TARIFNAME BIYOLOJIK SIVILARDA DIAMIN OKSIDAZ AKTIVITESI TAYIN KITI Bulusun Ilgili Oldugu Alan Bulus, saglik alaninda kullanilmaya yönelik olup özellikle gida alerjisi ve/veya histamin intoleransinin tanisi ile ilgilidir. Diamin oksidaz enzimi histaminin parçalanmasini saglayan bir enzimdir. Bu enzimin aktivitesinin yetersiz veya bloke olmasi sonucunda, histamin içerigi yüksek olan besinler alindiginda histamin parçalanamaz ve birikir. Bulus, biyolojik sivilarda Diamin Oksidaz enzim aktivitesinin spektrofotometrik olarak hizli ve ucuz bir sekilde belirlenmesini saglamaktadir. Bulusla ilgili Teknigin Bilinen Durumu (Önceki Teknik) Diamin oksidaz (EC , üç ila alti karbon atomlu diaminlerin, bunlarin bazilarinin türevlerinin ve ikame bir siklik diamin olarak kabul edilebilecek histaminin oksidatif deaminasyonunu katalize eden bakir içeren bir enzimdir. Serum diamin oksidaz (DAO) aktivitesi belirlenmesi için kolorimetrik, fluorometrik, kromotografik yöntemler literatürde sunulmus ve bazillari için patent alinmistir. Literatürde kullanilan kolorimetrik tahlil, peroksidaz ve bir kromojen, 10-(karboksimetil-aminokarbonil)-3,7-bis(dimetilamino) fenotiyazin sodyum tuzu (DA-67) ile birlestirilmis bir reaksiyona dayanmaktadir. Peroksit ve DA-67 varliginda peroksidaz, 668 nm'de maksimum absorpsiyona sahip metilen mavisi olusumunu katalize eder ve spektrofotometrik ölçüm gerçeklestirilir. Ayrica ticari olarak kullanilan Diamin Oksidaz kitleri de mevcuttur. Piyasada satilan kitler, yayinlar ve patentler taranmis olup asagidaki tablolarda sunulmustur. Piyasada bulunan kitler 1. Diamine Oxidase Activity Assay Kit(Fluorometric) Sigma-Aldrich Katalog No: MAK351 Test Sayisi:100 assays Yöntem: fluorometrik Örnek tipi: doku lizatlari Diamine Oksidaz Aktivite Test Kiti, doku lizatlarinin DAO aktivitesinin yani sira rekombinant enzimlerin DAO aktivitesini belirlemek için 1 pmol/dakika aktiviteden daha az bir tespit limiti ile basit bir yöntem saglar. Tahlilde, DAO saglanan substrati dönüstürerek bir ara ürün ve HZOZ verir. HZOZ daha sonra DAO Enzim Karisimi tarafindan DAO Probundan floresan (Ex/Em = 535/587 nm) olusturmak için kullanilir. Fluorometrik yöntem olup fluorometri cihazina gereksinim duyar. 2. Diamine Oxidase Assay Kit ABNOVA.ABCAM Katalog No : ab241004 Test Sayisi:100 assay Ücret: E390.00 Yöntem: Fluorometrik Örnek tipi: Tükürük, Doku, Gida örnekleri Tahlilde, DAO saglanan substrati dönüstürerek bir ara ürün ve HZOZ verir. HZOZ daha sonra DAO Enzim Karisimi tarafindan DAO Probundan floresan (Ex/Em = 535/587 nm) olusturmak için kullanilir. Fluorometrik yöntem olup fluorometri cihazina gereksinim duyar. 3. Diamine oxidase (DAO) Activity Assay Kit Elabscience Katalog No: E-BC-K524-M Test Sayisi: 96 assays Yöntem: Kolorimetrik Örnek Tipi: serum, plazma, idrar, hayvan doku ve hücre örnekleri Diamin oksidaz, hidrojen peroksit üretmek için amin maddeleri katalize edebilir ve hidrojen peroksit, 460 nm'de karakteristik bir absorpsiyon zirvesine sahip olan kromojenik madde üretmek için kromojenik madde ile reaksiyona girebilir. Diamin oksidazin aktivitesi, birim zaman basina absorbans degisim hizi ölçülerek hesaplanabilir. Kullanilan kromojenik madde belirtilmemistir, ancak dalga boyuna göre o-dianisidin kullanilmis olmasi muhtemeldir. 4. Diamine Oxidase assay kit BioVision Katalog No : K496-100 Test Sayisi: 100 Tests Ücret: E663.00 Yöntem: Florometrik Örnek Tipi: Hücre kültürü kaynakli materyal ,Sinovyal sivi, idrar, Kikirdak, Bag dokusu / tümörler Bu radyoaktif olmayan, florimetrik Diamin Oksidaz Testi (DAO) testi, putresinin pirolin arti NH3 ve H202'ye oksidasyonuna dayanir. Üretilen HZOZ daha sonra HRP tarafindan bir boyayi floresan haline getirmek için oksitlemek için kullanilir. Ex/Em = 530/585 nm'de floresandaki artis, enzim aktivitesi ile dogru orantilidir. Fluorometrik yöntem olup fluorometri cihazina gereksinim duyar. PATENTLER 1 . Applicants: UNIV ANTWERPEN [BE]; UNI ZIEKENHUIS ANTWERPEN [BE] Enzim-afinite yöntemine dayalidir. Mast hücrelerinde ve/veya bazofillerde hücre içi histamin ve histamin salinimini saptamak ve ölçmek için kulanilir. Inventors: BRIDTS CHRIS [BE]; EBO DIDIER [BE] Classifications: IPC G01N; Hücre içi histamin ve histamin salinimini saptamak ve ölçmek için yöntem ve kit Mevcut bulus, histaminaz diamin oksidazin (DAO) lazerle uyarilabilir bir florokroma baglandigi bir enzim- afinite yöntemine dayali sitometri ile bireysel mast hücrelerinde ve/veya bazofillerde hücre içi histamin ve histamin salinimini saptamak ve ölçmek için yöntemler ve kitler saglar. Enzim afinite yöntemi olup bizim önerimizdeki kolorimetrik yöntemden tamamen farklidir. 2. Applicants JARISCH REINHARD DR [AT]; MISSBICHLER ALBERT DR [AT]; REICHL HERWIG MAG [AT] Classifications: C12Q; Bir diaminin siklizasyonuna ve ardindan ürünün etil asetat ile özütlenmesine dayanan, histamin intoleransinin saptanmasi için yararli olan diamino-oksidaz aktivitesini belirleme yöntemi. Diamino-oksidaz aktivitesinin belirlenmesi (DAO; EC 1.4.3.6). Diamino-oksidaz aktivitesinin belirlenmesi (DAO; EC içeren sulu bir solüsyonla reaksiyona sokulmasi, böylece bir amino grubu formile dönüstürüldükten sonra intramoleküler siklizasyona ugramasi DAO'nun etkisiyle, burada siklizasyon ürünü (II), etil asetatta (EA) (I)'den daha yüksek çözünürlüge sahiptir; (b) (II)'nin EA'ya çikarilmasi ve (c) (II) miktarinin belirlenmesi için bir test kiti Enzim aktivitesi belirlenmesi ön asamalarinda bir özütleme ve ekstraksiyon içermektedir. Kolorimetrik yöntemden bahsedilmemektedir. 3. Applicants FUJIMOTO SEIYAKU KK Classifications: Published as JP55831999A KANDAKI HISTAMIN ÖLÇÜMÜ 4. Applicants Classifications: Published as: Histamin intoleransi sendromundan muzdarip oldugundan süphelenilen bir kiside histamin intoleransi tani yöntemidir. Izotop etiketli histamin metabolitleri, yani imidazol asetik asit ve metilimidazol asetik asit, LC-MS/MS teknigi kullanilarak bir hidrazinokinolin türevlendirme maddesiyle türevlendirmenin ardindan serum, plazma, idrar ve diger vücut sivilarinda tanimlanir ve ölçülür. Histamin intoleransinin ayirici tanisi için izotop etiketli oral histamin yükünün güvenli kullanimina ve ayrica salgilanan ve zarla iliskili DAO enzimlerinin toplam aktivitesinin zamana bagli ölçümlerine izin verir. LC MS/MS cihazinda çalisilan kromotografik bir yöntemdir. . Applicants UNIV BARCELONA [ES] Inventors: COMAS BASTE ORIOL [ES]; LATORRE MORATALLA MARIA LUZ [ES]; VECIANA NOGUES MARIA TERESA [ES]; VIDAL CAROU MARIA DEL CARMEN [ES] Classifications: IPC C12Q1/26; C12Q; Published as: E52877873A1 DIAMIN OKSIDAZ (DAO) AKTIVITESININ IN VITRO TAYINI IÇIN PROSEDÜR Mevcut bulus, diamin oksidaz (DAO) aktivitesinin in vitro belirlenmesi için bir yöntemle ilgilidir. Mevcut bulus, bu ihtiyaci karsilayan ve özellikle asagidaki yönleri iyilestiren, substrat türevlendirmesi olmadan LC-MS/MS saptamasina dayali bir yöntem sunulmustur. 6. AT502850 VERFAHREN ZUR BESTIMMUNG DER AKTIVITÄT VON DIAMINOOXIDASE Applicant SCIOTEC Diagnostic Technologies GmbH Pub.Date 15.08.2007 EP1948819DIAMINE OXIDASE DETERMINATION Applicant SCIOTEC DIAGNOSTIC TECH NOLOGIE Pub.Date 30.07.2008 Applicant Sciotec Diagnostic Technologies GmbH Pub.Date 18.09.2008 CA2629061DIAMINE OXIDASE DETERMINATION Applicant MISSBICHLER, ALBERT Pub.Date 24.05.2007 Applicant Sciotec Diagnostic Technologies GMBH Pub.Date 05.06.2008 Applicant MAYER, Isabella Pub.Date 24.05.2007 E52323553DETERMINACION DE DIAMINOOXIDASA. Applicant SCIOTEC DIAGNOSTIC TECHNOLOGIES GMBH Pub.Date 20.07.2009 AT428799BESTIMMUNG VON DIAMINOOXIDASE Applicant SCIOTEC DIAGNOSTIC TECH NOLOGIE Classification: IPC DIAMIN OKSIDAZ ÖLÇÜLMESI Bir numunede histaminaz (DAO; EC 1.4.3.6) aktivitesini belirlemek için bir yöntem açiklanmaktadir. Bahsedilen yöntem asagidaki adimlari içerir: önceden tanimlanmis miktarda bir diamin içeren sulu bir solüsyon saglanir; sulu solüsyon karistirilir ve diaminin numunede muhtemelen mevcut olan bir DAO ile reaksiyona girebilecegi kosullarda belirli bir süre boyunca numune ile inkübe edilir; diamin türetilir; türetilmis diamin miktari belirlenir; önceden tanimlanmis diamin miktari, türevlendirilmis diamin miktari ile karsilastirilir; ve muhtemelen mevcut olan histaminaz aktivitesi belirlenir. Bu yöntemde türevlendirilmis diamin oksidaz uygun bir antikor ile eslestirilerek ELISA prensibine dayali bir yöntem ile belirlenmektedir. Yukarida bahsedilen patent belgelerinde kullanilan yöntemler florometrik ölçüm, ELISA yöntemi veya LC MS/MS cihazlarinin kullanilmasini gerektirmektedir. Bizim önerimiz hizli bir spektrofotometrik yöntemi içermektedir. Spektrofotometrik yöntemlerle ilgili yayinlar bulunmakla birlikte, bu yayinlarda kullanilan renk maddeleri önerimizden tamamen farklidir. YAYINLAR 1. Sensitive colorimetric assay of serum diamine oxidase Takagi, K., Nakao, M., Ogura, Y., Nabeshima, T; Kunii, A. (1994). Sensitive colorimetric assay of serum Serum diamin oksidaz (DAO) aktivitesi için kolorimetrik tahlil, peroksidaz ve yeni bir kromojen, IO (karboksimetilaminokarbonil)-3,7_bis(dimetilamino) fenotiyazin sodyum tuzu (DA-67) ile birlestirilmis bir reaksiyona dayanir. Peroksit ve DA-67 varliginda peroksidaz, maksimum 668 nm absorpsiyona sahip metilen mavisi olusumunu katalize eder. Önerilen yöntem, askorbat oksidaz kullanimi ile serumda meydana gelen enterferanslari ortadan kaldirir ve sodyum dietilditiokarbamat ile reaksiyonu durdurur, reaksiyon karisimindaki metilen mavisini yaklasik 2 saat stabil birakir. Serum DAO'in düsük normal bazal degerleri 2.8-9.0 birim/l araliginda belirlenebilir. DA-67 ve metlien mavisi kullanan spektrofotometrik bir yöntem olup kullanilan renk maddeleri yöntemimizden farklidir. 2. Determination of diamine oxidase activity by liquid scintillation counting Okuyama, T., Kobayashi, Y. (1961). Determination of diamine oxidase activity by liquid scintillation counting.Archives of Biochemistry and Biophysics, 242-250. anlatilmistir. Yöntem, diamin oksidazin kadaverin-Cl* üzerindeki etkisinden radyoaktif toluen ile ekstrakte edilebilir son ürün(ler)in olusumuna dayanmaktadir. Nihai ürünler dogrudan toluen içine ekstrakte edilir ve bir sivi sintilasyon spektrometresinde analiz edilir. Yöntem, substrat olarak radyoaktif putresin kullanilarak uygulanabilir, ancak radyoaktif histamin ile çalismaz. Radyoaktif isaretli substrat kullanilmis, RIA yöntemidir. 3. Determination of Diamine Oxidase Activity in Normal Human Blood Serum Tufvesson, G.; Tryding, N. (2009). Determination of Diamine Oxidase Activity in Normal Human Blood (histaminaz) ve mola hidatidoza (koryonepitelioma) rutin ölçümü için substrat olarak 14 C-putresin ile yöntem modifiye edilmistir. Normal insan kan serumunda diamin oksidaz (DAO) aktivitesinin belirlenmesi için optimal deneysel kosullar verilmistir. Serum DAO için normal aralik 0 ila 20 mU/l'dir. Isaretli karbon atomu kullanan bir RIA yöntem gelistirilmistir. 4. Quantification of human diamine oxidase Boehm, T., Pils, S., Gludovacz, E., Szoelloesi, H., Petroczi, K., Majdic, O., Quaroni, A., Borth, N., Valent, P.; Insan DAO'sunun saptanmasi için bir poliklonal tavsan serum IgG fraksiyonu ve yakalama için bir fare monoklonal antikoru kullanilarak bir arastirma prototipi ELISA olusturulmustur . Bos limit (LoB), tespit limiti (LoD) ve tahmini miktar tayin limiti (eLoQ) ve serum ve plazmadaki normal DAO konsantrasyonlari belirlenmistir. ELISA yöntemidir. . Serum diamine oxidase activity as a diagnostic test for histamine intolerance Music, E., Korosec, P., Silar, M., AdamiG, K., Kosnik, M.,Rijavec, M. (2013). Serum diamine oxidase activity as a diagnostic test for histamine intolerance. Wiener Klinische Wochenschrift, (9-10),239-243. Serum DAO aktivitesi, serum ve EDTA-plazmada Di-Amine Oxidase (DAO) ile histamin- degradasyon aktivitesinin kantitatif tespiti için enzim immünoassay ile ölçüldü. Degerlendirme için referans degerleri: DAO 80 HDU/ml normal aktivite, DAO 40-80 HDU/ml azaltilmis aktivite ve DAO < 40 HDU/ml yüksek oranda azaltilmis aktivite. Bir HDU (histamin bozundurucu birim), 1 pmol/ml (0.11 ng/ml) histamin bozan DAO aktivitesine karsilik ELISA-immun assay yöntemidir. Yukarida belirtilen tüm yayinlar laboratuvar kosullarinda diamin oksidaz aktivitesini belirlemeye yönelik yöntemleri açiklamaktadir. Bulusun Amaçlari Gida Alerjileri bebeklik ve erken çocukluk (<3 yas kadar) çaginda gelisir ve ömür boyu sürebilir. Bununla birlikte, mevcut testler yeterli öngörü degerine sahip olmadigi ve/veya alerjen gidanin tüketilmesi esasina dayali oldugu için potansiyel ciddi olumsuz reaksiyonlara neden olabilir, bu nedenle gida alerji testleri rutin olarak yapilmamaktadir. Gida alerjileri genellikle yasami tehdit edebilecek olumsuz bir reaksiyondan sonra tanimlanir. Son yillarda yapilan çalismalarda Diamin oksidaz enzim eksikliginin gida alerjisi ve histamin intoleransinin en önemli nedeni oldugu gösterilmistir. Bu bulusun amaci, biyolojik sivilarda Diamin oksidaz enzim aktivitesi belirlenmesi için kolay ve ucuz bir tani kiti gelistirilmesidir. Bulusu Açiklayan Sekillerin Tanimlari Bulusumuzda DAO tayini için kullanilan yöntem asagida açiklanmistir. Bulusun Ayrintili Açiklamasi YÖNTEM TEST ILKESI Substrat + H20 + 02 _ 4-Aminobutan aldehyde + NH3+ HZO2 HZO2 + kromojen POD (EC 1'11'1'7li ZHZO + renkli kromojen Substrat DAO enzimi ile reaksiyona girer ve hidrojen peroksit olusur. Olusan hidrojen peroksit peroksidaz enzimi ile okside olarak kromojen ile renk açiga çikar. Yukaridaki test prensibine göre reaksiyon karisimi hazirlanir. tribromo-3-hidroksibenzoik asit) içerir. Test yöntemi: Asagidaki tabloya göre reaktifler sirasiyla eklenerek toplam hacimde karisim olusturulur: TEST karisimi KÖR karisimi Tampon-1 - 10 ul Tampon-2 100 ul 100 ul PUTRESIN 10 ul - 290 PEROKSIDAZ 20 Ml 20 ul 4-AMINOANTIPIRIN 10 Ml 10 ul 2,3,6 tribromo-3- hidroksi 10 ul 10 ul benzoik asit Toplam hacim (KARISIM) 150 ul 150 ul 295 Tablo da verilen hacimler bir test içindir. Test sayisi kadar reaktifler eklenir. 1. Plak okuyucunun kuyucuklarina 20 ul örnek (serum, plazma veya diger vücut sivilari) pipetlenir. Test için örnegin üzerine, test karisimi 150 ul pipetlenir. Kör için örnegin üzerine kör karisimi 150 ul pipetlenir. 37 9C' de 60 dakika karistiricida inkübe ediIir. Spektrofotometrede 510 nm 'de kör ve test kuyucuklarinin absorbansi okunur. 91.4›9'!" Hidrojen peroksit ile yapilan optimizasyon çalismasi sonucunda ele edilen renk için yapilan spektrumda en yüksek okuma 510 nm dalga boyunda tespit edilmistir. Farkli konsantrasyonda DAO enzimi ölçümü için 4 farkli konsantrasyonda Putresin ile çalisma yapilmistir (Grafik.1). Putresin için Km degeri: 11,36mM olarak bulunmustur, kit reaktifinde bunun 5 kati konsantrasyonda Putresin kullanilmistir. Zamana karsi farkli konsantrasyonlarda DAO ile yapilan çalismada en iyi linearite sonucu 1 saatte elde edilmis ollmakla birlikte 30 dakikada da basarili sonuçlar bulunmustur (Grafik.2) Bir ünite, pH 6,0 da, 37 9C'de, saatte 1.0 umol putresini oksitleyecektir. Olusan Hidrojen peroksit miktari stoikiometrik olarak yikilan putresin ile koreledir. Degisen hidrojen peroksit konsantrasyonlari ile elde edilen absorbans degerleri ve purifiye edilmis DAO enzimi ile elde edilen absorbans degerleri arasinda korelasyon gösterilmistir. Ticari olarak satin alinan DAO enziminin seruma eklenmesi ile yapilan geri elde çalismasinda geri elde degeri %933 olarak bulunmustur. Within run CV: %8.8 olarak bulunmustur. Bulusun sanayiye uygulanma biçimi Bulusumuz ile tip/saglik alaninda kullanmaya yönelik hazir kit üretimi yapilabilecektir. Hazir kitler tüm dünyadaki tibbi biyokimya laboratuvarlari, acil servislerde kullanilabilecektir. Absorbans degisimleri lineer oldugu için hazirlanacak kit ile 1 saatten çok daha kisa sürelerde sonuç verilebilecektir. Bu özelligi ile kit otomatik analizörlerde kullanima uygundur. TR TR TR TR TR TR TR TRDESCRIPTION DIAMIN OXIDASE ACTIVITY DETERMINATION KIT IN BIOLOGICAL FLUIDS Relevance of the Invention The invention is intended for use in the field of healthcare and is particularly relevant to the diagnosis of food allergy and/or histamine intolerance. Diamine oxidase is an enzyme that breaks down histamine. When this enzyme is insufficient or blocked in activity, histamine cannot be broken down and accumulates when foods high in histamine are consumed. The invention enables rapid and inexpensive spectrophotometric determination of Diamine Oxidase enzyme activity in biological fluids. Prior Art Related to the Invention Diamine oxidase (EC) is a copper-containing enzyme that catalyzes the oxidative deamination of diamines with three to six carbon atoms, some of their derivatives, and histamine, which can be considered as a substituted cyclic diamine. Colorimetric, fluorometric, chromatographic methods for the determination of serum diamine oxidase (DAO) activity have been presented in the literature, and some of them have been patented. The colorimetric assay used in the literature is based on a coupled reaction of peroxidase and a chromogen, 10-(carboxymethyl-aminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt (DA-67). In the presence of peroxide and DA-67, peroxidase catalyzes the formation of methylene blue, which has an absorption maximum at 668 nm, and spectrophotometric measurement is performed. is performed. Commercially available Diamine Oxidase kits are also available. Commercially available kits, publications, and patents have been searched and are presented in the tables below. Commercially available kits 1. Diamine Oxidase Activity Assay Kit(Fluorometric) Sigma-Aldrich Catalog No: MAK351 Number of Tests: 100 assays Method: Fluorometric Sample Type: Tissue Lysates The Diamine Oxidase Activity Assay Kit provides a simple method for determining the DAO activity of tissue lysates as well as the DAO activity of recombinant enzymes with a detection limit of less than 1 pmol/min activity. In the assay, DAO converts the provided substrate to give an intermediate product and HZOZ. HZOZ is then used by the DAO Enzyme Mix to generate fluorescence (Ex/Em = 535/587 nm) from the DAO Probe. This is a fluorometric method and requires a fluorometry device. 2. Diamine Oxidase Assay Kit ABNOVA.ABCAM Catalog No: ab241004 Number of Tests: 100 assays Fee: E390.00 Method: Fluorometric Sample Type: Saliva, Tissue, Food Samples In the assay, DAO converts the provided substrate to yield an intermediate product and HZOZ. HZOZ is then used by the DAO Enzyme Mix to generate fluorescence (Ex/Em = 535/587 nm) from the DAO Probe. This is a fluorometric method and requires a fluorometry device. 3. Diamine oxidase (DAO) Activity Assay Kit Elabscience Catalog No: E-BC-K524-M Number of Tests: 96 assays Method: Colorimetric Sample Type: Serum, plasma, urine, animal tissue, and cell samples Diamine oxidase can catalyze amines to produce hydrogen peroxide and hydrogen peroxide can react with the chromogenic substance to produce a chromogenic substance that has a characteristic absorption peak at 460 nm. Diamine oxidase activity can be calculated by measuring the rate of change of absorbance per unit time. The chromogenic substance used is not specified, but based on the wavelength it is likely that o-dianisidine was used. 4. Diamine Oxidase assay kit BioVision Catalog No: K496-100 Number of Tests: 100 Tests Price: E663.00 Method: Fluorometric Specimen Type: Cell culture-derived material, Synovial fluid, Urine, Cartilage, Connective tissue/tumors This non-radioactive, fluorimetric Diamine Oxidase Assay (DAO) assay is based on the oxidation of putrescine to pyrroline plus NH3 and H2O2. The produced H2O2 is then used by HRP to oxidize a dye to make it fluorescent. Ex/Em = The increase in fluorescence at 530/585 nm is directly proportional to the enzyme activity. It is a fluorometric method and requires a fluorometry device. PATENTS 1 . Applicants: UNIV ANTWERPEN [BE]; UNI ZIEKENHUIS ANTWERPEN [BE] It is based on an enzyme-affinity method. It is used to detect and measure intracellular histamine and histamine release in mast cells and/or basophils. Inventors: BRIDTS CHRIS [BE]; EBO DIDIER [BE] Classifications: IPC G01N; Method and kit for detecting and measuring intracellular histamine and histamine release The present invention relates to methods and kits for detecting and measuring intracellular histamine and histamine release in individual mast cells and/or basophils by cytometry based on an enzyme-affinity method in which histaminase diamine oxidase (DAO) is coupled to a laser-excitable fluorochrome. kits are provided. It is an enzyme affinity method, which is completely different from the colorimetric method we propose. 2. Applicants JARISCH REINHARD DR [AT]; MISSBICHLER ALBERT DR [AT]; REICHL HERWIG MAG [AT] Classifications: C12Q; Method for determining diamino-oxidase activity, useful for the determination of histamine intolerance, based on the cyclization of a diamine and subsequent extraction of the product with ethyl acetate. Determination of diamino-oxidase activity (DAO; EC 1.4.3.6). Determination of diamino-oxidase activity (DAO; EC) is achieved by reaction with an aqueous solution containing formyl, whereby an amino group is converted to formyl and subsequently undergoes intramolecular cyclization under the influence of DAO, wherein the cyclization product (II) has a higher solubility in ethyl acetate than (EA) (I); (b) A test kit for the extraction of (II) into EA and (c) quantification of (II) It includes an extraction and extraction in the preliminary steps of enzyme activity determination. The colorimetric method is not mentioned. 3. Applicants FUJIMOTO SEIYAKU KK Classifications: Published as JP55831999A MEASUREMENT OF HISTAMINE IN BLOOD 4. Applicants Classifications: Published as: It is a method for the diagnosis of histamine intolerance in a person suspected of suffering from histamine intolerance syndrome. Isotope-labeled histamine metabolites, namely imidazole acetic acid and methylimidazole acetic acid, are identified and quantified in serum, plasma, urine and other body fluids after derivatization with a hydrazinoquinoline derivatizing agent using the LC-MS/MS technique. The differential diagnosis of histamine intolerance is based on the following: The present invention relates to a method for the in vitro determination of diamine oxidase (DAO) activity. The present invention relates to a method for the in vitro determination of diamine oxidase (DAO) activity. The present invention meets this need and is particularly suited for the A method based on LC-MS/MS detection without substrate derivatization is presented, which improves the following aspects. 6. AT502850 VERFAHREN ZUR BESTIMMUNG DER AKTIVITÄT VON DIAMINOOXIDASE Applicant SCIOTEC Diagnostic Technologies GmbH Pub.Date 15.08.2007 EP1948819DIAMINE OXIDASE DETERMINATION Applicant SCIOTEC DIAGNOSTIC TECH NOLOGIE Pub.Date 30.07.2008 Applicant Sciotec Diagnostic Technologies GmbH Pub.Date 18.09.2008 CA2629061DIAMINE OXIDASE DETERMINATION Applicant MISSBICHLER, ALBERT Pub.Date 24.05.2007 Applicant Sciotec Diagnostic Technologies GMBH Pub.Date 05.06.2008 Applicant MAYER, Isabella Pub.Date 24.05.2007 E52323553DETERMINACION DE DIAMINOOXIDASA. Applicant SCIOTEC DIAGNOSTIC TECHNOLOGIES GMBH Pub.Date 20.07.2009 AT428799BESTIMMUNG VON DIAMINOOXIDASE Applicant SCIOTEC DIAGNOSTIC TECHNOLOGY Classification: IPC MEASUREMENT OF DIAMIN OXIDASE A method is described for determining the activity of histaminase (DAO; EC 1.4.3.6) in a sample. The method comprises the following steps: an aqueous solution containing a predefined amount of a diamine is provided; the aqueous solution is stirred and subjected to conditions such that the diamine can react with a DAO possibly present in the sample. The sample is incubated for a certain period of time; the diamine is derivatized; the amount of derivatized diamine is determined; the pre-defined amount of diamine is compared with the amount of derivatized diamine; and the possibly present histaminase activity is determined. In this method, derivatized diamine oxidase is determined by a method based on the ELISA principle by pairing it with a suitable antibody. The methods used in the above-mentioned patent documents require the use of fluorometric measurement, ELISA method or LC MS/MS devices. Our recommendation includes a rapid spectrophotometric method. Although there are publications on spectrophotometric methods, the colorants used in these publications are completely different from our recommendation. PUBLICATIONS 1. Sensitive colorimetric assay of serum diamine oxidase Takagi, K., Nakao, M., Ogura, Y., Nabeshima, T; Kunii, A. (1994). Sensitive colorimetric assay of serum The colorimetric assay for serum diamine oxidase (DAO) activity is based on a coupled reaction between peroxidase and a novel chromogen, IO (carboxymethylaminocarbonyl)-3,7_bis(dimethylamino) phenothiazine sodium salt (DA-67). In the presence of peroxide and DA-67, peroxidase catalyzes the formation of methylene blue, which has a maximum absorption at 668 nm. The proposed method eliminates interferences occurring in serum by the use of ascorbate oxidase and stops the reaction with sodium diethyldithiocarbamate, leaving methylene blue in the reaction mixture stable for approximately 2 hours. Low normal baseline values of serum DAO can be determined in the range of 2.8-9.0 units/L. It is a spectrophotometric method using DA-67 and methylene blue, and the dyes used are different from our method. 2. Determination of diamine oxidase activity by liquid scintillation counting is described in Okuyama, T., Kobayashi, Y. (1961). Determination of diamine oxidase activity by liquid scintillation counting. Archives of Biochemistry and Biophysics, 242-250. The method is based on the formation of extractable end product(s) with radioactive toluene from the action of diamine oxidase on cadaverine-Cl*. The end products are extracted directly into toluene and analyzed in a liquid scintillation spectrometer. The method can be applied using radioactive putrescine as a substrate, but it does not work with radioactive histamine. It is a RIA method using a radiolabeled substrate. 3. Determination of Diamine Oxidase Activity in Normal Human Blood Serum Tufvesson, G.; Tryding, N. (2009). Determination of Diamine For the routine measurement of Oxidase Activity in Normal Human Blood (histamine) and mola hydatidosis (chorionepithelioma), the method was modified with 14C-putrescine as a substrate. Optimal experimental conditions for the determination of diamine oxidase (DAO) activity in normal human blood serum are given. The normal range for serum DAO is 0 to 20 mU/L. A RIA method using a labeled carbon atom was developed. 4. Quantification of human diamine oxidase Boehm, T. , Pils, S. , Gludovacz, E. , Szoelloesi, H. , Petroczi, K. , Majdic, O. , Quaroni, A. , Borth, N. , Valent, P.; A research prototype ELISA was established for the detection of human DAO using a polyclonal rabbit serum IgG fraction and a mouse monoclonal antibody for capture. Limit of blank (LoB), limit of detection (LoD), estimated limit of quantification (eLoQ) and normal concentrations of DAO in serum and plasma were determined. It is an ELISA method. . Serum diamine oxidase activity as a diagnostic test for histamine intolerance Music, E., Korosec, P., Silar, M., AdamiG, K., Kosnik, M.,Rijavec, M. (2013). Serum diamine oxidase activity as a diagnostic test for histamine intolerance. Wiener Klinische Wochenschrift, (9-10),239-243. Serum DAO activity was measured by enzyme immunoassay for the quantitative determination of histamine-degrading activity by Di-Amine Oxidase (DAO) in serum and EDTA-plasma. Reference values for evaluation: DAO 80 HDU/ml for normal activity, DAO 40-80 HDU/ml reduced activity and DAO < 40 HDU/ml highly reduced activity. One HDU (histamine degrading unit) is an ELISA immunoassay method corresponding to 1 pmol/ml (0.11 ng/ml) histamine-degrading DAO activity. All the above-mentioned publications describe methods for determining diamine oxidase activity under laboratory conditions. Aims of the Invention Food allergies develop in infancy and early childhood (up to <3 years of age) and can last a lifetime. However, because current tests do not have sufficient predictive value and/or are based on the consumption of allergenic foods, they can cause potentially serious adverse reactions, therefore food allergy tests are not routinely performed. Food allergies are usually identified after a potentially life-threatening adverse reaction. In recent years, studies have shown that diamine oxidase deficiency is a risk factor for food allergy and histamine intolerance has been shown to be the most important cause of intolerance. The aim of this invention is to develop an easy and inexpensive diagnostic kit for determining diamine oxidase enzyme activity in biological fluids. Definitions of the Figures Explaining the Invention The method used for the determination of DAO in our invention is explained below. Detailed Description of the Invention METHOD TEST PRINCIPLE Substrate + H2O + O2 _ 4-Aminobutane aldehyde + NH3 + HZO2 HZO2 + chromogen POD (EC 1'11'1'7li ZHZO + colored chromogen The substrate reacts with DAO enzyme and hydrogen peroxide is formed. The hydrogen peroxide formed is oxidized by peroxidase enzyme and the color is revealed with the chromogen. The reaction mixture is prepared according to the above test principle. contains tribromo-3-hydroxybenzoic acid). Test method: The reagents are added in the order shown in the table to form a total volume mixture: TEST mixture BLIND mixture Buffer-1 - 10 ul Buffer-2 100 ul 100 ul PUTRESSINE 10 ul - 290 PEROXIDASE 20 ml 20 ul 4-AMINOANTIPYRINE 10 ml 10 ul 2,3,6 tribromo-3-hydroxy 10 ul 10 ul benzoic acid Total volume (MIXTURE) 150 ul 150 ul 295 The volumes given in the table are for one test. Reagents are added according to the number of tests. 1. Pipette 20 ul of sample (serum, plasma or other body fluids) into the wells of the plate reader. For the test, pipette 150 ul of test mixture onto the sample. For the blank, pour the blank mixture onto the sample. 150 ul are pipetted. It is incubated at 37 9 C for 60 minutes on a shaker. The absorbance of the blank and test wells is read at 510 nm on the spectrophotometer. 91.4›9'!" As a result of the optimization study with hydrogen peroxide, the highest reading in the spectrum for the color obtained was detected at 510 nm wavelength. For the measurement of DAO enzyme at different concentrations, studies were carried out with 4 different concentrations of Putrescine (Graphic 1). The Km value for Putrescine was found to be 11.36 mM, and Putrescine at a concentration five times this was used in the kit reagent. In the study conducted with DAO at different concentrations against time, the best linearity result was obtained in 1 hour, although successful results were also found in 30 minutes (Graphic 2). One unit, at pH 6.0, 37 At 9°C, it will oxidize 1.0 µmol of putrescine per hour. The amount of hydrogen peroxide formed correlates stoichiometrically with the putrescine degraded. A correlation has been demonstrated between absorbance values obtained with varying hydrogen peroxide concentrations and absorbance values obtained with purified DAO enzyme. In a recovery study conducted by adding commercially purchased DAO enzyme to serum, the recovery value was found to be 933%. Within-run CV was found to be 8.8%. Industrial Application of the Invention: With our invention, it is possible to produce ready-made kits for use in medicine/healthcare. These ready-made kits can be used in medical biochemistry laboratories and emergency rooms worldwide. Because absorbance changes are linear, the prepared kit can provide results in much less than an hour. This feature makes the kit suitable for use in automated analyzers. TR TR TR TR TR TR TR TR