WO2010049726A1 - Extraction de protéines, microréseau de protéines et son utilisation - Google Patents

Extraction de protéines, microréseau de protéines et son utilisation Download PDF

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Publication number
WO2010049726A1
WO2010049726A1 PCT/GB2009/051442 GB2009051442W WO2010049726A1 WO 2010049726 A1 WO2010049726 A1 WO 2010049726A1 GB 2009051442 W GB2009051442 W GB 2009051442W WO 2010049726 A1 WO2010049726 A1 WO 2010049726A1
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sample
protein
microarray
individual
proteins
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PCT/GB2009/051442
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English (en)
Inventor
Marcos Alcocer
Franco Harald Falcone
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The University Of Nottingham
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Priority claimed from GB0819666A external-priority patent/GB0819666D0/en
Priority claimed from GB0820487A external-priority patent/GB0820487D0/en
Application filed by The University Of Nottingham filed Critical The University Of Nottingham
Publication of WO2010049726A1 publication Critical patent/WO2010049726A1/fr

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention relates to a protein microarray and in particular to a method of obtaining proteins for use in a microarray, and to the use of a protein microarray.
  • the invention relates to the use of a protein microarray to determine the general immunoglobulin profile of an individual to proteins they have been exposed to, and in particular, for the in vitro diagnosis of an allergy in an individual.
  • An allergic reaction is a reaction produced by the body's immune response to a substance that would normally be considered to be harmless. Allergic reactions can be caused by a multitude of environmental factors, including certain food products, pollen, animal dander, drugs, other chemicals, insect venom, parasites and commensal bacteria.
  • Allergic reactions can cause a variety of symptoms, including, irritation of the nose, sneezing, itching and redness of the eyes, narrowing of the airways (bronchoconstriction) , increased production of mucus in the lungs, shortness of breath (dyspnea), coughing, wheezing, abdominal pain, bloating, vomiting, diarrhoea, itchy skin, swelling of the skin, cutaneous reactions, oedema, hypotension, anaphylaxis, coma, and even death.
  • the severity of the allergic response varies depending on the allergen and the individual.
  • Sera of immune competent individuals contain antibodies of the IgG, IgA, IgM, IgD and IgE immunoglobulin classes. Despite their very short half -life, average of 2.5 days for IgE and about 3 weeks for the other classes, the overall concentration of antibodies in serum is remarkably stable during late childhood- adulthood, with only small variations at old age. Regarding their origin, "natural" antibodies of the IgM, IgA and IgG3 subclasses are usually derived from Bl antibody-secreting cells. In general they possess a low affinity to multiple antigens, a basic requirement for their regulatory role in innate immunity, and are maintained throughout life.
  • the gut immune system is bombarded daily by a plethora of protein antigens present in our normal diet.
  • the expected outcomes from these exposures are mainly (i) induction of tolerance; (ii) systemic priming; or (iii) induction of local immunoglobulins. Breakdown or failure attributed to these responses result in yet not well understood immunological adverse reactions to foods, such as food allergy, food intolerance and inflammatory bowel diseases.
  • the present invention provides a method for determining the specific immunoglobulin profile of an atopic or a non-atopic individual, comprising
  • the sample is contacted with the microarray under conditions which will allow antibodies in the sample which are specific for proteins on the microarray to bind thereto.
  • the method can be used to detect more than one type of immunoglobulin.
  • the method of the invention may be used to detect the binding of one or more of IgG, IgA, IgM, IgE or IgD immunoglobulins or subclasses thereof.
  • the subclasses may be the subclasses of IgG, for example, IgGl, IgG2, IgG3 or IgG4 or subclasses of IgA, for example IgAl and IgA2.
  • the method of the invention allows at least two, preferably at least three, preferably at least four different immunoglobulin classes or subclasses to be detected.
  • Preferably more than one type of immunoglobulin can be detected simultaneously or separately.
  • antibody and “immunoglobulin” are used interchangeably herein.
  • the method of the invention may allow multiple potential allergens to be assayed at once, preferably the microarray includes at least 50, 100, 200, 300, 400 or more different proteins or protein samples. Accordingly, the method may be used in high throughput screening.
  • the proteins in the protein microarray may be proteins extracted from a foodstuff, parasite, aeroallergen, commensal bacteria or another source of potential environmental allergen. Alternatively the proteins may be synthetically produced or recombinantly produced.
  • the microarray may comprise a mixture of two or more of proteins wherein one or more of the proteins is extracted from an allergen is a synthetic protein or is a recombinant protein, or a mixture thereof.
  • the microarray may contain protein extracts representative of most of the food products/ingredients consumed in the UK. This may represent about 16 different groups of food such as spices, seeds, meat, fish and dairy amongst others. Such an array may be used to determine simultaneously the presence in the sample of immunoglobulins from two or more different immunoglobulin classes, or subclasses, for example, at least two of IgA, IgM, IgG and IgE, directed against one or more potential allergens.
  • allergens refers to a composition comprising a mixture of compounds including proteins which has the capacity to induce an immune response in an allergic individual.
  • the allergens are protein.
  • the immune response may cause the production of allergen specific antibodies, the antibodies may be one or more of IgG, IgA, IgD, IgM and IgE immunoglobulins or subclasses thereof.
  • the proteins in the microarray are immobilised on a nitrocellulose base slide (Fast slide) .
  • the sample is a sample of bodily fluid such as milk, saliva, blood or serum, preferably blood or serum.
  • the bound immunoglobulins may be detected by any suitable method.
  • the bound immunoglobulins are detected using labelled secondary antibodies or antibody fragments, specific to a particular immunoglobulin class.
  • FITC-labelled anti-IgA antibodies will allow the detection of bound IgA antibodies.
  • different classes of immunoglobulin bound to the microarray may be identified.
  • the labels may be detected by spectrophotoscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • the label may be selected from the group comprising biotin, a fluorescent dye such as fluorescein, rhodamine or the green fluorescent protein, a radiolabel, and an enzyme such as horseradish peroxidise.
  • secondary antibodies directed to different immunoglobulin classes have different labels, for example, different fluorescent labels.
  • An anti-IgG antibody may have a rhodamine label
  • an anti-IgE antibody may have a GFP label
  • an anti-IgA antibody may have a fluorescein label.
  • the secondary antibodies are fluorescently labelled they can be detected using a spectrophotometer which is capable of measuring at more than wavelength at the same time, for example has two or more lasers. This will allow rapid throughput and screening.
  • the present invention provides a protein microarray comprising at least one extract of protein sample from at least 100 different foodstuffs, preferably at least 200, preferably at least 300, preferably at least 400 different foodstuffs.
  • the one or more protein extracts for each foodstuff are preferably crude extracts of the foodstuffs.
  • the microarray may also comprise protein extracts obtained from human parasites, or bacteria, such as pathogenic and/or commensal bacteria.
  • the microarray may further comprise proteins obtained from pollen, animal dander and insect venom, preferably for each foodstuff, bacteria or other potential allergen included in the array there is more than one protein extract.
  • an immunoglobulin profile contains information related to the classes/subclasses of immunoglobulin's in a sample to which are proteins or protein extracts in the microarray. This profile may then be used to diagnose whether an individual is allergic to a specific potential allergen or a family of allergens or their degree of susceptibility to certain diseases, such as inflammatory bowel disease, Crohn's disease or other gut related immunodeficiency.
  • the immunoglobulin profile of an individual provides information on the presence of one or more immunoglobulin specific to one or more protein sample.
  • the immunoglobulin profile contains information about two or more, three or more, four or more, five or more, six or more, seven or more different immunoglobulin classes or subclasses.
  • the immunoglobulin classes or subclasses are selected from the group comprising IgA, IgG, IgM, IgE, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • the method of the invention also has the advantage that small sample volumes may be used. Much smaller than the sample volumes needed in most commercially available tests.
  • the method of the invention may be performed on samples, for example of blood or serum, as small as lOO ⁇ l, 50 ⁇ l, 25 ⁇ l or less.
  • the protein microarray system has the advantage that the results are highly reproducible.
  • the method of the invention may be used to diagnose an allergy in an individual or to obtain the immunoglobulin profile of an individual.
  • the invention provides a method of obtaining protein extracts from a sample, said method comprising the steps of a) obtaining a sample; b) homogenising the sample; c) adding a first buffer comprising a detergent to the sample; d) fractionating the product of c) to recover the extracted proteins; e) adding a second buffer comprising a chaotropic agent to the sample remaining after step d) . f) fractionating the product of e) to recover the extracted proteins.
  • the sample is preferably a sample of a potential allergen.
  • the sample may be of a food product, or human parasites, or it may be bacterial sample, or a pollen sample, or an insect venom sample, or a sample of animal dander, or a sample of any other potential allergen.
  • the sample may be freeze dried before use, and may be stored frozen before use.
  • the sample may be subjected to hexane extraction prior to step b), this may be required if the food sample contains 25% or more lipid.
  • the sample may be homogenised by mechanical grinding, this may be automatic or manual, for example, with a pestle-and-mortar.
  • the detergent in the first buffer may be a non-ionic, an anionic, or a zwitterionic detergent.
  • the buffer comprises about 0.05 to 3% w/v of detergent.
  • the detergent is Triton X, preferably the Triton X is used at a concentration of 0.5% w/v.
  • the second buffer preferably comprises a chaotropic agent at a concentration of about 4 to 8M.
  • the chaotropic agent is urea, preferably the urea is used at about 6M.
  • the first and second buffer may also comprise one or more of the following: PBS; dithiothreitol; glycerol and phenylmethylsulfonyl fluoride (PMSF) or any other protease inhibitor.
  • PBS dithiothreitol
  • glycerol glycerol
  • PMSF phenylmethylsulfonyl fluoride
  • the PBS is used at a concentration of 0.5X or IX PBS (wherein 0.5X PBS is 69 mM NaCl, 5mM Phosphate, 1.35 mM KCl, pH 7.4; and IX PBS is 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4).
  • IX PBS is used if the food sample contains meat.
  • the first and/or second buffer contain dithiothreitol, it is used at a concentration of ImM dithiothreitol.
  • dithiothreitol Preferably if glycerol is used, it is used at a concentration of 20% w/v glycerol.
  • PMSF Preferably if PMSF is used, it is used at a concentration of ImM PMSF.
  • steps c) and e) Preferably in steps c) and e) at least some of the protein is solubilised.
  • the sample and buffer are mixed to solubilise the proteins to allow them to be extracted.
  • the sample and buffer are mixed by applying a mechanical shear force. This may be achieved by using ball bearings, or a pestle, or by mechanical grinders.
  • the mechanical mixing is performed at a temperature of at least about 5 0 C or less than room temperature.
  • the mechanical mixing step may be carried out only once, or it be repeated several times.
  • the sample and buffer are subjected to mechanical mixing twice.
  • the sample comprises the pellet remaining after at least some of the protein solubilised in step c) has been recovered by the fractionation in step d) .
  • the solubilised protein is extracted by recovering the supernatant.
  • fractionated sample containing the protein extract is further clarified by centrifugation. This may be achieved by centrifugation at about 13000g for about 20 minutes.
  • the fractionated sample containing the protein extract may be further purified by passing it through a filter.
  • a filter Preferably a 0.45 ⁇ m filter is used.
  • the fractionated sample may be further enriched in its protein content by using anion exchange chromatography, for example, by using a Q-sepharose column.
  • anion exchange chromatography for example, by using a Q-sepharose column.
  • the solubilised protein is recovered by fractionating the sample and retaining the supernatant containing the soluble protein extract, at the end of the process the insoluble pellet is preferably discarded.
  • extracts of protein are recovered after some or all steps of the method.
  • Protein recovered after some or all steps of the method may be used to make the microarray of the invention.
  • the protein extracts recovered in the method of the invention are used to produce a microarray.
  • the total volume of the extract used may be in the range of nanolitres.
  • the invention provides a protein microarray comprising extracts of protein obtained from at least 10, 20, 30, 40, 50, 60, 100, 150, 200, 250, 300, 350, 400 or more potential allergens. More than one extract from each potential allergen may be included in the array.
  • the potential allergens may be food stuffs, human parasites, bacteria, dander, venom or any other protein containing potential allergen.
  • the proteins in the protein microarray may be extracted from the potential allergen using the aforementioned method of the invention, and then used to produce a protein microarray according to the invention.
  • the invention provides a protein microarray comprising at least one protein extracted according to the aforementioned method of the invention.
  • the protein microarray comprises at least 10, 20, 30, 40, 50, 60, 100, 150, 200, 250, 300, 350, 400 or more protein extracts obtained using aforementioned method of the invention.
  • the invention provides a method for diagnosis of an allergy or for immunoglobulin profile comprising combining basophil degranulation with a protein microarray according to the invention.
  • the basophile degranulation is performed as described in Lin et al in Clinical and Experimental Allergy (2007) 37, 1854-1862, modified to use the microarray of the invention.
  • the method of this aspect of the invention comprises incobating basophils isolated from a patient, or basophils which have been stripped of immunoglobulins and resensitised with the serum of a patient, with a protein microarray and then determining where on the array the basophils bind, and using this data to produce on immunoglobulin profile and/or to determine the allergic status of the patient.
  • the invention provides a kit which comprises a microarray according to the invention and instructions to use the kit.
  • the kit also includes at least one immunoglobulin detecting reagent.
  • the immunoglobulin detecting reagent is preferably a secondary antibody (an antiimmunoglobulin antibody), specific to a particular class and/or subclass of immunoglobulin .
  • the kit also provides a control known antibody at a known concentration which is specific for a control protein on the microarray, preferably this can be used as a positive control when using the kit.
  • the kit may also include a second known antibody, again at a known concentration, which does not bind to any of the proteins in the array, this may be used as a negative control.
  • the kit includes a reagent for detecting the first known antibody, and/or a reagent for detecting the second known antibody.
  • Figure 1 - shows the SDS-PAGE profile of a crude extract containing either triton/urea before sample loading, compared to elution fractions from MonoD ⁇ and MonoQ -Sepharose columns, using cod extracts as an example.
  • MonoQ-Sepharose was found to have superior performance for all food extracts in terms of binding and elution yields.
  • Figure 2 - shows immunoglobulin-specific IgA (A), IgM (B), IgG (C),
  • Figure 3 - illustrates the relative immunoglobulin cross reactivity of a panel of human sera to food products.
  • the patients are designated: (1)
  • Figure 4 - illustrates the correlations between immunoglobulin classes within the human serum panel.
  • the correlation values have been mapped to colours from blue ⁇ 0 to red ⁇ 0.8 (see colourbar on the right).
  • the vertical axis denotes for which patient and the horizontal axis for which array the correlation has been calculated.
  • Figure 6 - shows a comparison of total responses for IgG, IgE, IgA and IgM, grouped based on food type, as a comparison between non- atopic (black) and atopic (white) patients. Significance for each immunoglobulin is shown within each section by an asterisk.
  • Figure 7 - shows the results of competition of a food-specific immunoglobulin-A (non-atopic mother) micro-ELISA to food extracts of macadamia and sour cream. Plates were coated with approximately lO ⁇ g/ml of food extract. Food extract along with negative control proteins (PBS, BSA, rSFA8) were added to various diluted samples from the non-atopic mother to allow competition between binding immunoglobulins with supplemented proteins.
  • PBS negative control proteins
  • Figure 8 - shows the partial least square (PLS) results for prediction of UniCAP grade by IgE.
  • PLS partial least square
  • Figure 9 - shows cluster analysis of the four classes of immunoglobulins and the serum samples (15). The main classes of food products are indicated.
  • Control antibodies (IgG, IgM and IgA) were purchased from Calbiochem (Merck Biosciences, UK); IgE was purchased from Abd (Serotec, UK) and control serum from the ELISA IgA/IgG quantitation kits (Bethyl, Texas, USA). All other food samples were produced by the method outlined below. Nitrocellulose coated glass slides (FAST slides) were supplied by Whatman Schleicher & Schuell (Dassel, Germany). If not otherwise stated, all chemical reagents are from Sigma UK.
  • the antibodies were purchased from: Cy3-conjugated Goat anti-human IgA (Jackson Immuno Reseach, Baltimore, USA), Alexa 488-conjugated Goat anti-human IgM (Molecular Probes Europe BV, Holland), biotinylated Goat anti-human IgE (Vector Laboratories, Burlingame, USA), Goat anti-human IgG (Zymed, USA) was labelled in-house using Alexa 594 Protein Labelling kit (Molecular Probes) .
  • Caper Carrot, Cauliflower, Grapefruit (Red), Guava, Fennel Seed, Fragrance spice,
  • Mushroom Mushroom (Oyster), Pumpkin, Raisins,
  • Cannalini Bean (Cooked), Chestnut, Hazelnut, Oats, Pasta, Pearl Barley,
  • Table 1 List of all food ingredients (about 400) included in the "all-diet" protein microarray.
  • Sample Processing Food samples were freeze-dried and then immediately returned to a freezer in sealed airtight containers in the presence of silica gel to avoid rehydration. Batches of samples were then homogenised based on food type. Some food samples were amenable to mechanical grinding either by hand, or in a pestle-and- mortar whilst others, whose lipid levels were more than 25% (meat, fish, nuts and some seeds), required a hexane extraction prior to grinding.
  • the proteins were extracted using two buffers (A & B) containing 0.5x Phosphate Buffered Saline, (0.5xPBS- 69 mM NaCl, 5mM Phosphate, 1.35 mM KCl, pH 7.4); for a meat sample Ix PBS was used (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4) .
  • Both buffers also contained ImM dithiothreitol, 20% (w/v) glycerol (both from Sigma, UK) and ImM phenylmethylsulfonyl fluoride (PMSF - Perbio Science, UK). PMSF was added just prior to use due to its relatively short half- life.
  • Buffer A contained 0.5% w/v Triton-100 (Sigma, UK) and Buffer B 6M urea (Fisher, UK). Protein were extracted mechanically using a Mixer/Miller 8000 (Glen Creston Ltd, UK) and 1.2ml 96-well format 8-strip microtubes (Starlab, UK), each containing three 3mm stainless steel ball bearings (Midland Bearings Ltd, UK) and either (1.2ml) of Buffer A or B. The conditions were optimised to avoid generation of excessive heat, i.e. a short time (10 min) and temperatures of 5°C below room temperature prior to and after milling. After two rounds of extractions and centrifugations (MSE Mistral 300Oi, Sanyo, UK) 250Og - 10 minutes with Buffer A, supernatants were collected and the pellet re- extracted twice with Buffer B using the same procedure.
  • the slides were then washed three times for 2 minutes in PBS containing 0.05% (w/v) Tween-20, followed by five times 60 seconds washes with purified water, and finally dried by centrifugation (MSE Mistral 3000i, Sanyo, UK) at 30Og for ten minutes at room temperature.
  • the mothers were skin tested to a panel of allergens: cow's milk, hen's egg, house dust mite, cat dander, grass pollen mix, tree pollen mix, dog dander, Alternaria, Aspergillus on the velar surface of the forearm using a 1 mm lancet (Soluprick, ALK, Denmark). Saline was used as a negative control and histamine (10 mg/ml) as positive control. Skin prick tests were considered positive if larger than 3mm.
  • Microarray hybridizations were carried out using a Lucidea Slide Pro Hybridiser (Amersham, UK), optimised in-house for the protein array. Essentially the overall protocol involves multiple washes with PBS + T (0.2% w/v Tween-20) containing 1% BSA and incubations of diluted sera (1:15) in the same buffer for two hours at 26 0 C. After another round of washings, the second antibody was then added and incubated for a further 1 hour at 26 0 C. Final washes were carried out, three times with PBS + T 0.2% and five times with water for two and one minute duration respectively. The slides were then dried by centrifugation at 300g for 10 minutes.
  • the dried microarray slides were scanned in a four colour Axon 4200AL autoloader scanner (Axon Instruments, USA) with a PMT gain set to 100% and an adjustable PMT voltage and power (percentage variable) to vary detection sensitivity.
  • the maximal laser excitation wavelengths used were 488, 532, 594 and 635nm. While emission filters with a narrow bandwidth between 15-20nm as specified by the manufacturer (Axon Instruments) ensured a reduction in spillover from other fluorophores. Slides were scanned at lO ⁇ m resolution with
  • PLS Partial Least Squares Regression
  • Total human-IgA, IgG and IgE content were determined by ELISA using commercial kits (Bethyl Laboratories, USA) following the manufacturer's instructions and adapted to a 384 well scale.
  • Competitive micro ELISAs were carried out essentially as previously described (Lin J et al, International Archives of Allergy and Immunology 2006; 141: 91-102) with some modifications.
  • Micro- ELISA plates were coated with a range of food extracts (squid, sour cream, macadamia, egg white and others) diluted 1:100 in coating buffer, except for macadamia which was diluted 1:1000.
  • Serum was preincubated with a 1:10 serial dilution series of the competitive proteins from 0.0053 ⁇ g/ml to 500 ⁇ g/ml for one hour at RT.
  • the food extracts produced using these procedures were of a complex nature and were aimed to be representative of the natural exposure of the digestive tract to food products.
  • a chaotropic agent was used to solubilise proteins otherwise insoluble in the phosphate-detergent buffer.
  • Four ion exchange chromatographic resins were evaluated and MonoQ-Sepharose in particular demonstrated superior performance and yields at the physiological pH employed as shown by SDS- PAGE analysis ( Figure 1). After extraction, a filtration step (0.45 ⁇ m filter) was deemed preferable due to column fouling in subsequent chromatographic steps.
  • the food extracts were printed on the 3D-polymeric solid surface of FAST slides, previously shown (Lin J et al, Clin Exp Allergy 2007;37: 1854-1862; Pang S et al, Journal of Immunological Methods 2005;302: 1-12) to provide a stable matrix for protein arrays.
  • Several techniques were tried for the printing of the array with the food extracts amongst them split pins, solid pins and inkjet printing. The results presented here were all the product of an automated protocol using array slides printed using solid pins only.
  • spots of food extracts and purified fractions represented in the array were complex mixtures of proteins of discrete composition.
  • the protein array data are quantifiable and reproducible
  • PCA principal component analysis
  • Figure 5 includes the data for non-atopic volunteer control sera which is shown a triangles connected by solid lines.
  • Figure 5 shows that the IgE class has the greatest discrimination power between atopic and non-atopic subjects.
  • One interesting feature of the IgE cross reactivity concerned the pattern of response exemplified by patients PEI007, 016 and 023. These patients, clinically characterized as soy milk allergic subjects, were characterized in a previous study and did not cross-react with the ubiquitous major allergenic family of 2S albumins (Lin J et al, International Archives of Allergy and Immunology 2006; 141: 91-102). In the more comprehensive array used here, these three subjects clearly show a typical polyclonal response. This difference is even more accentuated if their IgE responses were compared to mono-specific subjects such as PEIOOl. Interestingly, IgG has also shown some degree of discrimination for some of the atopic sera.
  • the array binding data can be confirmed by ELISA Competitive ELISAs were performed to establish whether the antibodies detected to food extracts were specific rather than cross-reactive. Extracts ranging from squid, macadamia, sour cream and hen egg yolk were immobilized and used to confirm the specificity of various immunoglobulins to diluted food extracts. As exemplified in Figure 7 the strong positive array results obtained with total extracts of macadamia and sour cream were specifically competed out in an ELISA technique.
  • UniCAP classifications are negative (0) 0-0.35 kUA/L; borderline (1) 0.35-0.69 kUA/L; positive (2) 0.70-3.49 kUA/L; strong positive (3) 3.5-17.49 kUA/L; highly positive (4) 17.5-49 kUA/L; highly positive (5) 50-99 kUA/L; highly positive (6) > 99 kUA/L.
  • PEI 023 also returned very high UniCAP results for egg (yolk) (class 4), grass pollen (class 6) and hazelnut (class 5) . Both polyspecific patients showed a whole range of high IgE reactions in the microarray analysis ( Figure 3d, patients 14 and 15). Therefore, although only for few samples, the new UniCAP re-analysis did confirm the polyclonal nature of the PEIO 16/023 samples as suggested by the array. Some groups of food are recognized by all patients
  • the results presented here demonstrate that the method of the invention can be used to produce an individual's immunoglobulin profile.
  • the serum samples analysed in this work represented a well characterised cohort with a variety in reactivity, gender and age for which previous results were available.
  • the low UniCAP grade of some samples, although borderline for atopy, reflects the reality of many clinical practices.
  • the microarray of the invention has been shown to be versatile and able to profile simultaneously the four major immunoglobulins classes found in human sera from samples as small as a drop of blood from a finger prick. It is clear from the results that one of the great advantages of the technique described is a very small volume of sera is required for the analysis ( ⁇ 25 ⁇ l) .
  • the data presented shows that using the method of the invention it has been demonstrated that food specific IgA and IgM do correlate to each other and that IgE has a low correlation with any of the other immunoglobulin classes.
  • Specific food groups such as dairy /eggs and fish were recognized by most subjects by IgG and IgM respectively.
  • the array system has shown a great discrimination between atopic and non atopic individuals for IgE and surprisingly has shown a reduction in the overall IgG content for atopic patients. Poly and mono-specific IgE responders were easily identified.
  • the results show that the "all-diet" protein microarray can compete with present standard IgE techniques.
  • the array system possesses many advantages over traditional systems such as requirement of low sample volume, high sensitivity and a global view of the immune response.
  • the "all-diet" protein microarray is a viable and useful tool for the global profiling of the four major human food-specific immunoglobulins and their subclasses.
  • One advantage is the global view of the immune response, or "fingerprinting" of the immune response to the abundant food proteins which the microarray allows.
  • Another advantage is the requirement for low sample volumes. The inclusion in the array of proteins from parasites and commensal bacteria, or other allergens, would further expand this "fingerprinting" concept of the immune response. Due to its high sensitivity, the technique is also successful on some otherwise intractable samples.
  • the method of the invention may be used as a substrate for basophil degranulation, for maternal milk profiling, for post-mortem assessment and for investigating aspects of food preference, as well as allergy diagnosis.

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Abstract

La présente invention concerne un procédé permettant de déterminer le profil d’immunoglobuline spécifique d’un sujet atopique ou non atopique, comprenant: i) la mise à disposition d’un échantillon prélevé sur le sujet; ii) la mise en contact de l’échantillon avec un microréseau de protéines contenant des protéines ou des extraits de protéines provenant d’un ou de plusieurs allergènes potentiels; iii) la détection d’une ou de plusieurs immunoglobulines liées au microréseau. L’invention concerne également un procédé permettant d’obtenir des extraits de protéines à partir d'un échantillon, et des microréseaux de protéines associés, des trousses et des procédés correspondants.
PCT/GB2009/051442 2008-10-27 2009-10-27 Extraction de protéines, microréseau de protéines et son utilisation WO2010049726A1 (fr)

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GB0819666.9 2008-10-27
GB0819666A GB0819666D0 (en) 2008-10-27 2008-10-27 Microarray and use thereof
GB0820487.7 2008-11-08
GB0820487A GB0820487D0 (en) 2008-11-08 2008-11-08 Protein extraction, protein microarray and uses thereof

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AU2020233774B2 (en) * 2014-11-14 2021-11-25 Biomerica, Inc. Compositions, devices, and methods of ibs sensitivity testing
US10788498B2 (en) 2014-11-14 2020-09-29 Biomerica, Inc. IBS sensitivity testing
WO2016182835A1 (fr) * 2015-05-14 2016-11-17 Bioscreening And Diagnostics Llc Systèmes et procédés de prédiction de l'autisme avant le déclenchement de symptômes comportementaux et/ou de diagnostic de l'autisme
CN108593933A (zh) * 2018-05-03 2018-09-28 沈阳汇敏源生物科技有限责任公司 基于IgG1抗体检测食物过敏原的ELISA试剂盒
CN108593914A (zh) * 2018-05-03 2018-09-28 沈阳汇敏源生物科技有限责任公司 基于IgA抗体捕获食物过敏原的ELISA试剂盒
CN108845137A (zh) * 2018-05-03 2018-11-20 沈阳汇敏源生物科技有限责任公司 基于IgG1抗体捕获食物过敏原的ELISA试剂盒
CN108845135A (zh) * 2018-05-03 2018-11-20 沈阳汇敏源生物科技有限责任公司 基于IgA抗体检测食物过敏原的ELISA试剂盒
CN108845122A (zh) * 2018-05-03 2018-11-20 辽宁汇普源生物医学科技开发有限责任公司 基于IgG3抗体检测气传过敏原的ELISA试剂盒
CN108802359A (zh) * 2018-07-06 2018-11-13 沈阳汇敏源生物科技有限责任公司 基于IgG2抗体检测食物过敏原的ELISA试剂盒
CN108872597A (zh) * 2018-07-06 2018-11-23 沈阳汇敏源生物科技有限责任公司 基于IgD抗体检测食物过敏原的ELISA试剂盒
CN109283338A (zh) * 2018-09-21 2019-01-29 锦州医科大学附属第医院 基于IgD抗体捕获食物过敏原的ELISA试剂盒
CN109283336A (zh) * 2018-09-21 2019-01-29 锦州医科大学附属第医院 基于IgD抗体捕获气传过敏原的ELISA试剂盒

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