WO2019119087A1 - Kit e processo para detecção de imunoglobulinas e e imunoglobulinas g1 - Google Patents
Kit e processo para detecção de imunoglobulinas e e imunoglobulinas g1 Download PDFInfo
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- WO2019119087A1 WO2019119087A1 PCT/BR2018/050219 BR2018050219W WO2019119087A1 WO 2019119087 A1 WO2019119087 A1 WO 2019119087A1 BR 2018050219 W BR2018050219 W BR 2018050219W WO 2019119087 A1 WO2019119087 A1 WO 2019119087A1
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- WIPO (PCT)
- Prior art keywords
- immunoglobulins
- food
- pbs
- saliva
- allergy
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Definitions
- the present invention relates to the detection of specific immunoglobulins E (IgE) and immunoglobulins G1 (IgG1) in biological samples using the indirect ELISA technique.
- the present invention is in the field of medicine and pharmacy, nursing, nutrition, biomedicine and bioengineering, more specifically, biological material analysis.
- AA Food allergy
- AA is more common in children, it is estimated that a prevalence of approximately 6% to 8% in children under three years and of 3.5% in adults. This higher prevalence in childhood may be mainly because the immune system does not present complete maturation (Batista et al., 2009;
- the determination of total IgE is usually required in the investigation of allergy, but numerous factors (genetic predisposition, sex, age, infections, pollution and smoking) contribute to the variation of the serum IgE level and must be considered for its correct interpretation .
- the serum level of total IgE is detected by enzyme-linked immunosorbent assay (ELISA) showing quantitative results and is variable according to age, being undetectable in the neonate and reaching maximum concentrations in young adults (ASBAI, 2009).
- the elevated IgE level does not indicate the presence of allergic disease, since it only evaluates its momentary quantity and does not specify the antigen causing the manifestations.
- the high IgE results may also be associated with intestinal or cutaneous parasitoses, myeloma, hyper IgE syndrome, among others (BOYCE et al, 2010).
- US201154401 uses body fluids to diagnose food allergy by means of a KIT called FIXANAL which detects the presence of IgE and IgG immunoglobulins. Unlike this document, the present invention discloses a process for investigating food allergy using crude extracts of the foods to be investigated by the ELISA technique which detects the presence of IgE and IgG1 immunoglobulins in biological samples from a patient.
- Document RU2442980 uses IgA immunoglobulin in saliva after 5 days of contact with the allergen to diagnose food allergy by the ELISA technique. Differently from this document, this In the present invention, IgE and IgG1 immunoglobulins are detected in the saliva by the ELISA technique, and examination can be performed on the same day of contact with the allergen.
- Document PI0114343-3 discloses the diagnosis of food allergy by detecting IgE and IgG in the blood of patients using the microarray technique. Unlike this document, the present invention provides a process for detecting IgE and IgG1 from saliva or serum using the ELISA technique and crude extract from the investigated foods.
- WO2016094961 discloses the diagnosis of food allergy through the Methylation Patter technique which detects methylated regions / genes in the DNA of various body fluids, having saliva as one of them.
- This patent does not carry out immunoenzymatic assays and therefore does not detect immunoglobulins, which makes it different from the present invention, which provides an investigation of food allergy by detecting IgE and IgG1 immunoglobulins in saliva and serum of the patient using the technique of ELISA and the crude extract of the food investigated.
- US2014315749 discloses the diagnosis of food allergy through the detection of B lymphocytes in body fluids using the flow cytometry technique.
- the present invention provides the investigation of food allergy in patients by detecting IgE and IgG1 immunoglobulins in saliva using crude extract from the investigated feed and the ELISA technique.
- the present invention aims to solve the known problems in the state of the art from the development of the development of a method to detect food-specific IgE and IgG1 immunoglobulins involving the Enzyme Linked Immuno Sorbent Assay (ELISA) in a non-invasive, more reliable in their results, fast results, in a single test can be tested various foods.
- ELISA Enzyme Linked Immuno Sorbent Assay
- the method described does not require the patient to be fasted and can be applied at any age.
- the present invention describes a kit for detecting immunoglobulins E and immunoglobulins G1, comprising (1) a saliva collector, (2) components for preparing a food extract;
- said component (2) should have at least 1X PBS, wherein the 1X PBS is composed of sodium chloride, dibasic potassium phosphate, dibasic sodium phosphate and distilled water.
- the present invention describes a method for the detection of immunoglobulins E and immunoglobulins G1 carried out in an indirect enzyme-linked immunosorbent assay (ELISA) having results collected and comprising the components:
- an antigen in said antigen is obtained from a food or medicine which may cause allergies;
- said biological sample selected from serum or saliva
- the analysis of the results involves an analysis of the absorbances obtained at the end of the indirect immunoenzymatic assay performed to determine the values of immunoglobulins E and immunoglobulins G1,
- cut-off point is defined by a statistical analysis of the sum of the mean absorbance of the negative control samples, with addition of three times the standard deviation.
- inventive concept common to all claimed protection contexts is the detection of immunoglobulins E and G1 immunoglobulins, for the investigation of allergic food reactions.
- Figure 1 shows an Enzyme linked immunosorbent assay (ELISA) with serum of allergic and non-allergic children at the 1: 100 dilution, revealing the presence of IgE for food proteins (maize, papaya, cow, egg white, soy, peanut) in the group of allergic patients when compared to non-allergic patients.
- ELISA Enzyme linked immunosorbent assay
- Figure 2 shows an Enzyme linked immunosorbent assay (ELISA) with serum of allergic and non-allergic children at a 1: 100 dilution, revealing the presence of IgG1 for food proteins (maize, papaya, cow's milk, egg white, soy, peanut) in the group of allergic patients when compared to non-allergic patients.
- ELISA Enzyme linked immunosorbent assay
- Figure 3 shows an Enzyme linked immunosorbent assay (ELISA) with saliva from allergic and non-allergic children at the dilution of 1: 100, revealing the presence of IgE for food proteins (corn, papaya, cow, egg white, soy, peanut) in the group of allergic patients when compared to non-allergic patients.
- ELISA Enzyme linked immunosorbent assay
- Figure 4 shows a comparison of the Enzyme linked immunosorbent assay (ELISA) with serum and saliva of children at a 1: 100 dilution, revealing the presence of IgE in the food proteins (corn, papaya, cow's milk, egg white, soy, peanut) in the group of allergic patients.
- ELISA Enzyme linked immunosorbent assay
- Figure 5 shows a comparison of the Enzyme linked immunossorbent assay (ELISA) with serum and saliva of children at a 1: 100 dilution, revealing the presence of IgG1 for food proteins (corn, papaya, cow's milk, egg white, soy, peanut) in the group of allergic patients.
- ELISA Enzyme linked immunossorbent assay
- Figure 6 shows a comparison of the Enzyme linked immunosorbent assay (ELISA) with serum at 1: 100 dilution, revealing the presence of IgE and IgG1 specific for food proteins (maize, papaya , cow's milk, egg white, soy, peanut) in the group of allergic patients.
- ELISA Enzyme linked immunosorbent assay
- Figure 7 shows a comparison of the Enzyme linked immunosorbent assay (ELISA) with saliva from children at a 1: 100 dilution, revealing the presence of specific IgE and IgG1 for food proteins (maize, papaya , cow's milk, egg white, soy, peanut) in the group of allergic patients
- ELISA Enzyme linked immunosorbent assay
- the present invention relates to a process which uses a sample biological samples collected from human candidates for the investigation of specific food allergy markers from laboratory tests. It is noteworthy that once the biological sample is collected, the process described herein does not involve any other in vivo step. More precisely, the present invention describes the development of a method for detecting immunoglobulins E (IgE) and immunoglobulins G1 (IgG1) from biological samples of human candidates, preferably using saliva as a biological material. The objective of the process is to promote the investigation of food allergic reactions through the presence of IgE and IgG1, using crude food extract, collected saliva and the ELISA technique.
- IgE immunoglobulins E
- IgG1 immunoglobulins G1
- the biological samples collected in the present invention may be diverse. Since for this purpose various methods of collecting material already known in the art can be used in the present invention.
- the biological material used in the present invention is saliva.
- the present invention provides a solution with respect to a process for investigating food allergy by detecting specific IgE and IgG1 immunoglobulins for certain foods. Since the determination of total IgE, used in existing diag- (genetic predisposition, sex, age, infections, pollution, and smoking), which does not make the results reliable. In addition, current diagnostic tests, except for oral challenge, can only detect immunoglobulin E by diagnosing only one type of allergy (IgE-mediated), or only diseases associated with its elevation of serum intestinal or cutaneous parasitoses, myeloma, hyper- -lgE, among others.
- the present invention will be more reliable as it is the detection of specific immunoglobulins, in addition to being able to diagnose the three types of existing allergies (IgE-mediated, non-IgE-mediated and mixed) through the detection of lgG1 immunoglobulin, which is present in allergic individuals in general and do not suffer variations with external factors.
- the technology used in the present invention is the enzyme-linked immunosorbent assay (ELISA) already used today because it is a rapid result. In the present invention, with only one test it is possible to investigate various foods at the same time.
- the extract of food used may be in its raw form or lyophilized.
- the test can also be used to diagnose allergic reactions caused by medications (antibiotics such as penicillin, dipyrone, etc.).
- the present invention describes a diagnostic kit, skilled at production on an industrial scale, to enable the test to be present in hospitals and even clinics and Health Units, as the present invention will be self-explanatory and can be performed by health professional, which facilitates the diagnosis of food allergy in the population.
- the kit comprises a biological material collector (1) which aims to collect the saliva or serum for analysis.
- Said biological material collector (1) may have numerous shapes and structures.
- the biological material collector (1) is a saliva collector.
- the kit further comprises a component for the preparation of the antigen (2), the function being to treat a food extract or medicament to provide the antigens present.
- the antigen preparation component (2) is preferably a PBS solution, more preferably a 1X PBS solution, said solution being distinguished by comprising sodium chloride, potassium phosphate dibasic sodium phosphate, and distilled water.
- the present invention describes a kit for detecting immunoglobulins E and immunoglobulins G1, comprising collecting biological material (1), (component for the preparation of the antigen (2);
- said component (2) comprises a PBS providing antigens from a food extract, or an X providing antigens from a medicament.
- the kit may be used in conjunction with an indirect enzyme-linked immunosorbent assay (ELISA) to enable the detection of immunoglobulins E and immunoglobulins G1.
- ELISA enzyme-linked immunosorbent assay
- KIT stands out to be used for the execution of the process for the detection of immunoglobulins E (IgE) and immunoglobulins G1 (IgG1).
- IgE immunoglobulins E
- IgG1 immunoglobulins G1
- the present invention describes a process for the detection of immunoglobulins E and immunoglobulins G1 carried out in an indirect enzyme-linked immunosorbent assay (ELISA), the results of which are collected and comprising the components:
- an antigen in said antigen is obtained from a food or medicine which may cause allergies;
- said biological sample selected from serum or saliva
- the analysis of the results involves an analysis of the absorbances obtained at the end of the indirect immunoenzymatic assay performed to determine the values of immunoglobulins E and immunoglobulins G1,
- cut-off point is defined by a statistical analysis of the sum of the mean absorbance of the negative control samples, with addition of three times the standard deviation.
- the allergen is a food extract selected from: cow's milk, corn, soy, peanut, egg white, and papaya.
- the step of preparing the indirect ELISA immunoenzymatic assay comprises the step of sensitizing the indirect ELISA immunoenzymatic assay with allergen extracts.
- the process comprises the following steps:
- step viii repetition of step vii twice;
- step ix the rest of the material obtained in step ix for 1 h at
- step xii repetition of step xi twice;
- the present invention is notable for having a non-invasive technique, which detects specific antigens, more reliable in its results, fast results, in a single test can be tested various foods, easy access and low cost. Also, it does not require fasting and can be applied at any age. When compared to the current test, these characteristics are not found.
- Saliva samples were collected with a disposable plastic pipette in the morning, not requiring fasting. After the collection, the materials were placed in plastic tubes of 1, 5mL properly closed, identified and taken to a styrofoam containing ice. The saliva samples were centrifuged for 10 minutes and supernatants removed for then storing them at 20 Q C until analyzes.
- the protein extracts were obtained from foods previously macerated and centrifuged to obtain the supernatant, containing the proteins soluble in phosphate buffer pH 7.4. Proteins from the supernatant were precipitated with 10% acetone TCA (trichloroacetic acid). The protein precipitate was resuspended in phosphate buffer pH 7.4 and used for plate sensitization
- the indirect Enzyme Linked Immunosorbent Assay (ELISA) technique was used to evaluate the levels of IgE and IgG1 present in serum and saliva of allergic and non-allergic patients.
- ELISA Enzyme Linked Immunosorbent Assay
- the plates were sensitized with the extracts of the food under study (5 pg / well). All antigens were diluted in 50 mM sodium carbonate buffer, pH 9.6, using 100 mI in each well.
- the plates were incubated overnight at 4 ° C. The plates were then washed with PBS (NaCl, KH 2 PO 4 , Na 2 HPO 4 .12H 2 O and KCl) -Tween (0.05%) and blocked for 2 h at 37 ° C with 200 pL of 1% PBS gelatin. After discarding the blocking solution, the plates were incubated with samples of serum and saliva of the patients also diluted in PBS gelatine in the ratio 1: 100 pg / ul, where they remained for 2 hours at 37 Q C according to the methodology cited by Paschoal (2010).
- PBS NaCl, KH 2 PO 4 , Na 2 HPO 4 .12H 2 O and KCl
- the absorbance averages of the blood and saliva samples of the control group served to establish the cutoff, which was calculated by adding the average to three times the standard deviation, according to Oliveira et al. (2006).
- the values found above cuttof were considered positive for food allergy.
- Serum and saliva from allergic and non-allergic children were collected and tested with the food extracts produced.
- the sample studied was composed of children being allergic to cow's milk protein (APLV) and lacking allergic symptoms.
- the study population consisted of 55% of boys and 45% of girls with a mean age of 14.7 months ( ⁇ 1.1 months) and the diagnosis of APLV at 3.6 months ( ⁇ 4.1 months).
- the symptoms presented were diarrhea, urticaria, abdominal distension, vomiting, bleeding, angioedema, atopic dermatitis, low weight gain and abdominal pain.
- ABBAS Abul K .; LICHTMAN, Andrew H .; PILLAI, Shiv. Cellular and Molecular Immunology. ⁇ -edition. Rio de Janeiro, Elsevier: 2012.
- BRAZILIAN ASSOCIATION OF ALLERGY AND IMMUNOPATHOLOGY AND BRAZILIAN SOCIETY OF FOOD AND NUTRITION A practical guide to the diagnosis and treatment of Cow's Milk Protein Allergy mediated by immunoglobulin E. Rev. bras. allerg. immunopatol. 2012; 35 (6).
- BRAZIL Ministry of Health. Secretariat of Health Care. Department of Basic Attention. Guidelines for the collection and analysis of anthropometric data in health services: Technical Standard of the Food and Nutrition Surveillance System - SISVAN / Ministry of Health, Ministry of Health Health Care, Department of Primary Care. Bras ⁇ lia: Ministry of Health, 201 1. 76 p .: il. - (Series G. Statistics and Health Information).
- FRISANCHO A. R. Antropometric standards for the assessment of growth and nutrition status. Ann Arbor: The University of Michigan Press, 1990.
- HALKEN S. Clinical symptoms of food allergy / intolerance in children.
- SABR ⁇ A., DEL CASTILLO, R "SABR ⁇ , S” MADI, K. Food Allergy. In: Subjects of Pediatrics, 1995. n.59, page: 56, Nestlé.
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Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
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US20030087320A1 (en) * | 2001-08-14 | 2003-05-08 | Aristo Vojdani | Saliva test for detection of food allergy, candidiasis, microflora imbalance, intestinal barrier dysfunction and humoral immunodeficiencies |
US20030143627A1 (en) * | 2001-08-14 | 2003-07-31 | Aristo Vojdani | Saliva test for detection of food allergy and intolerance |
EP1322960B1 (en) * | 2000-10-03 | 2005-01-05 | VBC-Genomics Bioscience Research GmbH | Allergen-microarray assay |
US20070122840A1 (en) * | 2003-05-08 | 2007-05-31 | Cousins Peter D G | Assay panel comprising food allergens |
JP2008164523A (ja) * | 2006-12-28 | 2008-07-17 | Nippon Medical Soken:Kk | 唾液を利用したアレルギー診断薬 |
US20090253154A1 (en) * | 2008-04-02 | 2009-10-08 | Immunosciences Lab., Inc. | Blood and saliva test for detection of delayed food allergy and intolerance against modified foods |
WO2010114912A1 (en) * | 2009-03-31 | 2010-10-07 | University Of North Carolina At Greensboro | Minimally invasive assessment of ige mediated allergy |
US20110195401A1 (en) * | 2010-02-10 | 2011-08-11 | Selinfreund Richard H | Systems and methods for determining food allergies |
US20160320403A1 (en) * | 2015-01-30 | 2016-11-03 | Aristo Vojdani | SIMULTANEOUS CHARACTERIZATION OF IgG AND IgA ANTIBODIES TO MULTIPLE FOOD ANTIGENS AND C1Q-FOOD PROTEIN IMMUNE COMPLEXES |
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2017
- 2017-12-20 BR BR102017027544-2A patent/BR102017027544A2/pt unknown
-
2018
- 2018-06-29 WO PCT/BR2018/050219 patent/WO2019119087A1/pt active Application Filing
- 2018-06-29 BR BR112020012702-7A patent/BR112020012702A2/pt unknown
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
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EP1322960B1 (en) * | 2000-10-03 | 2005-01-05 | VBC-Genomics Bioscience Research GmbH | Allergen-microarray assay |
US20030087320A1 (en) * | 2001-08-14 | 2003-05-08 | Aristo Vojdani | Saliva test for detection of food allergy, candidiasis, microflora imbalance, intestinal barrier dysfunction and humoral immunodeficiencies |
US20030143627A1 (en) * | 2001-08-14 | 2003-07-31 | Aristo Vojdani | Saliva test for detection of food allergy and intolerance |
US20070122840A1 (en) * | 2003-05-08 | 2007-05-31 | Cousins Peter D G | Assay panel comprising food allergens |
JP2008164523A (ja) * | 2006-12-28 | 2008-07-17 | Nippon Medical Soken:Kk | 唾液を利用したアレルギー診断薬 |
US20090253154A1 (en) * | 2008-04-02 | 2009-10-08 | Immunosciences Lab., Inc. | Blood and saliva test for detection of delayed food allergy and intolerance against modified foods |
WO2010114912A1 (en) * | 2009-03-31 | 2010-10-07 | University Of North Carolina At Greensboro | Minimally invasive assessment of ige mediated allergy |
US20110195401A1 (en) * | 2010-02-10 | 2011-08-11 | Selinfreund Richard H | Systems and methods for determining food allergies |
US20160320403A1 (en) * | 2015-01-30 | 2016-11-03 | Aristo Vojdani | SIMULTANEOUS CHARACTERIZATION OF IgG AND IgA ANTIBODIES TO MULTIPLE FOOD ANTIGENS AND C1Q-FOOD PROTEIN IMMUNE COMPLEXES |
Non-Patent Citations (2)
Title |
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BIRMINGHAM, N. ET AL.: "An ELISA-based method for measurement of food-specific IgE antibody in mouse serum: an alternative to the passive cutaneous anaphylaxis assay", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 275, no. 1-2, 2003, pages 89 - 98, XP004416754, DOI: doi:10.1016/S0022-1759(03)00008-5 * |
BOTTCHER, M. E. ET AL.: "Total and allergen-specific immunoglobulin A levels in saliva in relation to the development of allergy in infants up to 2 years of age", CLIN EXP ALLERGY, vol. 32, no. 9, 2002, pages 1293 - 1298, XP008147351, DOI: doi:10.1046/j.1365-2222.2002.01470.x * |
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BR102017027544A2 (pt) | 2021-11-09 |
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