NO138182B - ANTIBODIES SPECIFICALLY AIMED AT IMMUNGLOBULIN E (IGE) OR AGAINST FRAGMENT OF IGE - Google Patents
ANTIBODIES SPECIFICALLY AIMED AT IMMUNGLOBULIN E (IGE) OR AGAINST FRAGMENT OF IGE Download PDFInfo
- Publication number
- NO138182B NO138182B NO247772A NO247772A NO138182B NO 138182 B NO138182 B NO 138182B NO 247772 A NO247772 A NO 247772A NO 247772 A NO247772 A NO 247772A NO 138182 B NO138182 B NO 138182B
- Authority
- NO
- Norway
- Prior art keywords
- reagin
- antibodies
- allergen
- ige
- directed against
- Prior art date
Links
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- GOLCXWYRSKYTSP-UHFFFAOYSA-N arsenic trioxide Inorganic materials O1[As]2O[As]1O2 GOLCXWYRSKYTSP-UHFFFAOYSA-N 0.000 description 1
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical class O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
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- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
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- SRRKNRDXURUMPP-UHFFFAOYSA-N sodium disulfide Chemical compound [Na+].[Na+].[S-][S-] SRRKNRDXURUMPP-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0058—Antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
- A61K49/0043—Fluorescein, used in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1015—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against material from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1018—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against material from animals or humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1078—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody the antibody being against an immunoglobulin, i.e. being an (anti)-anti-idiotypic antibody
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig
- C07K16/4291—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an allotypic or isotypic determinant on Ig against IgE
-
- 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
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2121/00—Preparations for use in therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Optics & Photonics (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Botany (AREA)
- Endocrinology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Description
Oppfinnelsen.vedrører antilegemer spesifikt rettet The invention relates to antibodies specifically directed
mot immunglobulin E (IgE) eller mot fragment av IgE inneholdende klasse-spesifikke determinanter, hvilke antilegemer er beregnet til å anvendes in vitro ved immunkjemiske bestemmelsesmetoder basert på reaksjoner, hvori et allergen bundet til en i vann uoppløselig polymer, IgE rettet mot allergenet og antilegemer rettet mot IgE eller fragment herav er innbefattet, karakterisert ved at antilegemene rettet mot IgE eller fragment herav er gitt evne til å utsende radioaktiv stråling eller fluorescens-stråling ved merking med en eller flere radioaktive isotoper eller med en gruppe inneholdende en radioaktiv isotop resp. med en fluorescerende gruppe. against immunoglobulin E (IgE) or against fragments of IgE containing class-specific determinants, which antibodies are intended to be used in vitro by immunochemical determination methods based on reactions, in which an allergen is bound to a water-insoluble polymer, IgE directed against the allergen and antibodies directed against IgE or fragment thereof is included, characterized in that the antibodies directed against IgE or fragment thereof are given the ability to emit radioactive radiation or fluorescence radiation by labeling with one or more radioactive isotopes or with a group containing a radioactive isotope resp. with a fluorescent group.
Det er kjent at en del immunogene stoffer, såkalte allergener, av hvilke mange er av protein-, peptid- eller kull-hydratnatur kan gi grunn til allergiske sykdomstilstander, f.eks. astma, høysnue. (Slike allergener forekommer generelt og kan stamme fra f.eks. hår, vekstpollen, mikroorganismer, en del parasitter etc.). Herved forekommer en spesiell type antilegemer, såkalte reaginer, som utgjøres av immunglobuliner som er rettet spesifikt mot allergenet eller allergenene, vanligvis i meget lave konsentrasjoner, i f.eks. pasientens blod og kan på-vises i blodserum. Disse spesielle immunglobuliner kalles i det følgende reagin-immunglobulin eller reagin-Ig. (Se f.eks. Advances in Immunology, bind 3, (1963) side l8l - 183). Med reagin-Ig forstås her og i det følgende således den gruppe immunglobuliner som kan reagere med allergener og som derved, It is known that some immunogenic substances, so-called allergens, many of which are of a protein, peptide or carbohydrate nature, can give rise to allergic disease states, e.g. asthma, hay fever. (Such allergens occur in general and can originate from e.g. hair, plant pollen, micro-organisms, some parasites etc.). This results in a special type of antibodies, so-called reagins, which are made up of immunoglobulins that are directed specifically against the allergen or allergens, usually in very low concentrations, in e.g. the patient's blood and can be detected in blood serum. These special immunoglobulins are called reagin-immunoglobulin or reagin-Ig in the following. (See, e.g., Advances in Immunology, Vol. 3, (1963) pp. l8l - 183). Reagin-Ig is understood here and in the following to mean the group of immunoglobulins that can react with allergens and which thereby,
når reaksjonen finner sted in vivo, forårsaker allergiske reaksjoner hos mennesker eller dyr.' (Nå betegnes ofte disse immunglobuliner med IgE). when the reaction takes place in vivo, causes allergic reactions in humans or animals.' (Now these immunoglobulins are often referred to as IgE).
Foreliggende oppfinnelse er av betydning ved påvis-.. ning av spesifikke reagin-Ig rettet mot allergener i vannholdige prøver. Å påvise reagin-Ig har spesielt betydning i forbindelse med diagnostisering av overfølsomhet hos mennesker og dyr. The present invention is of importance in the detection of specific reagin-Ig directed against allergens in aqueous samples. Detecting reagin-Ig is particularly important in connection with the diagnosis of hypersensitivity in humans and animals.
For dette formål har det tidligere blitt anvendt såkalte hudprøver, idet prøveallergen tilføres prøveobjektet, f.eks. intrakutant. Om overfølsomhet (allergi) foreligger, opp-står en reaksjon in vivo mellom allergenet og reagin-Ig rettet mot det omtalte allergenet, som får til følge visse symptomer som rødming eller hovning av det sted hvor injiseringen foregikk, hvilke iakttas og bedømmes med hensyn på graden av virkningen. For this purpose, so-called skin tests have previously been used, as the test allergen is added to the test object, e.g. intracutaneously. If hypersensitivity (allergy) exists, a reaction occurs in vivo between the allergen and reagin-Ig directed against the mentioned allergen, which results in certain symptoms such as redness or swelling of the place where the injection took place, which are observed and assessed with regard to the degree of impact.
In vivo-undersøkelser er også såkalte provokasjons-prøver, idet pasienten får innånde et allergen i aerosolform. Hvis overfølsomhet mot dette foreligger, fåes en reaksjon, som f.eks. kan gi astmasymptom. Om begge prøvetyper kan sies at de er omstendelige og tidskrevende for pasienten samt i visse tilfeller forbundet med ubehag eller fare for dette. I en del tilfeller går det ikke å utføre dem. Videre kreves også en mang-foldig in vivo-prøver for at man skal kunne diagnostisere en overfølsomhet mot et visst eller visse allergener. In vivo examinations are also so-called provocation tests, as the patient inhales an allergen in aerosol form. If hypersensitivity to this exists, a reaction is obtained, such as e.g. can cause asthma symptoms. Both test types can be said to be cumbersome and time-consuming for the patient, and in certain cases associated with discomfort or the risk of this. In some cases it is not possible to carry them out. Furthermore, multiple in vivo tests are also required in order to be able to diagnose a hypersensitivity to a certain or certain allergens.
Tidligere prøvede in vitro-metoder savner praktisk betydning. Previously tried in vitro methods lack practical significance.
Det er riktignok fra J. Immunology 97 (1966), 840-853 og 98 (1967), 490-501 kjent å påvise Ig-E med en strål-ningsimmunologisk metode, hvor anvendelsen av merket allergen er et vesentlig trekk. Det merkede allergen bringes ved denne metode til å reagere med Ig-E i oppløsning, hvoretter det dannede kompleks utfelles fra oppløsningen ved tilsetning av antilegemer mot Ig-E. Det allergen som herved ble anvendt er allergen fra "ragweed", som er et av de få unntakstilfeller der allergenet kan isoleres i høy renhet. Allergenholdig ekstrakt av annen opprinnelse, f.eks. fra hund- eller hesteepitel eller bjerkpollen har ikke tilstrekkelig renhet til å kunne utnyttes ved denne fremgangsmåte, som derfor har et spesielt begrenset anvendelsesområde. Videre krever fremgangsmåten en merknings-prosedyre for hvert prøveallergen, som blir både kostbart og om-stendelig. Ved denne metode er videre å bemerke at allergenet bare reagerer med Ig-E spesifikt rettet mot dette allergen, mens man ved utfelling av komplekset med antilegemene mot Ig-E sam-tidig får en utfelling av reaksjonsproduktene mellom antilegemene og andre reaginimmunglobuliner (andre Ig-E) enn det som er spesifikt rettet mot det aktuelle allergen. Hvis man i dette system istedenfor å merke allergenet, skulle merke antilegemene, skulle man riktignok også få en fast fase som avgir radioaktiv stråling, men det er i dette tilfelle umulig å avgjøre om allergenet inngår i noen del av denne fase eller ikke. Følgelig skulle man i dette tilfellet ikke få den ønskede informasjon om eventuelt nærvær av et Ig-E spesifikt rettet mot allergenet. It is admittedly known from J. Immunology 97 (1966), 840-853 and 98 (1967), 490-501 to detect Ig-E with a radiation immunological method, where the use of labeled allergen is an essential feature. The labeled allergen is caused by this method to react with Ig-E in solution, after which the formed complex is precipitated from the solution by the addition of antibodies against Ig-E. The allergen used here is allergen from "ragweed", which is one of the few exceptional cases where the allergen can be isolated in high purity. Allergen-containing extract of other origin, e.g. from dog or horse epithelium or birch pollen does not have sufficient purity to be utilized by this method, which therefore has a particularly limited area of application. Furthermore, the method requires a labeling procedure for each test allergen, which is both costly and time-consuming. With this method, it is also worth noting that the allergen only reacts with Ig-E specifically directed against this allergen, while precipitation of the complex with the antibodies against Ig-E simultaneously results in a precipitation of the reaction products between the antibodies and other reagin immunoglobulins (other Ig- E) than that which is specifically directed against the allergen in question. If, in this system, instead of labeling the allergen, you were to label the antibodies, you would of course also get a solid phase that emits radioactive radiation, but in this case it is impossible to determine whether the allergen is included in any part of this phase or not. Consequently, in this case one would not get the desired information about the possible presence of an Ig-E specifically directed against the allergen.
Formålet med oppfinnelsen er å få frem et reagens som kan anvendes for en enkel og tilforlatelig in vitro-metode, hvorved ovennevnte ulemper er fjernet og som kan utføres uten ubehag eller risiko for pasienten. The purpose of the invention is to produce a reagent which can be used for a simple and reliable in vitro method, whereby the above-mentioned disadvantages are removed and which can be carried out without discomfort or risk for the patient.
I norsk patent nr. 127-685 er beskrevet en metode ifølge hvilken den for prøvningen beregnede prøve, f.eks. en kroppsvæske som blodserum eller blodplasma, in vitro bringes i berøring med en i vann uoppløselig polymer, hvortil er bundet et prøveallergen idet en reaksjon mellom prøveallergenet og polymeren og mot dette rettet reagin-Ig finner sted, således at reagin-Ig bindes til prøveallergenet på den uoppløselige polymer, og at polymere med vedsittende prøveallergen og reagin-Ig bringes i berøring med enten a) antilegemer mot reagin-Ig, hvilke antilegemer er merket med et strålingsavgivende atom eller gruppe, Norwegian patent no. 127-685 describes a method according to which the sample calculated for the test, e.g. a body fluid such as blood serum or blood plasma, in vitro, is brought into contact with a water-insoluble polymer, to which a test allergen is bound, as a reaction between the test allergen and the polymer and reagin-Ig directed against this takes place, so that reagin-Ig binds to the test allergen on the insoluble polymer, and that polymers with adherent test allergen and reagin-Ig are brought into contact with either a) antibodies against reagin-Ig, which antibodies are labeled with a radiation-emitting atom or group,
eller or
b) antilegemer mot reagin-Ig og med reagin-Ig, idet reagin-Ig er merket med et strålingsavgivende b) antibodies against reagin-Ig and with reagin-Ig, the reagin-Ig being labeled with a radiation-emitting
atom eller gruppe atom or group
hvoretter den uoppløselige polymer med vedsittende stoffer skil-les fra væsken og derettermåler strålingen fra den uoppløselige polymer med vedsittende stoffer eller fra den fraskilte væske. after which the insoluble polymer with adherent substances is separated from the liquid and then the radiation from the insoluble polymer with adherent substances or from the separated liquid is measured.
Hvis således reagin-Ig rettet mot allergenet finnes tilstede i prøven, bindes det merkede reagens til den uoppløse-lige fase som derpå utsender stråling. Sistnevnte øker med økende konsentrasjon av reagin-Ig i prøven. Den flytende fases stråling minsker i stedet med økende konsentrasjon av reagin-Ig, da større mengder merket reagens bindes til den uoppløselige fasen. De målte strålingsverdier for prøven kan sammenlignes med verdier på kontrollprøven. Thus, if reagin-Ig directed against the allergen is present in the sample, the labeled reagent binds to the insoluble phase, which then emits radiation. The latter increases with increasing concentration of reagin-Ig in the sample. The radiation of the liquid phase instead decreases with increasing concentration of reagin-Ig, as larger amounts of labeled reagent are bound to the insoluble phase. The measured radiation values for the sample can be compared with values on the control sample.
Som bærere av prøveallergenet anvendes i vann uopp-løselige polymere. De kan eksempelvis foreligge i form av partikler av varierende form og størrelse. Som bærere kan også anvendes polymere i form av f.eks. plane gjenstander som skiver eller bånd eller også f.eks. i form av veggen i et rør, f.eks. innersiden av et prøverør. Bindingen mellom den polymere og prøveallergenet skal være slik at prøveallergenet ved normale vaskningsoperasjoner ikke kan løsgjøres fra den polymere. Water-insoluble polymers are used as carriers of the test allergen. They can, for example, be in the form of particles of varying shape and size. Polymers in the form of e.g. flat objects such as disks or bands or also e.g. in the form of the wall of a pipe, e.g. the inside of a test tube. The bond between the polymer and the test allergen must be such that the test allergen cannot be detached from the polymer during normal washing operations.
For dette formål kan den f.eks. være av kjemisk eller eventuelt også av fysikalsk natur. En egnet kjemisk bindingsform er å tilveiebringe broer av kovalent karakter mellom den polymere og prøveallergenet. For this purpose, it can e.g. be of a chemical or possibly also of a physical nature. A suitable form of chemical bonding is to provide bridges of a covalent nature between the polymer and the test allergen.
For dette formål velges den polymere således at For this purpose, the polymer is chosen so that
den har egnede reaktive grupper, f.eks. aminogrupper, hydroksylgrupper og karboksylgrupper for lett å muliggjøre en binding av prøveallergenet til den polymere. Herved velges fortrinnsvis en kjemisk binding med broer med bindinger av kovalent karakter. it has suitable reactive groups, e.g. amino groups, hydroxyl groups and carboxyl groups to easily enable a binding of the test allergen to the polymer. Hereby, a chemical bond with bridges with bonds of a covalent nature is preferably chosen.
Spesielt egnet er det å velge polymere bestående It is particularly suitable to choose polymers consisting of
av et tredimensjonalt nettverk sammenholdt av bindinger av kovalent karakter. Slike polymere er, selv om de er svellbare i vann eller vannoppløselige medier, helt uoppløselige og kan derved ikke slippe ifra seg noe av polymermaterialet eller derpå fastbundet stoff, f.eks. ved vaskeprosedyre. Eksempel på slike polymere er polymerisat dannet ved tverrbinding av stoffer inneholdende et flertall hydroksylgrupper som kullhydrater og sukkeralkoholer, f.eks. dekstran, stivelse, dekstriner og andre polysakkarider samt polyvinylalkohol med et bifunksjonelt stoff, f.eks. et bifunksjonelt stoff av typen X - R - Z, hvor eksempelvis X og Z er halogen eller epoksygrupper og R..resten av det bi-funksj onelle stoff, f.eks. et alifatisk radikal inneholdende 3-10 karbonatomer. Andre eksempler på uoppløselige polymer-materiale er cellulose, agarosegel, polyaminostyren. of a three-dimensional network held together by bonds of a covalent nature. Such polymers, even if they are swellable in water or water-soluble media, are completely insoluble and thereby cannot release any of the polymer material or substances bound to it, e.g. by washing procedure. Examples of such polymers are polymers formed by crosslinking substances containing a majority of hydroxyl groups such as carbohydrates and sugar alcohols, e.g. dextran, starch, dextrins and other polysaccharides as well as polyvinyl alcohol with a bifunctional substance, e.g. a bifunctional substance of the type X - R - Z, where, for example, X and Z are halogen or epoxy groups and R.. the rest of the bi-functional substance, e.g. an aliphatic radical containing 3-10 carbon atoms. Other examples of insoluble polymer material are cellulose, agarose gel, polyaminostyrene.
For formålet kan det anvendes korn av det kommersielt tilgjengelige produkt "Sephadex", som består av dekstran tverrbundet med glyceroleterbroer, dannet ved behandling av dekstran med epiklorhydrin. Produktet kan anvendes f.eks. i form av små korn. "Sephadex" og på lignende måte oppnådde pro-dukter er i vann svellbare, men uoppløselige gelprodukter, f.eks. i kornform. De inneholder hydroksylgrupper og kan derved lett substitueres også med andre grupper, f.eks. slike inneholdende aminogrupper eller karboksylgru<p>per egner seg derigjennom godt for brodannelse med bindinger av kovalent karakter til prøve-allergenet. For this purpose, grains of the commercially available product "Sephadex" can be used, which consists of dextran cross-linked with glycerol ether bridges, formed by treating dextran with epichlorohydrin. The product can be used e.g. in the form of small grains. "Sephadex" and similarly obtained products are water-swellable but insoluble gel products, e.g. in grain form. They contain hydroxyl groups and can therefore easily be substituted also with other groups, e.g. such containing amino groups or carboxyl groups are therefore well suited for bridging with bonds of a covalent nature to the test allergen.
Hensiktsmessig velges en s'lik form av den polymere Appropriately, such a form of the polymer is chosen
at det oppnås en stor kontaktoverflate, f.eks. små partikler. that a large contact surface is achieved, e.g. small particles.
Til denne bærepolymer bindes prøveallergenet under milde betingelser, således at allergenets immunkjemiske reak-tivitet ikke vesentlig minsker. Ved kjemisk binding mellom allergenet og polymeren utnyttes reaktive grupper i disse som aminogrupper, hydroksylgrupper, merkaptogrupper, amidgrupper og karboksylgrupper, idet det slåes en bro med kjemisk binding, fortrinnsvis av kovalent karakter, fra prøveallergenet til den polymere, f.eks. av typen: The test allergen is bound to this carrier polymer under mild conditions, so that the allergen's immunochemical reactivity does not significantly decrease. In the case of chemical bonding between the allergen and the polymer, reactive groups are utilized in these such as amino groups, hydroxyl groups, mercapto groups, amide groups and carboxyl groups, as a bridge is built with a chemical bond, preferably of a covalent nature, from the test allergen to the polymeric one, e.g. of type:
Broen mellom allergenet og polymeren behøver ikke å være strukturbestemt, og kan derfor velges av meget skiftende type da den bare har til formål å hindre at allergenet vaskes bort fra den uoppløselige polymer. The bridge between the allergen and the polymer does not need to be structurally determined, and can therefore be chosen of a very variable type as it only has the purpose of preventing the allergen from being washed away from the insoluble polymer.
Bestemmelsesmetoden for reagin-Ig er basert på at reagin-Ig kan bindes til såvel det allergen som det er spesielt rettet mot som til antilegemer generelt rettet mot reagin-Ig. Slike antilegemer er minst bivalente, dvs. de kan binde minst to molekyler reagin-Ig. Antilegemer rettet mot reagin-Ig og også reagin-Ig selv kan merkes med et strålingsavgivende atom eller gruppe, f.eks. med radioaktiv isotop, eller en fluorescerende gruppe. The determination method for reagin-Ig is based on the fact that reagin-Ig can bind both to the allergen that it is specifically directed against and to antibodies generally directed against reagin-Ig. Such antibodies are at least bivalent, i.e. they can bind at least two molecules of reagin-Ig. Antibodies directed against reagin-Ig and also reagin-Ig itself can be labeled with a radiation-emitting atom or group, e.g. with a radioactive isotope, or a fluorescent group.
Sammenlignet med de kjente metoder med utførelse av. hudprøver og provokasjonsprøver har ovenstående metode den fordel at man ved en blodprøve fra pasienten på en for denne helt ufarlige måte in vitro med en meget følsom og enkel metode kan bestemme nærvær av og også mengden av reagin-Ig rettet mot en bestemt type allergen eller en viss gruppe allergener, f.eks. dyreallergen og vekstallergen. Vesentlig for metoden er at det prøveallergen mot hvilket det reagin-Ig som skal bestemmes er rettet er meget fast bundet til en i vann uoppløselig bærer. Reagin-Ig som reagerer med allergenet kan således lett separeres fra ikke-allergenbundne reaginimmunglobuliner. Under metodens ytterligere arbeidstrinn kan videre de antilegemer resp. det reagin-Ig som ikke er bundet til den polymere, meget enkelt separeres fra dem som er bundet til den polymere. Compared to the known methods with execution of. skin tests and provocation tests, the above method has the advantage that with a blood sample from the patient in a completely harmless way in vitro with a very sensitive and simple method, the presence of and also the amount of reagin-Ig directed against a specific type of allergen or a certain group of allergens, e.g. animal allergen and plant allergen. Essential to the method is that the test allergen against which the reagin-Ig to be determined is directed is very firmly bound to a water-insoluble carrier. Reagin-Ig that reacts with the allergen can thus be easily separated from non-allergen-bound reagin immunoglobulins. During the further working steps of the method, the antibodies or the reagin-Ig that is not bound to the polymer is very easily separated from those that are bound to the polymer.
Separering av den polymere med vedsittende stoffer fra væsken kan lett utføres. Hvis den polymere foreligger i partikkelform, kan separeringen skje gjennom f.eks. enkel sentrifugering eller filtrering. Separeringen er ufølsom for vari-asjoner i salt- og proteinkonsentrasjon hos væsken innen fysio-logiske grenser. Hele bestemmelsesfremgangsmåten innbefattende separasjonen av frie merkede antilegemer resp. reagin-Ig og polymerbundne merkede antilegemer resp. reagin-Ig kan f.eks. ut-føres i samme prøverør under tilsetning av utfellingsmiddel eller lignende. Separation of the polymer with adherent substances from the liquid can be easily carried out. If the polymer is present in particulate form, the separation can take place through e.g. simple centrifugation or filtration. The separation is insensitive to variations in salt and protein concentration in the liquid within physiological limits. The entire determination procedure including the separation of free labeled antibodies resp. reagin-Ig and polymer-bound labeled antibodies resp. reagin-Ig can e.g. carried out in the same test tube with the addition of a precipitating agent or similar.
Ovenstående metode forutsetter tilgang til isolert The above method requires access to isolated
reagin-Ig samt antilegemer mot disse. reagin-Ig and antibodies against these.
Reagin-Ig kan utvinnes fra serum fra allergiske pasienter og i spesielle tilfeller fra serum fra pasienter med tumor i de plasmaceller som danner reagin-Ig. Reagin-Ig kan renses ved forskjellige metoder for separering av proteiner som gelfiltrering, ionebyttekromatografering og elektroforese. I likhet med andre immunglobuliner kan reagin-Ig spaltes kjemisk eller enzymatisk i en antigenbindende del og en del som er bærer av de klassespesifikke determinantene. Reagin-Ig can be extracted from serum from allergic patients and in special cases from serum from patients with tumors in the plasma cells that form reagin-Ig. Reagin-Ig can be purified by different methods for separating proteins such as gel filtration, ion exchange chromatography and electrophoresis. Like other immunoglobulins, reagin-Ig can be cleaved chemically or enzymatically into an antigen-binding part and a part that carries the class-specific determinants.
Oppfinnelsen vedrører antilegemer mot reagin-Ig eller fragment herav, hvilke antilegemer ved merkning har blitt gitt evnen til å utsende stråling og er beregnet til å anvendes ved strålings-immunologiske metoder, eksempelvis som angitt for bestemmelse av reagin-Ig.. The invention relates to antibodies against reagin-Ig or fragments thereof, which antibodies have been labeled with the ability to emit radiation and are intended to be used in radiation-immunological methods, for example as indicated for the determination of reagin-Ig..
Antilegemer mot reagin-Ig kan fremstilles ifølge i og for seg kjente metoder for immunisering av forsøksdyr ved f.eks. gjentatte subkutane injeksjoner av små mengder reagin-Ig eller fragment av reagin-Ig i blanding med et egnet adjuvant, f.eks. Freunds mineraloljesuspensjon. De i dyrene, f.eks. kanin, dannede antilegemer kan utvinnes fra dyrenes blodserum. Antibodies against reagin-Ig can be produced according to methods known per se for immunization of laboratory animals by e.g. repeated subcutaneous injections of small amounts of reagin-Ig or fragment of reagin-Ig in mixture with a suitable adjuvant, e.g. Freund's mineral oil suspension. Those in the animals, e.g. rabbit, antibodies formed can be recovered from the animals' blood serum.
Spesifikke antilegemer, dvs. antilegemer rettet mot de klassespesifikke determinantene i reagin-Ig, kan fåes ved immunisering med spaltstykker av reagin-Ig-molekylene inneholdende disse determinanter eller også ved "isolering av disse spesifikke antilegemer fra en blanding av antilegemer dannet ved immunisering med hele reagin-Ig. Specific antibodies, i.e. antibodies directed against the class-specific determinants in reagin-Ig, can be obtained by immunization with fragments of the reagin-Ig molecules containing these determinants or also by "isolation of these specific antibodies from a mixture of antibodies formed by immunization with whole reagin-Ig.
Spesifikke antilegemer rettet mot reagin-Ig kan, hvis ønsket, isoleres fra antiserumet ved konvensjonell imrnuno-sorbentteknikk, idet reagin-Ig koples til f.eks. bromacetylcellulose og de antilegemer som blir bundet til koplet reagin-Ig, frigjøres ved senkning av pH. Specific antibodies directed against reagin-Ig can, if desired, be isolated from the antiserum by conventional immunosorbent technique, reagin-Ig being coupled to e.g. bromoacetyl cellulose and the antibodies that are bound to coupled reagin-Ig are released by lowering the pH.
Det er en fordel å anvende spesifikke antilegemer mot de klassespesifikke determinantene i reagin-Ig for å oppnå høy sikkerhet og nøyaktighet ved bestemmelsene. It is an advantage to use specific antibodies against the class-specific determinants in reagin-Ig in order to achieve high safety and accuracy in the determinations.
Merkning med radioaktiv isotop av reagin-Ig resp. antilegemer mot reagin-Ig kan gjøres på vanlig måte idet en for formålet egnet isotop velges, f.eks. <125>I (se f.eks. Labeling with a radioactive isotope of reagin-Ig resp. antibodies against reagin-Ig can be made in the usual way, choosing an isotope suitable for the purpose, e.g. <125>I (see e.g.
metoden ifølge Hunter og Greenwood, Nature, bind 194 (1962) the method of Hunter and Greenwood, Nature, vol. 194 (1962)
side 495). Likeledes kan merkningen med en fluorescensav-givende gruppe gjøres på vanlig måte, f.eks. med et fluores-cein-derivat som fluorescein-isotiocyanat.• page 495). Likewise, the labeling with a fluorescence-emitting group can be done in the usual way, e.g. with a fluorescein derivative such as fluorescein isothiocyanate.•
Allergenholdige ekstrakter, f.eks. fra hund- eller heste-epitel, timotei- eller bjerkepollen, finnes kommersielt tilgjengelig. Allergen-containing extracts, e.g. from dog or horse epithelium, timothy or birch pollen, are commercially available.
Radioaktivitetsbestemmelsene kan skje med vanlige metoder, f.eks. ved hjelp av scintillasjonsdetektorer. Likeledes kan fluorescensmålinger skje med vanlige metoder. The radioactivity determinations can be carried out using usual methods, e.g. using scintillation detectors. Likewise, fluorescence measurements can be carried out using common methods.
De merkede antilegemer ifølge oppfinnelsen kan anvendes i et _hjelpemiddel for bestemmelse av spesifikke reagin-Ig rettet mot allergener i vannholdige prøver, omtalt i norsk patent nr. 127.685, hvilke omfatter et første reagens, hvori inngår en i vann uoppløselig polymer hvortil ved hjelp av kovalente bindinger, ionebindinger eller adsorbsjonsbindinger det er bundet et prøveallergen et annet reagens hvori inngår antilegemer mot reagin-Ig, idet antilegemene mot reagin-Ig ved merking er gitt evne til å utsende stråling, idet det første reagens er beregnet til in vitro å komme i berøring med en vannholdig prøve inneholdende reagin-Ig for å binde•reagin-Ig til prøveallergenet på den uoppløselige polymer, og idet det andre reagenset er beregnet til å bringes i berøring med den uoppløselige polymer med det vedsittende prøveallergen og reagin-Ig for dannelse av en blanding inneholdende uoppløselig materi-ale som stråler og som består av uoppløselig polymer og ved denne fastsittende stoffer og strålende væskefase, idet det oppløse-lige materialets og væskefasens stråling hver er en funksjon av konsentrasjonen av reagin-Ig rettet mot prøveallergenet i prøven. The labeled antibodies according to the invention can be used in an aid for the determination of specific reagin-Ig directed against allergens in aqueous samples, described in Norwegian patent no. 127,685, which comprise a first reagent, which includes a water-insoluble polymer to which, by means of covalent bonds, ionic bonds or adsorption bonds a test allergen is bound another reagent containing antibodies against reagin-Ig, since the antibodies against reagin-Ig are given the ability to emit radiation by labeling, since the first reagent is intended to come into contact in vitro with an aqueous sample containing reagin-Ig to bind•reagin-Ig to the test allergen on the insoluble polymer, and the second reagent being intended to contact the insoluble polymer with the adjacent test allergen and reagin-Ig to form a mixture containing insoluble material that radiates and which consists of insoluble polymer and by this stuck substances and radiant liquid phase, the radiation of the soluble material and the liquid phase each being a function of the concentration of reagin-Ig directed against the test allergen in the sample.
Den uoppløselige polymer foreligger hensiktsmessig The insoluble polymer is conveniently present
i form av partikler. in the form of particles.
Reagenset kan inngå i en reagenskombinasjon som The reagent can form part of a reagent combination which
er beregnet til å anvendes for prøving av prøver fra aller-gikere . is intended to be used for testing samples from allergists.
I reagenskombinasjonen kan det inngå en ampulle The reagent combination may include an ampoule
eller lignende med merkede antilegemer mot reagin-Ig, fortrinnsvis i tørket, f.eks. lyofilisert form. or similar with labeled antibodies against reagin-Ig, preferably in dried form, e.g. lyophilized form.
Bestemmelsesmetoden for reagin-Ig skal i det følg-ende beskrives med eksempel, som mere detaljert viser fremstil-lingen av merkede antilegemer mot reagin-Ig og hvorledes de kan anvendes ved bestemmelse av reagin-Ig. The determination method for reagin-Ig shall in the following be described with an example, which shows in more detail the production of labeled antibodies against reagin-Ig and how they can be used for the determination of reagin-Ig.
Eksempel 1. Example 1.
Bestemmelse av reagin- Ig rettet mot et vekstallergen ( Bjerkepollen) A. Fremstilling av partikler med kjemisk bundet allergen. Determination of reagin-Ig directed against a plant allergen (Bjerkepollen) A. Production of particles with chemically bound allergen.
Som utgangsmateriale anvendes finkornede partikler bestående av dekstran tverrbundet med glyceroleter-broer ("Sephadex G 25", superfine) og som substitueres med isotiocya-natofenoksyhydroksypropylgrupper (R. Axén og J. Porath, Acta Chem. Sean., 18 (1964), side 2193). Til en suspensjon av 100 mg av partiklene i en vannoppløsning av natriumhydrogenkarbonat ble det satt 1 ml allergenoppløsning (kommersielt tilgjengelig allergenekstrakt) med en omtrentlig konsentrasjon 0,2 mg allergen pr. ml. Blandingen ble inkubert 3 døgn ved værelsestemperatur under konstant langsom vertikal rotasjon. Partiklene ble deretter sentrifugert og vasket to ganger med hver gang 0,5 M NaHCO^, 0,1 M acetatpuffer, 0,1 M trispuffer med 1% bovint serumalbumin. Partiklene ble homogenisert og suspendert i 0,1 M trispuffer med 1% bovint serumalbumin. As starting material, fine-grained particles consisting of dextran cross-linked with glycerol ether bridges ("Sephadex G 25", superfine) and substituted with isothiocyanatophenoxyhydroxypropyl groups are used (R. Axén and J. Porath, Acta Chem. Sean., 18 (1964), page 2193). To a suspension of 100 mg of the particles in a water solution of sodium hydrogen carbonate was added 1 ml of allergen solution (commercially available allergen extract) with an approximate concentration of 0.2 mg of allergen per ml. The mixture was incubated for 3 days at room temperature under constant slow vertical rotation. The particles were then centrifuged and washed twice each time with 0.5 M NaHCO 3 , 0.1 M acetate buffer, 0.1 M Tris buffer with 1% bovine serum albumin. The particles were homogenized and suspended in 0.1 M Tris buffer with 1% bovine serum albumin.
B. Fremstilling av reagin- Ig B. Preparation of reagin-Ig
Prøve av opptinet eller nylig oppsamlet plasma fra Sample of thawed or recently collected plasma from
en pasient med myelomatosis ble fortynnet med 0,15 M natrium-klorid til et innhold på omkring 15 mg/ml med hensyn på M-komponenten (reagin-Ig). Utfelling med vannfritt natriumsulfat (18 g/100 ml) ble gjort ved 25°C. Etter en time oppsamles fel-lingen ved sentrifugering (10.000 x g, 20 minutter, 25°C). Sedimentet ble tatt vare på, vasket med natriumsulfatoppløsning a patient with myelomatosis was diluted with 0.15 M sodium chloride to a content of about 15 mg/ml with respect to the M component (reagin-Ig). Precipitation with anhydrous sodium sulfate (18 g/100 ml) was done at 25°C. After one hour, the precipitate is collected by centrifugation (10,000 x g, 20 minutes, 25°C). The sediment was preserved, washed with sodium sulfate solution
(18 g/100 ml) og ble oppløst i 0,8 volum 0,1 M natriumfosfatpuffer, pH 7,5- Materialet ble gjenutfelt en gang, som'omtalt. Etter siste utfelling ble sedimentet oppløst i 0,1 M tris-HCl-puffer, pH 8,0 (20°C) og anbragt på en kolonne (3,2 x 30 cm) (18 g/100 ml) and was dissolved in 0.8 volume of 0.1 M sodium phosphate buffer, pH 7.5 - The material was reprecipitated once, as mentioned. After the last precipitation, the sediment was dissolved in 0.1 M tris-HCl buffer, pH 8.0 (20°C) and placed on a column (3.2 x 30 cm)
med DEAE-"Sephadex" A-50 ekvilibrert.med samme puffer. Eluering av materialet ble utført med en kontinuerlig gradient fra 0,1 M til 1 M tris-HCl ved konstant pH på 8,0 ved 20°C. Fraksjoner inneholdende reagin-Ig ble oppbevart og konsentrert og anbragt på en kolonne (3,2 x 95 cm) med "Sephadex" G 150 ekvi-librert med 0,1 M tris-HCl- 0,2 M NaCl - 0,02 M EDTANa2 pH 7,7 og inneholdende 0,02% natriumazid. Materialet fikk passere gjennom kolonnen tre ganger (resirkulasjonskromatografimetoden): Fraksjoner inneholdende reagin-Ig ble tatt vare på og konsentrert ved ultrafiltrering (Visking tubing 8/32") ved 4°C. with DEAE-"Sephadex" A-50 equilibrated.with the same buffer. Elution of the material was performed with a continuous gradient from 0.1 M to 1 M tris-HCl at a constant pH of 8.0 at 20°C. Fractions containing reagin-Ig were saved and concentrated and applied to a column (3.2 x 95 cm) of "Sephadex" G 150 equilibrated with 0.1 M tris-HCl - 0.2 M NaCl - 0.02 M EDTANa2 pH 7.7 and containing 0.02% sodium azide. The material was passed through the column three times (recirculation chromatography method): Fractions containing reagin-Ig were saved and concentrated by ultrafiltration (Visking tubing 8/32") at 4°C.
Reagin-Ig fremstilt som beskrevet var forurenset med mindre enn 0, 1% av totalt protein med andre immunglobuliner (IgA, IgD, IgG og IgM). Renset reagin-Ig ga en eneste topp Reagin-Ig prepared as described was contaminated with less than 0.1% of total protein by other immunoglobulins (IgA, IgD, IgG and IgM). Purified reagin-Ig gave a single peak
ved elektroforese pH 8,6 I = 0,05 vandre i B-a-området. Ultra-sentrifugeringsstudier viste en eneste grenselinje og sedimen-tas j onskonstanten S°£o. U n i W, ble beregnet til 8,20s. Molekylvekten for reagin-Ig ble beregnet til 200.000 ± 5000 ved anvendelse by electrophoresis pH 8.6 I = 0.05 wander in the B-a area. Ultra-centrifugation studies showed a single boundary line and the sedimentation constant S°£o. U n i W, was calculated at 8.20s. The molecular weight of reagin-Ig was calculated to be 200,000 ± 5,000 using
av et partielt spesifikt volum på 0,713 cm^/g bestemt på basis av aminosyre- og kullhydratsammensetningen for reagin-Ig. Dif-fusjonskonstanten, D°on . ble beregnet til 3,71 x 10 cm /sek. of a partial specific volume of 0.713 cm^/g determined on the basis of the amino acid and carbohydrate composition of reagin-Ig. The diffusion constant, D°on . was calculated at 3.71 x 10 cm/sec.
du ,w you, w
Reduksjonseksperimentet viste at reagin-Ig består av to typer av polypeptidkjeder og således ligner andre serum-immunglobuliner. Bevis har fremkommet for nærvær av to lette polypeptidkjeder med molekylvekter 22600 i reagin-Ig. Molekylvekten for de tunge polypeptidkjedene innbefattende dets pros-tetiske kullhydratgruppe(r) ble beregnet til å være omkring 77»^00 under antagelse av molforhold på 1 : 1 for tunge og lette peptidkjeder i nativt reagin-Ig. Aminosyre- og kullhydratsammensetningen vises i tabell 1. The reduction experiment showed that reagin-Ig consists of two types of polypeptide chains and thus resembles other serum immunoglobulins. Evidence has emerged for the presence of two light polypeptide chains with molecular weights of 22600 in reagin-Ig. The molecular weight of the heavy polypeptide chain including its prosthetic carbohydrate group(s) was calculated to be about 77%, assuming a molar ratio of 1:1 for heavy and light peptide chains in native reagin-Ig. The amino acid and carbohydrate composition is shown in Table 1.
C. Isolering av fragment av reagin-Ig inneholdende klasse- spesifikke determinanter ( såkalt Fc- fragment) C. Isolation of fragment of reagin-Ig containing class-specific determinants (so-called Fc fragment)
100 mg reagin-Ig fremstilt ifølge eksempel 1 B i 0,1 M natriumfosfatpuffer (pH 7,0) med 0,1 M cystein ble inkubert ved 37°C i 4 timer med 1 mg papain, hvorpå reaksjonen ble avbrutt ved tilsetning av jodacetamidoppløsning til en slutt-konsentrasjon på 0,05 M. Reaksjonsblandingen ble fraksjonert ved gelfiltrering ("Sephadex G 150"), idet det ble anvendt et 100 mg of reagin-Ig prepared according to example 1 B in 0.1 M sodium phosphate buffer (pH 7.0) with 0.1 M cysteine was incubated at 37°C for 4 hours with 1 mg of papain, after which the reaction was stopped by the addition of iodoacetamide solution to a final concentration of 0.05 M. The reaction mixture was fractionated by gel filtration ("Sephadex G 150"), using a
sampolymerisat av dekstran og epiklorhydrin med vannopptag-ningsevne 15 g pr. g tørrstoff og eluering av gelmassen foregikk ved 0,1 M tris-HCl, 0,2 M NaCl-puffer (pH 7,7). Ved ana-lyse ble det fastslått den fraksjon som inneholdt Fc-fragment, som deretter ble isolert og anvendt for immunisering. D. Fremstilling av antilegemer mot Fc-fragmentet av reagin- Ig. copolymer of dextran and epichlorohydrin with a water absorption capacity of 15 g per g dry matter and elution of the gel mass took place with 0.1 M tris-HCl, 0.2 M NaCl buffer (pH 7.7). By analysis, the fraction containing the Fc fragment was determined, which was then isolated and used for immunization. D. Production of antibodies against the Fc fragment of reagin-Ig.
Kaniner ble sprøytet intramuskulært med hver en blanding av 0,5 mg Fc-fragment av reagin-Ig i 0,2 ml, 0,15 M natriumkloridoppløsning og 0,8 ml komplett Freunds adjuvant. Immuniseringen ble gjentatt etter 14 dager og deretter hver uke i tre uker. Etter ytterligere 10 dager ble det tappet blod av kaninene og antiserum ble fremstilt fra blodet ved at dette fikk koagulere og blodkoagulatet derpå fraskiltes. Rabbits were injected intramuscularly with each a mixture of 0.5 mg Fc fragment of reagin-Ig in 0.2 ml, 0.15 M sodium chloride solution and 0.8 ml complete Freund's adjuvant. The immunization was repeated after 14 days and then every week for three weeks. After a further 10 days, the rabbits were bled and antiserum was prepared from the blood by allowing it to coagulate and the blood coagulum was then separated.
Antiserumet ble gjort spesifikt ved absorbsjon i under 1 time ved +37°C og 16 timer ved + 4°C ved tilsetning av en del normalt humanserum til en del av antiserumet. Etter sentrifugering ble det prøvet spesifikiteten hos dette antiserum ved immun-elektroforese mot normalt humanserum og med Ochterlonygeldiffusjonsanalyse mot rene immunglobuliner A, G og M, såkalt lette polypeptidkjeder fra normalt polart immunglobulin G samt Bence-Jones-proteiner, hvorved det viste seg å være spesifikt for reagin-Ig. The antiserum was made specific by absorption for less than 1 hour at +37°C and 16 hours at +4°C by adding one part of normal human serum to one part of the antiserum. After centrifugation, the specificity of this antiserum was tested by immuno-electrophoresis against normal human serum and with Ochterlony gel diffusion analysis against pure immunoglobulins A, G and M, so-called light polypeptide chains from normal polar immunoglobulin G as well as Bence-Jones proteins, whereby it proved to be specific for reagin-Ig.
Fra dette antiserum ble det isolert antilegemer rettet mot Fc-fragmentet på følgende måte: 2 gram av et kompleks mellom bromacetylcellulose og reagin-Ig ble blandet med 34 ml oppløsning inneholdende ca. 95 mg immunglobulin isolert fra antiserum rettet mot Fc-fragment ved ionoutvekslerkromato-grafi. Den dannede suspensjonen ble aktivert ved pH 7 og 4°C From this antiserum, antibodies directed against the Fc fragment were isolated in the following way: 2 grams of a complex between bromoacetyl cellulose and reagin-Ig was mixed with 34 ml of solution containing approx. 95 mg immunoglobulin isolated from antiserum directed against Fc fragment by ion exchange chromatography. The resulting suspension was activated at pH 7 and 4°C
i 2 timer, hvoretter det ble sentrifugert med 20.000 xg i 20 minutter ved 10°C. Overvæsken ble fraskilt, cellulosen ble vasket med 0,15 M NaCl - 8 M karbamid, pH 6 - 7, sentrifugert for 2 hours, after which it was centrifuged at 20,000 xg for 20 minutes at 10°C. The supernatant was separated, the cellulose was washed with 0.15 M NaCl - 8 M carbamide, pH 6 - 7, centrifuged
og overvæsken fraskilt. Denne vaskningsprosedyre ble gjentatt til overvæsken ble fri for protein. Celluloseproteinkomplekset ble blandet med 9 ml 0,1 M glycin-HCl-puffer (pH 3,1) og inkubert under omrøring ved 37°C i 40 minutter. Det hele ble sentrifugert og overvæsken dialysert mot 1200 ml 0,01 M tris-HCl 0,1 M NaCl (pH 7,1) ved 4°C i 48 timer. Denne oppløsning inneholdt antilegemer i en mengde på ca. 1. mg pr. ml, som er spesifikke for Fc-fragmentet av reagin-Ig og således også mot and the supernatant separated. This washing procedure was repeated until the supernatant became free of protein. The cellulose protein complex was mixed with 9 ml of 0.1 M glycine-HCl buffer (pH 3.1) and incubated with agitation at 37°C for 40 minutes. The whole was centrifuged and the supernatant dialyzed against 1200 ml of 0.01 M tris-HCl 0.1 M NaCl (pH 7.1) at 4°C for 48 hours. This solution contained antibodies in an amount of approx. 1 mg per ml, which are specific for the Fc fragment of reagin-Ig and thus also against
hele reagin-Ig. Dette antilegememateriale rekker for noen 10-talls millioner bestemmelser. whole reagin-Ig. This antibody material is sufficient for some 10s of millions of determinations.
E. Merking av antilegemer spesifikke mot Fc-delen av reagin- Ig. E. Labeling of antibodies specific to the Fc portion of reagin-Ig.
Merkingen ble utført med I ifølge en fremgangsmåte omtalt av Hunter og Greenwood, Nature, bind 194 (1962), side 495- Labeling was carried out with I according to a method described by Hunter and Greenwood, Nature, vol. 194 (1962), pp. 495-
Merkingen kan også utføres på samme måte som beskrevet nedenfor i eksempel 2 for antilegemer mot reagin-Ig. The labeling can also be carried out in the same way as described below in example 2 for antibodies against reagin-Ig.
F. Bestemmelse: F. Provision:
1. 0,5 ml suspensjon av partikler med kjemisk bundet vekstallergen dannet i henhold til punkt A ovenfor, hvilken suspensjon inneholder 1 mg partikler pr. 1. 0.5 ml suspension of particles with chemically bound plant allergen formed according to point A above, which suspension contains 1 mg of particles per
ml ble innført i hvert og ett av to prøverør. ml was introduced into each of two test tubes.
2. 0,05 ml av det for prøving beregnede pasientserum 2. 0.05 ml of the patient serum intended for testing
ble satt til det ene av rørene. was added to one of the pipes.
3- 0,05 ml kontrollserum fra ikke-allergiker ble satt 3- 0.05 ml control serum from a non-allergic person was added
til det andre rør. to the other pipe.
4. Rørinnholdet ble inkubert i 5 timer ved værelsestemperatur idet rørene fikk rotere langsomt i 4. The tube contents were incubated for 5 hours at room temperature while the tubes were allowed to rotate slowly
vertikalplanet. the vertical plane.
5- Partiklene ble nedsentrifugert ved 3000 omdreininger pr. minutt i 1 minutt, hvorpå ovenstående 5- The particles were centrifuged down at 3000 revolutions per minute for 1 minute, after which the above
væske ble fjernet. liquid was removed.
6. Partiklene ble vasket tre ganger med 0,1 M trispuffer pH 7,4 med 1% bovint serum-albumin. Overvæsken ble frasuget etter hver sentrifugering. Ved hver avsugning ble det påsett at faste partikler ikke fulgte med. 7. 0,1 ml av den i henhold til punkt D ovenfor dannede oppløsning av antilegemer mot Fc-delen av 6. The particles were washed three times with 0.1 M tris buffer pH 7.4 with 1% bovine serum albumin. The supernatant was aspirated after each centrifugation. At each aspiration, it was ensured that solid particles did not follow. 7. 0.1 ml of the solution of antibodies against the Fc part of
125 125
reagm-Ig og merket med I ble tilsatt til alle reagm-Ig and labeled with I were added to all
rørene. the tubes.
8. Rørinnholdene ble inkubert 15 timer ved værelsestemperatur, idet rørene ble bragt til langsomt å 8. The tube contents were incubated for 15 hours at room temperature, the tubes being brought to a slow boil
rotere i vertikalplanet. rotate in the vertical plane.
9. Partiklene ble nedsentrifugert ved 3000 omdreininger pr. minutt i 1 minutt, hvorpå ovenstående 9. The particles were centrifuged down at 3000 rpm. minute for 1 minute, after which the above
væske ble fraseparert. liquid was separated.
10. Partiklene ble vasket tre ganger med 0,1 M tris- 10. The particles were washed three times with 0.1 M tris-
HCl-puffer (pH 7,4) med 1 vekt# bovint serumalbumin med nedsentrifugering og avsugning av overvæsken etter hver vasking. Ved hver avsugning ble HCl buffer (pH 7.4) with 1 wt# bovine serum albumin with centrifugation down and suction of the supernatant after each washing. At each sucking was
det påsett at faste partikler ikke medfulgte. it was ensured that solid particles did not accompany it.
11. Rørene ble plassert i scintillasjonsdetektor for måling av gammastråling. 11. The tubes were placed in a scintillation detector for measuring gamma radiation.
Por vurdering ble den oppnådde verdi sammenlignet med strålingen fra det ene røret med verdien på strålingen fra det annet rør som inneholdt kontrollserum fra ikke-allergiker. For evaluation, the value obtained was compared with the radiation from one tube with the value of the radiation from the other tube containing control serum from non-allergic patients.
Alternativt kan etter nedsentrifugering i henhold til punkt 9 et visst volum av overvæsken overføres i regnerør, hvoretter gammastrålingen fra merkede antilegemer mot Fc-delen fri i oppløsningen måles. Alternatively, after centrifugation according to point 9, a certain volume of the supernatant can be transferred into a rain tube, after which the gamma radiation from labeled antibodies against the Fc part free in the solution is measured.
For kvantitativ bestemmelse kan i punkt 2 ovenfor 0,05 ml prøveserum i forskjellige fortynninger tilsettes hvert og ett av et antall prøverør. Et antall rør utstyres på samme måte med forskjellige fortynninger av et standardserum. Aktivi-teten kan deretter uttrykkes i forhold til standardserumets. Eksempel 2. For quantitative determination, in point 2 above, 0.05 ml of sample serum in different dilutions can be added to each of a number of test tubes. A number of tubes are similarly equipped with different dilutions of a standard serum. The activity can then be expressed in relation to that of the standard serum. Example 2.
Merking av antilegemer mot reagin- Ig. Labeling of antibodies against reagin-Ig.
Antilegemer mot reggin- Ig. Antibodies against reggin- Ig.
Disse ble fremstilt ved immunisering av kaniner These were produced by immunization of rabbits
med reagin-Ig på tilsvarende måte som omtalt i eksempel 1 D. Radioaktivt jod ( 125 I). with reagin-Ig in a similar way as discussed in example 1 D. Radioactive iodine (125 I).
Jod-125, bærerfritt, ble oppløst som Nal i 0,1 M NaOH og anvendes fritt for reduksjonsmiddel. Iodine-125, carrier-free, was dissolved as Nal in 0.1 M NaOH and is used free of reducing agent.
Jodmonoklorid. Iodine monochloride.
Jodmonoklorid ble fremstillet ifølge J.L. Izzo, Iodine monochloride was prepared according to J.L. Izzo,
W.F. Bale, M.J. Izzo og A. Roncone, J. Biol. Chem., 239, nr. W. F. Bale, M.J. Izzo and A. Roncone, J. Biol. Chem., 239, no.
11 (1964) 3742 og jodinnholdet ble bestemt ved reduksjon av alt jod til jodid med arsenikk-trioksyd og etterfølgende ti-trering med 0,1 M AgNO-j. Stammoppløsningen av ICI inneholdt 2,54 mg I/ml i 0,02 M KC1, 2,0 M NaCl og 1,0 M HC1 og er stabil ved værelsestemperatur i det minste i 18 måneder. Før anvendelsen ble stammoppløsningen av ICI fortynnet til den nødvendige konsentrasjon. Som fortynningsmiddel ble det fremstilt en HC1-NaCl-oppløsning, hvis konsentrasjon ble justert individuelt for hvert enkelt -eksperiment for å være godt over de minimumskonsen-trasjoner av HC1 og NaCl hvor ICI er kjent for å være stabilt (R.W. Helmkamp, M.A. Conteras og W.F. Bale: Int. J. appl. Radiat. Isotopes, 18 (1967) 737). 11 (1964) 3742 and the iodine content was determined by reduction of all iodine to iodide with arsenic trioxide and subsequent titration with 0.1 M AgNO-j. The stock solution of ICI contained 2.54 mg I/ml in 0.02 M KCl, 2.0 M NaCl and 1.0 M HCl and is stable at room temperature for at least 18 months. Before use, the stock solution of ICI was diluted to the required concentration. As a diluent, an HC1-NaCl solution was prepared, the concentration of which was adjusted individually for each individual experiment to be well above the minimum concentrations of HC1 and NaCl at which ICI is known to be stable (R.W. Helmkamp, M.A. Conteras and W.F. Bale: Int. J. Appl. Radiat. Isotopes, 18 (1967) 737).
Merkningsfremgangsmåte. Marking procedure.
Joderingen er basert på Helmkamps modifikasjon av McFairlanes jodmonokloridmetode (A.S. McFairlane, Nautre, 182 The iodination is based on Helmkamp's modification of McFairlane's iodine monochloride method (A.S. McFairlane, Nautre, 182
(1958) 53) med ytterligere modifikasjoner (J.L. Izzo, W.F. Bale, M.J. Izzo og A. Roncone, J.Biol.Chem. 239, nr. 11 (1964). 3743, W.F. Bale, R.W. Helmkamp, T.P. Davis, M.J. Izzo, R.L. Gooland, M.A. Contreras og I.L. Spar, Proc.Soc. Exp.Med. 122 (1958) 53) with further modifications (J.L. Izzo, W.F. Bale, M.J. Izzo and A. Roncone, J.Biol.Chem. 239, No. 11 (1964). 3743, W.F. Bale, R.W. Helmkamp, T.P. Davis, M.J. Izzo , R.L. Gooland, M.A. Contreras, and I.L. Spar, Proc.Soc. Exp.Med. 122
(1966) 407 og R.W. Helmkamp, M.A. Contreras og W.F. Bale, Int. J. appl. Radiat. Isotopes, 18 (1967) 737). Antilegemer mot reagin-Ig (100 ,ug) ble merket med et molforhold på ICI til antilegemer mot reagin-Ig på 2 : 1 og ICI til 12^;I på 1 : 1. Følgende oppløsninger ble anvendt: Oppløsning.I: Antilegemer mot reagin-Ig ble oppløst i 0,2 M tris-HCl-puffer pH 8 til en konsentrasjon på 10,0 mg/ml. Oppløsning II: Fortynnings-midlet for stamoppløsningen av ICI ble fremstilt ved å oppløse 5,84 g NaCl i 35 ml 2,0 HC1 og 65 ml destillert vann. Oppløsning III: Den endelige jodmonokloridoppløsningen ble fremstilt ved å fortynne lOO^ul av stamoppløsningen av ICI med 20,2 ml av oppløsning II. Oppløsning IV: 44,29/Ul av radiojodidet (4,0 mCi) ble satt til 25/Ul av oppløsning III og hensatt i et isbad før anvendelse. Alle oppløsninger holdes kalde og jodineringsreaksjonen ble utført ved istemperatur. Reagensene ble tilsatt i følgende rekkefølge i et lite glassrør: Til 200^ul boratkarbonatpuffer (0,4 M, pH 9,1) og lO^ul oppløsning I (lOO^ug antilegemer mot reagin-Ig) ble satt 50^ul av oppløsning IV og blandet umiddel-bart kraftig. Etter 60 sekunder ble det tilsatt 20^,ul 0,1 M Na2S2°3' 5°/ul 2# Kl og 200/Ul 5% humanserumalbumin-blått (1966) 407 and R.W. Helmkamp, M.A. Contreras and W.F. Bale, Int. J. Appl. Radiate. Isotopes, 18 (1967) 737). Antibodies to reagin-Ig (100 µg) were labeled with a molar ratio of ICI to antibodies to reagin-Ig of 2 : 1 and ICI to 12^;I of 1 : 1. The following solutions were used: Solution.I: Antibodies to reagin-Ig was dissolved in 0.2 M tris-HCl buffer pH 8 to a concentration of 10.0 mg/ml. Solution II: The diluent for the stock solution of ICI was prepared by dissolving 5.84 g of NaCl in 35 ml of 2.0 HCl and 65 ml of distilled water. Solution III: The final iodine monochloride solution was prepared by diluting 100 µl of the stock solution of ICI with 20.2 ml of solution II. Solution IV: 44.29/µl of the radioiodide (4.0 mCi) was added to 25/µl of solution III and placed in an ice bath before use. All solutions are kept cold and the iodination reaction was carried out at ice temperature. The reagents were added in the following order in a small glass tube: To 200 µl borate carbonate buffer (0.4 M, pH 9.1) and 10 µl solution I (100 µg antibodies against reagin-Ig) 50 µl of solution was added IV and mixed immediately vigorously. After 60 seconds, 20 µl 0.1 M Na2S2°3' 5°/µl 2# Kl and 200/µl 5% human serum albumin blue were added
(HSA-blått). HSA-blått ble fremstilt ifølge Melani (F. Melani, K.M. Bartelt, R. Conrads, E.F. Pfeiffer, Z.klin.Chem. 4. årgang 1966, hefte 4). Dette ble tilsatt for å minske strålingsskader og adsorbsjon av antilegemer mot reagin-Ig mot glassveggene. (HSA blue). HSA blue was prepared according to Melani (F. Melani, K.M. Bartelt, R. Conrads, E.F. Pfeiffer, Z.klin.Chem. 4th year 1966, booklet 4). This was added to reduce radiation damage and adsorption of antibodies against reagin-Ig to the glass walls.
125 125
Preparat med I-joderte antilegemer mot reagm-Ig kan renses ved "Sephadex"-tverrbundet dekstran eller "Sepharose"-gelfiltrering (agarosegel) eller med "Dowex" - eller "Amberlite"-ionevekslere. Fraksjoner fra kolonnen ble tatt vare på omkring 4°C i 0,1 M tris-HCl-puffer pH 7,72 inneholdende 5% HSA. Fraksjonene inneholdende de merkede antilegemer mot reagin-Ig ble slått sammen og oppbevart i frosset tilstand. Preparations with I-iodinated antibodies against reagm-Ig can be purified by "Sephadex" cross-linked dextran or "Sepharose" gel filtration (agarose gel) or with "Dowex" or "Amberlite" ion exchangers. Fractions from the column were stored at about 4°C in 0.1 M tris-HCl buffer pH 7.72 containing 5% HSA. The fractions containing the labeled antibodies against reagin-Ig were pooled and stored frozen.
Denne metode ga 81,355 binding av den totale jod-mengde til antilegemer mot reagin-Ig. Det merkede immunoglo-binet inneholdt omkring 1 atom "'"^-I og omkring 1 atom <12>^-l pr. molekyl (molekylvekt 150.000) og hadde en spesifikk aktivitet rundt 16 mCi/mg. This method gave 81,355 binding of the total amount of iodine to antibodies against reagin-Ig. The labeled immunoglobulin contained about 1 atom "1"^-I and about 1 atom <12>^-1 per molecule (molecular weight 150,000) and had a specific activity around 16 mCi/mg.
Eksempel 3» Example 3»
Merking av antilegemer som er spesifikke mot Fc-fragmentet av reagin- Ig ( merking med en fluorescerende gruppe). Labeling of antibodies specific to the Fc fragment of reagin-Ig (labeling with a fluorescent group).
For merking med en fluorescerende gruppe kan antilegemer mot Fc-fragmentet av reagin-Ig fremstilt ifølge eksempel 1 D anvendes. 2 ml 0, 9%- ig vannoppløsning av NaCl og 2 ml 0,5 M vannoppløsning av natriumkarbonat-natriumhydrogenkarbonat-puffer med pH 9 ble blandet. Antilegemene ble oppløst i denne blanding i slik mengde at en enprosentig oppløsning av antilegemer fremkom. 3 mg fluoresceinisotiocyanat ble adsorbert på 30 mg kiselgur ("Ce.lite") tilsettes. Blandingen ble rystet i 3 minutter, hvoretter den ble sentrifugert ved 2000 omdreininger pr. minutt i 5. minutter. Den overstående klare oppløsning, hvilken inneholdt de merkede antilegemer, ble oppbevart. For å rense de merkede antilegemer som hadde reagert med fluoresceinisotio-cyanatet, fra ikke-reagert fluoresceinisotiocyanat, ble det ut-ført gelfiltrering for å separere de høymolekylære, merkede antilegemer fra lavmolekylære forurensninger. For dette formål ble det anvendt en kolonne (lengde 15 cm, diameter 1 cm), som var fylt med gelpartikler av et uoppløselig men i vann svell-bart gelfiltreringsmateriale bestående av dekstran tverrbundet med epiklorhydrin ("Sephadex G 25"), idet gélmassen ble bragt i likevekt med 0,9%-ig vannoppløsning av NaCl. Elueringen foregikk med 0, 1%- ig vandig oppløsning av NaCl. For labeling with a fluorescent group, antibodies against the Fc fragment of reagin-Ig prepared according to example 1 D can be used. 2 ml of 0.9% aqueous solution of NaCl and 2 ml of 0.5 M aqueous solution of sodium carbonate-sodium hydrogencarbonate buffer of pH 9 were mixed. The antibodies were dissolved in this mixture in such an amount that a one percent solution of antibodies appeared. 3 mg of fluorescein isothiocyanate was adsorbed on 30 mg of diatomaceous earth ("Ce.lite") is added. The mixture was shaken for 3 minutes, after which it was centrifuged at 2000 rpm. minute for 5 minutes. The supernatant clear solution, which contained the labeled antibodies, was saved. In order to purify the labeled antibodies that had reacted with the fluorescein isothiocyanate from unreacted fluorescein isothiocyanate, gel filtration was performed to separate the high molecular weight labeled antibodies from low molecular weight contaminants. For this purpose, a column (length 15 cm, diameter 1 cm) was used, which was filled with gel particles of an insoluble but water-swellable gel filtration material consisting of dextran cross-linked with epichlorohydrin ("Sephadex G 25"), the gel mass being brought into equilibrium with a 0.9% water solution of NaCl. The elution took place with a 0.1% aqueous solution of NaCl.
De merkede antilegemer som ble eluert i en fraksjon før de lavmolekylære forurensninger ble oppbevart, i frosset tilstand. De dannede, merkede antilegemer hadde kraftig fluorescens i ultrafiolett lys og kan anvendes for påvisning og bestemmelse av reagin-Ig. They labeled antibodies that were eluted in a fraction before the low-molecular-weight contaminants were stored, in a frozen state. The formed, labeled antibodies had strong fluorescence in ultraviolet light and can be used for the detection and determination of reagin-Ig.
Claims (2)
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE1229767A SE341239B (en) | 1967-09-06 | 1967-09-06 |
Publications (2)
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NO138182B true NO138182B (en) | 1978-04-10 |
NO138182C NO138182C (en) | 1978-07-26 |
Family
ID=20295495
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Application Number | Title | Priority Date | Filing Date |
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NO345868A NO127685B (en) | 1967-09-06 | 1968-09-05 | |
NO247772A NO138182C (en) | 1967-09-06 | 1972-07-11 | ANTIBODIES SPECIFICALLY AIMED AT IMMUNGLOBULIN E (IGE) OR AGAINST FRAGMENT OF IGE. |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
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NO345868A NO127685B (en) | 1967-09-06 | 1968-09-05 |
Country Status (13)
Country | Link |
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JP (1) | JPS50892B1 (en) |
AT (2) | AT295744B (en) |
BE (1) | BE720341A (en) |
CH (1) | CH534878A (en) |
CS (1) | CS191857B2 (en) |
DK (1) | DK131400B (en) |
ES (1) | ES357863A1 (en) |
FI (1) | FI48785C (en) |
FR (1) | FR1588874A (en) |
GB (1) | GB1248764A (en) |
NL (1) | NL162209C (en) |
NO (2) | NO127685B (en) |
SE (1) | SE341239B (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
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USRE31006E (en) * | 1968-09-24 | 1982-08-03 | Akzona Incorporated | Process for the demonstration and determination of reaction components having specific binding affinity for each other |
NL154600B (en) * | 1971-02-10 | 1977-09-15 | Organon Nv | METHOD FOR THE DETERMINATION AND DETERMINATION OF SPECIFIC BINDING PROTEINS AND THEIR CORRESPONDING BINDABLE SUBSTANCES. |
US3966897A (en) * | 1973-04-02 | 1976-06-29 | Marine Colloids, Inc. | Medium for use in bioassay and method of using same |
US4041146A (en) * | 1975-05-01 | 1977-08-09 | General Electric Company | Method for detection of biological particles |
GB1555142A (en) * | 1975-08-14 | 1979-11-07 | Sinai School Medicine | Blood cell typing and compatibility test procedure |
AT343822B (en) * | 1976-08-20 | 1978-06-26 | Immuno Ag | RADIOIMMUNOLOGICAL METHOD AND EQUIPMENT FOR DETERMINING ANTIGENES |
US4289748A (en) * | 1979-05-31 | 1981-09-15 | United States Of America | Ultrasensitive enzymatic radioimmunoassay method |
FR2502786B1 (en) * | 1981-03-24 | 1985-06-21 | Stallergenes Laboratoire | METHOD FOR FIXING ANTIGENS AND ANTIBODIES TO POLYSACCHARIDE SUPPORT, AND USE OF THE PRODUCT OBTAINED THEREFOR FOR IMMUNOASSAYS |
US4481298A (en) * | 1981-04-13 | 1984-11-06 | Amf Incorporated | Pre-precipitated double antibody immunoassay method |
NL8102178A (en) * | 1981-05-02 | 1982-12-01 | Stichting Centraal Lab | METHOD FOR DEMONSTRATING ANTI-ANTIGENS AGAINST CERTAIN ANTIGENS, EQUIPMENT AND REAGENTS FOR CARRYING OUT THIS METHOD |
JPS6199195U (en) * | 1984-11-30 | 1986-06-25 |
-
1967
- 1967-09-06 SE SE1229767A patent/SE341239B/xx unknown
-
1968
- 1968-08-28 FR FR1588874D patent/FR1588874A/fr not_active Expired
- 1968-08-30 DK DK418468A patent/DK131400B/en not_active IP Right Cessation
- 1968-09-02 CH CH1313868A patent/CH534878A/en not_active IP Right Cessation
- 1968-09-03 BE BE720341D patent/BE720341A/xx not_active IP Right Cessation
- 1968-09-03 GB GB4180168A patent/GB1248764A/en not_active Expired
- 1968-09-04 NL NL6812559A patent/NL162209C/en not_active IP Right Cessation
- 1968-09-05 NO NO345868A patent/NO127685B/no unknown
- 1968-09-05 FI FI249868A patent/FI48785C/en active
- 1968-09-06 CS CS626568A patent/CS191857B2/en unknown
- 1968-09-06 AT AT359370A patent/AT295744B/en not_active IP Right Cessation
- 1968-09-06 AT AT873168A patent/AT288593B/en not_active IP Right Cessation
- 1968-09-06 JP JP6421968A patent/JPS50892B1/ja active Pending
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1969
- 1969-09-05 ES ES357863A patent/ES357863A1/en not_active Expired
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1972
- 1972-07-11 NO NO247772A patent/NO138182C/en unknown
Also Published As
Publication number | Publication date |
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DK131400C (en) | 1975-11-24 |
CH534878A (en) | 1973-03-15 |
AT295744B (en) | 1972-01-10 |
FI48785C (en) | 1974-12-10 |
NL162209B (en) | 1979-11-15 |
SE341239B (en) | 1971-12-20 |
CS191857B2 (en) | 1979-07-31 |
DE1798170B1 (en) | 1972-04-27 |
FI48785B (en) | 1974-09-02 |
DK131400B (en) | 1975-07-07 |
NL162209C (en) | 1980-04-15 |
NO138182C (en) | 1978-07-26 |
ES357863A1 (en) | 1970-04-01 |
BE720341A (en) | 1969-02-17 |
AT288593B (en) | 1971-03-10 |
NL6812559A (en) | 1969-03-10 |
GB1248764A (en) | 1971-10-06 |
JPS50892B1 (en) | 1975-01-13 |
NO127685B (en) | 1973-07-30 |
FR1588874A (en) | 1970-03-16 |
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