for the control sera, two standard deviations of this average, so as to obtain a value likely to include 95 % of the values obtained in a population of normal individuals. The value thus calculated was divided by the average of the readings obtained in the normal control population, thereby obtaining a numerical coefficient which may be used in each subsequent experiment in order to calculate the threshold value by using a pool of normal control sera .
A blind analysis of 107 samples of serum (47 taken from individuals with gluten enteropathy and 60 from healthy control individuals) was then carried out. Figure 1 illustrates the average and the standard deviation of the optical density readings for the 107 serum samples analysed. The results of these experiments were positive: the average of the optical density values in the sera of individuals suffering from celiac disease (CE) was 0.680 (median =0.647, standard deviation =0.307- ) , whereas in the healthy sera (controls) the average was 0.179 (median =0.146, standard deviation =0.098). The difference between the two averages was highly significant (p<106) .
Figure 2 illustrates the distribution of the optical density readings of the 107 samples. By calculating the threshold value as described above (represented in the figure by a horizontal dashed line) only 4 CEs were negative in the test, while only 2 controls gave slightly higher values.
The ELISA test carried out as described above gave results equal to those obtained by immunofluoresence in 106 out of the 107 sera examined.
The preparation of the homogenate has also been carried out according to an alternative procedure, which is reported hereinbelow, obtaining comparable results.
The fragments of umbilical cord are put into an ice-cold buffer having the following composition: 0.35 M NaCl
10 mM Tris-HCl (pH 7.5) 5 mM EDTA
10 mM 2-mercaptoethanol 1 microgram/ml Leupeptine, Aprotinin, Pepstatine.
Then the tissue is homogenized by means of 3 cycles (30 seconds each one) ; the total volume is measured and ammonium sulphate is added to a final concentration of 0.7 M.
Two homogenization cycles of 30 seconds each one are carried out until the solution will clear; the solution is centrifuged for 20 minutes at 12000 r.p.m. at 4°C. The supernatant is taken and centrifuged again at 10000 r.p.m. for 150 minutes at 4°C. The volume of the supernatant is then measured and ammonium sulphate is added to a final concentration of 70°%.
Another centrifugation is carried out for 20 minutes at 12000 r.p.m. at 4°C. and the resulting pellet (dried with a blotting paper) is suspended in 3-4 ml of a buffer having the following composition: 50 mM Tris-HCl pH 8 5 mM 2-mercaptoethanol 50 mM NaCl 0.1 mM EDTA 10 % glycerol
Finally a dyalisis in the same buffer (4 1) is carried out overnight at 4°C and aliquots are made and kept at -80°C.
From the experiments carried out, the results show that some parameters of the process described above can be varied within certain limits while still obtaining satisfactory results:
The pH of the buffer used in the process may range between 7 and 8; various buffer systems which are conventionally used in this field such as, for example, the Tris/HCl system, may also be used.
The human serum may be used undiluted or diluted down to 1:100.
As an alternative to rabbit IgG anti -human IgA, anti -human IgA antibodies from other animals (mice, pigs, etc.) may be used.
Various measuring techniques other than the ELISA method may also be used; for example, it is possible to use RIA (radioimmunoassay) techniques in which a radioactive marker is used instead of an enzyme. In this case, the radioactivity will be measured rather than the enzymatic activity of complexes bound to the surface of the solid phase. As an alternative to alkaline phosphatase, other enzymes such as, for example, peroxidase, may be used when applying the ELISA method, without any substantial changes being observed in the results obtained.

Claims

1. Sandwich-type method for measuring the anti- endomysial human serai IgA (EMA) which may be present in a sample, this method comprising the stages of:
(a) immobilizing an antigen which reacts with anti- endomysial antibodies on a solid phase;
(b) bringing the solid phase obtained in stage (a) into contact with the said sample which may contain the said anti-endomysial human serai IgA;
(c) bringing the solid phase obtained in stage (b) into contact with an aqueous phase containing an anti -human IgA antibody bound to a marker;
(d) detecting the presence of the said marker in the solid phase obtained in stage (c) .
2. Method according to Claim 1, characterized in that the said marker is an enzyme.
3. Method according to Claim 2, characterized in that the said enzyme is alkaline phosphatase.
4. Method according to one of the preceding claims, characterized in that the said antigen which reacts with anti-endomysial antibodies is obtained from animal tissue .
5. Method according to Claim 4, characterized in that the said antigen which reacts with anti-endomysial antibodies is obtained from human umbilical cord tissue.
6. Method according to any one of the preceding claims, characterized in that the said anti -human IgA antibody bound to a marker is a rabbit IgG anti -human IgA antibody.
7. Diagnostic kit for carrying out the method according to the preceding claims, characterized in that it comprises the following components:
(a) a plate constituting a solid phase
(b) a liquid phase containing an antigen which reacts with anti-endomysial antibodies; (c) an anti -human IgA antibody, bound to a marker, in aqueous phase;
(d) a number of sera from healthy individuals;
(e) a number of sera from individuals suffering from celiac disease.
8. Diagnostic kit according to Claim 7, characterized in that it comprises washing buffers in aqueous phase .
9. Diagnostic kit according to Claim 7 or 8 , characterized in that the said marker is an enzyme.
10. Diagnostic kit according to Claim 9, characterized in that the said enzyme is alkaline phosphatase.
11. Diagnostic kit for carrying out the method according to Claims 1 to 6, characterized in that it comprises the following components:
(a) a plate constituting a solid phase, having an antigen which reacts with the anti-endomysial antibodies bound thereto;
(c) an anti-human IgA antibody, bound to a marker, in aqueous phase;
(d) a number of sera from healthy individuals;
(e) a number of sera from individuals suffering from celiac disease.
12. Diagnostic kit according to Claim 11, characterized in that it comprises washing buffers in aqueous phase .
13. Diagnostic kit according to Claim 11 or 12, characterized in that the said marker is an enzyme.
14. Diagnostic kit according to Claim 13, characterized in that the said enzyme is alkaline phosphatase.
PCT/EP1997/005420 1996-10-04 1997-10-02 Assay for determining anti-endomysial antibodies WO1998015836A1 (en)

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IT96MI002054A IT1285775B1 (en) 1996-10-04 1996-10-04 TEST FOR THE DETERMINATION OF ANTI-ENDOMISIO ANTIBODIES
ITMI96A002054 1996-10-04

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008976A3 (en) * 2001-07-21 2003-09-25 Univ Nottingham Trent Kit, zugehörige reagentien und methode zur diagnose von zoeliakie

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
D'CRUZ D P ET AL: "ANTIBODIES TO ENDOTHELIAL CELLS IN SYSTEMIC LUPUS ERYTHEMATOSUS: A POTENTIAL MARKER FOR NEPHRITIS AND VASCULITIS", CLINICAL AND EXPERIMENTAL IMMUNOLOGY, vol. 85, 1991, pages 254 - 261, XP000196751 *
LADINSER B ET AL: "ENDOMYSIUM ANTIBODIES IN COELIAC DISEASE: AN IMPROVED METHOD", GUT, vol. 35, 1994, pages 776 - 778, XP000196760 *
UNSWORTH: "Serological diagnosis of gluten sensitive eneteropathy", JOURNAL OF CLINICAL PATHOLOGY, vol. 49, no. 9, September 1996 (1996-09-01), LONDON, pages 704 - 711, XP002054388 *
VECCHI ET AL.: "A novel immunoenzymatic method for the detection of anti-endomysial antibodies using human umbilical cord tissue", GASTROENTEROLOGY, vol. 112, no. 4, April 1997 (1997-04-01), PHILADELPHIA, PA, pages A1113, XP002054389 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008976A3 (en) * 2001-07-21 2003-09-25 Univ Nottingham Trent Kit, zugehörige reagentien und methode zur diagnose von zoeliakie
US7575871B2 (en) 2001-07-21 2009-08-18 Aston University Method of diagnosis and kit of parts therefor

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IT1285775B1 (en) 1998-06-18

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