DK145286B - METHOD FOR APPLYING A MEASURED QUANTITY OF A WATER SOLUBLE COUNTER WATER DISPERSIBLE REAGENT TO A WATER SOLID MEDIUM AND RELATED PROCEDURE TO ANALYSIS OF A TEST - Google Patents
METHOD FOR APPLYING A MEASURED QUANTITY OF A WATER SOLUBLE COUNTER WATER DISPERSIBLE REAGENT TO A WATER SOLID MEDIUM AND RELATED PROCEDURE TO ANALYSIS OF A TEST Download PDFInfo
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Description
(19) DANMARK (8)(19) DENMARK (8)
W (12) FREMLÆGGELSESSKRIFT on 145286 BW (12) PUBLICATION NOTICE on 145286 B
DIREKTORATET FOR PATENT- OG VAREMÆRKEVÆSENETDIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM
(21) Ansøgning nr. l46l/76 (51) IntCI* Β 01 J 4/02 (22) Indleveringsdag 50 · mar. 1976 6 01 Ν 31/00 (24) Løbedag 50. mar. 1976 (41) Aim. tilgængelig 1 . okt. 1977 (44) Fremlagt 25 · okt. 1982 (86) International ansøgning nr. -(86) International indleveringsdag -(85) Videreførelsesdag -(62) Stamansøgning nr. -(30) Prioritet - (71) Ansøger FMC CORPORATION, Wilmington, US.(21) Application No. l46l / 76 (51) IntCI * Β 01 J 4/02 (22) Filing day 50 · Mar. 1976 6 01 Ν 31/00 (24) Race day 50 Mar 1976 (41) Aim. available 1. October 1977 (44) Posted 25 Oct. 1982 (86) International Application No. - (86) International Filing Day - (85) Continuation Day - (62) Stock Application No. - (30) Priority - (71) Applicant FMC CORPORATION, Wilmington, US.
(72) Opfinder Donald Walter Renn, US.(72) Inventor Donald Walter Renn, US.
(74) Fuldmægtig Internationalt Patent-Bureau.(74) International Patent Bureau.
(54) Middel til tilføring af en afmålt mængde af et van&opløsellgt eller i vand dispergerbart reagens til et vandholdigt fast medium og til= hørende fremgangsmåde til analyse af et prøvestof.(54) Agent for applying a metered amount of a water-soluble or water-dispersible reagent to an aqueous solid medium and associated method for analyzing a test substance.
Den foreliggende opfindelse angår et middel til tilføring af en afmålt mængde af et vandopløseligt eller i vand dispergerbart reagens til et vandholdigt fast medium til anvendelse ved molekular-2 diffusions- eller affinitetsseparationsfremgangsmåder, og opfindel- D sen angår desuden en tilhørende fremgangsmåde til analyse af et prø- ^ vestof.The present invention relates to a means for applying a metered amount of a water-soluble or water-dispersible reagent to an aqueous solid medium for use in molecular-2 diffusion or affinity separation methods, and the invention further relates to a method for analyzing a test- ^ vestof.
f) Der er udviklet mange forskellige analysefremgangsmåder til fraskillelse og identificering af forskellige i et prøvestof tilstedeværende molekylarter, hvorved man anbringer prøvestoffet i kon- ^ takt med et vandholdigt fast medium og fremkalder molekulardiffusionf) Many different assay methods have been developed for separating and identifying different molecular species present in a test substance, placing the test substance in contact with an aqueous solid medium and eliciting molecular diffusion.
JJ
af prøvestoffet gennem mediet. Der er navnlig blevet anvendt chroma-tografi- og elektroforesefremgangsmåder, herunder immunoelektrofore- 145286 2 sefremgangsmåder, hvilke fremgangsmåder alle giver adskillelse af forskellige molekylarter ved differentialdiffusion af et prøvestof gennem et vandholdigt fast medium. Ved sådanne fremgangsmåder kan der også til mediet tilføres mange forskellige reagenser, som reagerer med én eller flere af molekylarterne i prøvestoffet, før, under eller efter adskillelsesprocessen for at medvirke til adskillelse eller identificering af molekylarterne.of the test substance through the medium. In particular, chromatography and electrophoresis methods, including immunoelectrophoresis methods, have been used, all of which give rise to different molecular species by differential diffusion of a test substance through an aqueous solid medium. In such methods, many different reagents which react with one or more of the molecular species in the test substance may also be added to the medium before, during or after the separation process to assist in the separation or identification of the molecular species.
Disse reagenser er tidligere blevet indført i mediet på forskellige måder. I nogle tilfælde er de blevet indført i mediet på dettes fremstillingstidspunkt, men i de fleste tilfælde er de blevet tilført til overfladen af det vandholdige faste medium på det tidspunkt, hvor der har været brug for dem, enten ved anbringelse af en flydende opløsning eller dispersion af reagenset på overfladen af mediet og henstilling heraf eller ved neddypning af det faste medium i en opløsning eller en dispersion af reagenset i et passende flydende bæremiddel. I begge tilfælde er nøjagtig måling og regulering af mængden og anbringelsen i mediet, ind i hvilket reagenset diffunde-rer, vanskelig og ubestemt. Det er også blevet foreslået at disperge-re sådanne reagenser i et fast vandbestandigt bindemiddel, såsom af Rey et al. i USA-patentskrift nr. 3.630.957 og af Sherelis i USA-patentskrift nr.3.694.163, og at holde et reagensholdigt fast vandu-opløseligt bindemiddel i kontakt med overfladen af et vandholdigt eller absorberende medium, såsom papir, som af Verbeck i USA-patentskrift nr. 3.672.845. Reagenser i vandbestandige eller vanduopløselige bindemidler kan imidlertid ikke let ekstraheres og diffundere ind i et vandholdigt fast medium, hvilket gør regulering og måling af den i mediet indførte mængde uigennemførlig i praksis.These reagents have previously been introduced into the medium in various ways. In some cases, they have been introduced into the medium at the time of manufacture, but in most cases they have been applied to the surface of the aqueous solid medium at the time they have been needed, either by applying a liquid solution or dispersion. of the reagent on the surface of the medium and recommendation thereof, or by immersing the solid medium in a solution or dispersion of the reagent in a suitable liquid carrier. In both cases, accurate measurement and control of the amount and placement in the medium into which the reagent diffuses is difficult and undetermined. It has also been proposed to disperse such reagents in a solid water resistant binder such as by Rey et al. in U.S. Patent No. 3,630,957 and by Sherelis in U.S. Patent No. 3,694,163, and keeping a reagent-containing, water-insoluble binder in contact with the surface of an aqueous or absorbent medium, such as paper, as by Verbeck in U.S. Patent No. 3,672,845. However, reagents in water-resistant or water-insoluble binders cannot easily be extracted and diffused into an aqueous solid medium, making regulation and measurement of the amount introduced into the medium impracticable in practice.
Ifølge opfindelsen tilvejebringes der et middel og en fremgangsmåde, som letter nøjagtig og kvantitativ indføring af reagens i et fast vandholdigt medium til anvendelse ved molekulardiffusions- eller affinitetsseparationsfremgangsmåder.Opfindelsen er særlig anvendelig i forbindelse med et medium i form af et tyndt lag, dvs. et vandholdigt fast medium i form af et lag med en tykkelse på 0,1 til 2 mm.According to the invention, there is provided an agent and method which facilitates accurate and quantitative introduction of reagent into a solid aqueous medium for use in molecular diffusion or affinity separation methods. The invention is particularly useful in connection with a thin layer medium, i.e. an aqueous solid medium in the form of a layer having a thickness of 0.1 to 2 mm.
Midlet ifølge opfindelsen er ejendommeligt ved, at det omfatter en fast film bestående i det væsentlige af et filmdannende, fast, organisk, polymert bindemiddel, der er opløseligt i vand i et omfang på mindst 1 vægt% ved 20°C, og inkorporeret i nævnte bin-demiddelfilm en afmålt mængde af nævnte reagens, hvorhos filmen er af en sådan størrelse og form, at den er egnet til at anbringes i kontakt med nævnte medium til muliggørelse af, at nævnte reagens U5286 3 og bindemiddel diffunderer fuldstændigt ind i mediet.The composition of the invention is characterized in that it comprises a solid film consisting essentially of a film-forming solid organic polymeric binder which is soluble in water to at least 1% by weight at 20 ° C and incorporated in said binder film is a metered amount of said reagent wherein the film is of such size and shape that it is suitable for contact with said medium to enable said reagent U5286 3 and binder to completely diffuse into the medium.
En hensigtsmæssig udførelsesform for midlet ifølge opfindelsen består i, at det desuden omfatter en bærende bagklædning af vanduopløseligt syntetisk plast bundet til den ene overflade af nævnte film.A convenient embodiment of the agent according to the invention consists in that it further comprises a backing of water-insoluble synthetic plastic bonded to one surface of said film.
Ifølge opfindelsen tilvejebringes der også en fremgangsmåde til analyse af et prøvestof, ved hvilken man underkaster prøvestoffet en molekulardiffusionsseparationsproces i et vandholdigt fast medium og bringer bestanddele af prøvestoffet til at reagere med et reagens i nævnte medium, hvilken fremgangsmåde er ejendommelig ved, at man anbringer den ene side af det ovenfor beskrevne middel ifølge opfindelsen, med eller uden nævnte bagklædning, i kontakt med nævnte medium og holder den i kontakt i tilstrækkelig tid til at muliggøre, at nævnte reagens og bindemiddel diffunderer fuldstændigt ind i mediet.According to the invention there is also provided a method for analyzing a test substance by which the test substance is subjected to a molecular diffusion separation process in an aqueous solid medium and causes components of the test substance to react with a reagent in said medium which is characterized by applying it. one side of the above-described agent of the invention, with or without said backing, in contact with said medium and keeping it in contact for a sufficient time to allow said reagent and binder to completely diffuse into the medium.
Ved bindemidler, der er "opløselige i vand",skal der forstås sådanne materialer, som danner kolloide opløsninger eller dispersioner, såvel som sådanne, der danner ægte opløsninger.Binders which are "soluble in water" are to be understood as materials which form colloidal solutions or dispersions, as well as those which form true solutions.
De bindemidler, der kan anvendes i midlet ifølge opfindelse, omfatter forskellige polymere materialer, såsom dextran, vandopløseligt polyacrylamid, polyacrylsyre og vandopløselige metalsalte heraf, vandopløselig polyvinylalkohol, polyethylenglycol, poly-ethylenoxid, polyvinylpyrrolidon, klaret guargummi, vandopløselig carboxymethylcellulose, vandopløselig hydroxyethylcellulose, vandopløselig methylcellulose, algin, carrageenan, xanthangummi, stivelse, vandopløselige copolymere af maleinsyreanhydrid med forskellige vinylmonomere som beskrevet f.eks. i USA-patentskrift nr. 2.047.398, navnlig copolymere af maleinsyreanhydrid med vinylethere eller vinylestere eller deres tilsvarende salte. Sammen med bindemidlet kan der også være konventionelle fugtighedsbindende midler eller overfladeaktive ’ midler (dispergeringsmidler) til stede til opretholdelse af bindemidlets fleksibilitet og til lettelse eller fremskyndelse af dets dispergering eller opløsning i vand.The binders which may be used in the agent of the invention include various polymeric materials such as dextran, water-soluble polyacrylamide, polyacrylic acid and water-soluble metal salts thereof, water-soluble polyvinyl alcohol, polyethylene glycol, polyethylene oxide, polyvinylpyrrolidone, water-soluble carboxyl-soluble , algin, carrageenan, xanthan gum, starch, water soluble copolymers of maleic anhydride with various vinyl monomers as described e.g. in U.S. Patent No. 2,047,398, in particular copolymers of maleic anhydride with vinyl ethers or vinyl esters or their corresponding salts. Along with the binder, conventional moisture binding agents or surfactants (dispersants) may also be present to maintain the binder's flexibility and to facilitate or accelerate its dispersion or dissolution in water.
Filmen af polymert bindemiddel kan have en vilkårlig ønsket tykkelse, men er fortrinsvis fra 0,01 til 2 mm. Den kan være forsynet med et midlertidigt, fjerneligt bærelag eller bagklædning i form af papir eller vanduopløselig plastfilm, men en sådan bagklædning eller bærelag er kun af betydning for yderst svage eller skøre film og kan udelades i tilfælde af selvbærende film. Når der anvendes et bærelag eller en bagklædning, er det/den hensigtsmæssig transparent. Egnede 145286 4 bagklædninger omfatter bagklædninger dannet af sådanne plast som polyestere, polystyren, celluloseacetat, polyamider og lignende af varierende tykkelse. Tykkelsen af bagklædningen holdes sædvanligvis mindst mulig for at holde omkostningerne lavest mulige under samtidig tilvejebringelse af den ønskede mekaniske forstærkning eller styrke. Bindingsgraden af reagens-bindemiddel-filmen til bagklædningsfilmen er ikke væsentlig. Der opnås sædvanligvis en tilstrækkelig bindingsgrad, hvis bindemiddelfilmen dannes in situ på overfladen af bagklædningsfilmen ud fra en opløsning eller en smelte.The polymeric binder film may have any desired thickness, but is preferably from 0.01 to 2 mm. It may be provided with a temporary, removable backing or backing in the form of paper or water-insoluble plastic film, but such backing or backing is of importance only for extremely weak or brittle films and may be omitted in the case of self-supporting films. When a carrier or backing is used, it is suitably transparent. Suitable backings include backings formed of such plastics as polyesters, polystyrene, cellulose acetate, polyamides and the like of varying thickness. The thickness of the backing is usually kept as low as possible to keep costs down while providing the desired mechanical strength or strength. The degree of binding of the reagent-binder film to the backing film is not significant. A sufficient degree of bonding is usually obtained if the binder film is formed in situ on the surface of the backing film from a solution or melt.
De reagenser, som kan inkorporeres i filmen af polymert bindemiddel, kan være vilkårlige af de vandopløselige eller i vand dis-pergerbare stoffer, hvoraf mange er almindelig anvendt ved prøvningsmetoder, såsom antistoffer, antigener, enzymer, enzymsubstrater, precipitin-fluorescensstoffer, farvereagenser, fældningsmidler, mikrobiologiske organismer og/eller næringsstoffer herfor, puffere, salte og lignende, samt radioaktivt mærkede eller fluorescererde reagenser af de ovennævnte typer.The reagents which can be incorporated into the polymeric binder film can be any of the water-soluble or water-dispersible substances, many of which are commonly used in assay methods such as antibodies, antigens, enzymes, enzyme substrates, precipitin fluorescents, color reagents, precipitants. , microbiological organisms and / or nutrients thereof, buffers, salts and the like, as well as radiolabelled or fluorescent reagents of the above types.
De relative mængder af reagens og vandopløseligt polymert bindemiddel i det omhandlede middel kan varieres inden for vide grænser afhængig af størrelsen eller mængden af den afmålte mængde reagens, som ønskes t °<J er et spørgsmål om valg eller hensigstmæssig-hed. Sædvanligvis er det mest hensigtsmæssigt at anvende et middel, hvori mængden af vandopløseligt polymert bindemiddel beløber sig til ca. 10 til 95 vægt% af filmen, medens reagenset udgør den resterende del. Der kan benyttes to eller flere reagenser i blanding med hinanden. En blanding af to eller flere forskellige polymere bindemidler kan også anvendes, men der er dog sædvanligvis ikke nogen fordel ved at anvende et sådan blanding.The relative amounts of reagent and water-soluble polymeric binder in the present agent may be varied within wide limits depending on the size or amount of the metered amount of reagent desired to be ° is a matter of choice or suitability. Usually, it is most convenient to use an agent in which the amount of water-soluble polymeric binder amounts to approx. 10 to 95% by weight of the film, while the reagent constitutes the remaining portion. Two or more reagents may be used in admixture with each other. A mixture of two or more different polymeric binders can also be used, but there is usually no advantage in using such a mixture.
Reagenserne kan inkorporeres i filmen af polymert bindemiddel på mange forskellige måder, idet man tager i betragtning, at det sædvanligvis er vigtigt at have reagenset fordelt så ensartet som muligt gennem hele massen af polymert bindemiddel. Reagenset kan blandes med det polymere bindemiddel, medens sidstnævnte er i smeltet form eller i form af en opløsning i et flygtigt opløsningsmiddel, hvorefter man former blandingen til en film af den ønskede tykkelse og lader den tørre eller afkøle, således at den bliver fast. Filmen af vandopløseligt polymert bindemiddel kan også dannet særskilt ud fra en opløsning af bindemidlet eller ud fra en smelte, hvorefter man kan påføre en opløsning eller dispersion af reagenset i et egnet flydende bæremiddel på overfladen af filmen og lade den diffundere 145286 5 ind i filmen og derpå tørre filmen. I nogle tilfælde kan reagenset i tør, findelt partikelform adspredes på overfladen af filmen af vandopløseligt polymert bindemiddel, hvorefter man smelter sidstnævnte og lader det størkne påny. Medens der sædvanligvis kan benyttes tørring med tvungen luftcirkulation ved dannelse af filmen og/eller inkorporering af reagenset i filmen, kan der også benyttes vakuumtørring eller frysetørring i tilfælde af varmefølsomme materialer.The reagents can be incorporated into the polymeric binder film in many different ways, considering that it is usually important to have the reagent distributed as uniformly as possible throughout the mass of polymeric binder. The reagent may be mixed with the polymeric binder while the latter is in molten form or in the form of a solution in a volatile solvent, after which the mixture is formed into a film of the desired thickness and allowed to dry or cool so that it solidifies. The film of water-soluble polymeric binder may also be formed separately from a solution of the binder or from a melt, after which a solution or dispersion of the reagent in a suitable liquid carrier may be applied to the surface of the film and allowed to diffuse into the film and then dry the film. In some cases, the reagent, in dry, finely divided particulate form, can be dispersed on the surface of the film of water-soluble polymeric binder, after which the latter is melted and resuspended. While forced air circulation drying can usually be used to form the film and / or incorporate the reagent into the film, vacuum drying or freeze drying can also be used in the case of heat sensitive materials.
Størrelsen af midlet ifølge opfindelsen er et spørgsmål om valg, afhængig af arten af den prøvemetode der skal udføres. Almindeligvis er det ikke hensigtsmæssigt at anvende midler, hvor filmens 2 dimensioner er mindre end ca. 5 mm , men mindre midler kan dog anvendes i særlige tilfælde, hvor det ønskes at afgrænse reagenset til et lille lokaliseret område på mediet, og der kan anvendes større midler og herunder sådanne, som er store nok til at dække hele den blottede overflade af det medium, hvortil reagenset skal tilføres, 2 hvilken overflade kan være af størrelsesordenen 100 cm eller endog mere. Filmen kan have en vilkårlig ønsket form, og den kan f.eks. være rørformet eller perforeret.The size of the agent according to the invention is a matter of choice, depending on the nature of the test method to be performed. Generally, it is not appropriate to use agents where the 2 dimensions of the film are less than approx. However, 5 mm but smaller agents may be used in particular cases where it is desired to confine the reagent to a small localized area of the medium and larger agents, including those large enough to cover the entire exposed surface thereof, may be used. medium to which the reagent is to be added, 2 of which may be of the order of 100 cm or more. The film may have any desired shape, and it may e.g. be tubular or perforated.
Midlet ifølge opfindelsen kan anvendes til tilføring af reagenser til et vilkårligt vandholdigt fast medium, som anvendes ved molekulardiffusions- eller affinitetsseparationsfremgangsmåder,som f.eks. chromatografi og elektroforese, navnlig sådanne medier, som er i form af tynde lag (dvs. med en maksimal tykkelse på 2 mm), hvadenten de er i form af en gel, en membran, et cellulært produkt eller et væv. Af særlig betydning er hydratiserede geler dannet af agarose, agar, polyacrylamid (tværbundet til forhindring af opløsning) eller celluloseacetat, og papir og celluloseacetatmembraner er også af betydning.The agent of the invention can be used to supply reagents to any aqueous solid medium used in molecular diffusion or affinity separation methods such as e.g. chromatography and electrophoresis, in particular, such media which are in the form of thin layers (ie, with a maximum thickness of 2 mm), whether in the form of a gel, membrane, cellular product or tissue. Of particular importance are hydrated gels formed from agarose, agar, polyacrylamide (crosslinked to prevent dissolution) or cellulose acetate, and paper and cellulose acetate membranes are also of importance.
Ved anvendelse af midlet ifølge opfindelsen anbringer man simpelthen overfladen af den reagensholdige film i kontakt med det vandholdige faste medium og lader den blive der, indtil der sker fuldstændig opløsning eller diffusion af reagenset og det polymere bindemiddel, og reagenset og bindemidlet diffunderer ind i mediet. Dette trin udføres sædvanligvis ved stuetemperatur, men der kan dog anvendes andre temperaturer fra 1°C til 60°C. Når filmen har et vand-uopløseligt bagklædningslag eller bærelag som en del af det omhandlede middel, kan man lade dette forblive på plads på overfladen af det vandholdige medium, eller man kan om ønsket fjerne det, når diffusionen af reagenset og bindemidlet er fuldstændig. Det omhandlede 145286 6 middel kan også benyttes til at indføre reagenser i medier, der anvendes til dyrkning af bakterier, og det kan anvendes til histologisk farvning ved anbringelse af et farvereagensholdigt middel i kontakt med væv.Using the agent of the invention, the surface of the reagent-containing film is simply contacted with the aqueous solid medium and left there until complete dissolution or diffusion of the reagent and polymeric binder occurs and the reagent and binder diffuse into the medium. This step is usually carried out at room temperature, however other temperatures from 1 ° C to 60 ° C can be used. When the film has a water-insoluble backing layer or carrier layer as part of the present agent, it may be left in place on the surface of the aqueous medium or, if desired, removed when the diffusion of the reagent and the binder is complete. The present agent can also be used to introduce reagents into media used for culture of bacteria, and it can be used for histological staining by applying a color reagent-containing agent to tissue.
Midlet og fremgangsmåden ifølge opfindelsen belyses nærmere ved hjælp af de efterfølgende eksempler.The agent and method according to the invention are further illustrated by the following examples.
Eksempel 1Example 1
En opløsning af mælkesyre-dehydrogenase (LDH) lokaliseringsreagens fremstilledes ud fra et i handelen gående substratfarvestof og en pufferblanding. Til 1 ml af denne opløsning sattes 9 ml af en 0,5 vægt%'s vandig opløsning af polyethylenglycol-4000-bindemiddel, og blandingen omrørtes. 5 ml af denne blandede opløsning anbragtes derefter jævnt på en 8 1/4 x 10 cm bagklædning eller bærefilm af vanduopløseligt hydrofilt polyethylenterejiithalat solgt under handelsbetegnelsen "CronaP^' og blev tørret hurtigt ved 40°C i en ovn med tvungen luftcirkulation til dannelse af en bindemiddel-LDH-film af en tykkelse på ca. 0,02 mm på bagklædningen.A solution of lactic acid dehydrogenase (LDH) localization reagent was prepared from a commercially available substrate dye and a buffer mixture. To 1 ml of this solution was added 9 ml of a 0.5% by weight aqueous solution of polyethylene glycol-4000 binder and the mixture was stirred. 5 ml of this mixed solution was then evenly applied to a 8 1/4 x 10 cm backing or carrier film of water-insoluble hydrophilic polyethylene terephthalate sold under the trade designation "CronaP" and dried quickly at 40 ° C in a forced air circulation oven to form a binder LDH film of about 0.02 mm thickness on the backing.
Et konventionelt hydratiseret agarose-elektroforesemedium (et 1 ^il 2 mm tykt lag af hydratiseret agarose på et bærelag af "Cronar*"-film) blev ækvilibreret med vandig standardpufferopløsning, podedes med humant serum og underkastede elektroforese. Efter fuldendelse af elektroforesen anbragtes LDH-bindemiddel-filmen på agarose-mediet med bagklædningen øverst, og denne sandwichslignende kombination anbragtes i en inkubator ved 37°C i 1 time. Der fremkom synlige violette linier i mediet, som viste beliggenheden af de serumkomponenter, hvormed LDH havde reageret. Bagklædningsfilmen fjernedes derefter fra overfladen af mediet, sidstnævnte vaskedes kortvarigt i rindende vand og gennemvædes derefter i 5 vægt%'s vandig eddikesyre til fiksering af de farvede linier og tørredes til dannelse af en permanent optagelse.A conventional hydrated agarose electrophoresis medium (a 1 µl 2 mm thick layer of hydrated agarose on a carrier layer of "Cronar *" film) was equilibrated with standard aqueous buffer solution, inoculated with human serum, and subjected to electrophoresis. Upon completion of the electrophoresis, the LDH binder film was placed on the agarose medium with the backing at the top, and this sandwich-like combination was placed in an incubator at 37 ° C for 1 hour. Visible violet lines appeared in the medium showing the location of the serum components with which LDH had reacted. The backing film was then removed from the surface of the medium, the latter washed briefly in running water and then soaked in 5% by weight aqueous acetic acid to fix the stained lines and dried to form a permanent uptake.
Om ønsket kan reagens-bindemiddel-filmen efter fremstillingen oplagres ubegrænset før anvendelsen ved indeslutning i en pose af 0,075 mm polyethylen og opbevaring ved 4-8°C.If desired, the reagent binder film after manufacture can be stored indefinitely before use by enclosing it in a bag of 0.075 mm polyethylene and storing at 4-8 ° C.
Eksempel 2Example 2
Man fremstillede den samme type reagens-bindemiddel-film som i eksempel 1, bortset fra at opløsningen af polyethylenglycol 4000 anbragtes særskilt på bagklædningsfilmen og tørredes. LDH-Synliggø-relsesopløsningen (1 ml) fordeltes derefter jævnt på overfladen af 145286 7 det nævnte polyethylenglycol 4000 og tørredes. Det således fremstillede middel anvendtes derefter ved samme fremgangsmåde som beskrevet i eksempel 1.The same type of reagent binder film was prepared as in Example 1 except that the solution of polyethylene glycol 4000 was applied separately to the backing film and dried. The LDH visualization solution (1 ml) was then evenly distributed on the surface of said polyethylene glycol 4000 and dried. The agent thus prepared was then used by the same procedure as described in Example 1.
Eksempel 3Example 3
Til 10 ml af en 0, 5%'s vandig opløsning af polyvinylpyrrolidon-bindemiddel sattes som reagens 0,4 ml anti-human-helserum fremstillet i geder, og blandingen omrørtes kortvarigt. 5 ml af denne blanding fordeltes på en 8 1/4 x 10 cm bagklædningsfilm som i eksempel 1 og tørredes ved 25°C i en ovn med tvungen luftcirkulation til dannelse af en ca. 0,1 mm tyk reagens-bindemiddel-film på bagklædningsfilmen.To 10 ml of a 0.5% aqueous solution of polyvinylpyrrolidone binder was added as reagent 0.4 ml of anti-human health room made in goats and the mixture was stirred briefly. 5 ml of this mixture was distributed on an 8 1/4 x 10 cm backing film as in Example 1 and dried at 25 ° C in a forced air circulation oven to form a ca. 0.1 mm thick reagent binder film on the backing film.
Det således fremstillede middel anvendtes ved anbringelse af reagens-bindemiddel-filmen i kontakt med overfladen af et 8 1/4 χ 10 cm almindeligt hydratiseret agarose-elektroforesefilmmedium forsynet med de sædvanlige prøvehuller til elektroimmunodiffusion. Efter 30 minutters forløb ved stuetemperatur blev bagklædningsfilmen fjernet, prøvehullerne tømtes for tilbageværende væske, og prøvemængder af serum indførtes i hullerne. Efter elektroforese i 45 minutter ved 100 V var der udviklet de ønskede synlige "raket"-precipitinmønstre, hvor der var indtruffet reaktion af reagenset med serumbestanddelene.The agent thus prepared was used by contacting the reagent binder film with the surface of an 8 1/4 χ 10 cm plain hydrated agarose electrophoresis film medium provided with the usual electro-immunodiffusion test holes. After 30 minutes at room temperature, the backing film was removed, the sample holes were emptied of residual fluid and samples of serum were introduced into the holes. After 45 minutes of electrophoresis at 100 V, the desired visible "rocket" precipitin patterns had been developed in which the reaction of the reagent with the serum constituents had occurred.
Eksempel 4Example 4
En reagens-bindemiddel-film fremstillet som beskrevet i eksempel 3 anbragtes i kontakt med et frisk fremstillet 1 mm tykt 1%’s-vandig agarosegelmedium indeholdende 0,85% natriumchlorid og 0,05% natriumazid, i hvilket medium der blev udskåret to huller på 2 mm i diameter til radialimmunodiffusion. Efter 30 minutters forløb ved stuetemperatur fjernedes bagklædningsfilmen, og den tilbageværende væske fjernedes fra hullerne. Prøvemængder på 5 mikroliter af humant serum anbragtes i hullerne, og gelmediet anbragtes i et fugtigheds-kairaner. Efter 24 timers forløb viste gelen de ønskede precipitinringe omkring hullerne, hvilke ringe viste beliggenheden af reaktionsprodukterne af serummet og reagenset efter diffusion.A reagent binder film prepared as described in Example 3 was placed in contact with a freshly prepared 1 mm thick 1% aqueous agarose gel medium containing 0.85% sodium chloride and 0.05% sodium azide in which two holes were excised of 2 mm in diameter for radial immunodiffusion. After 30 minutes at room temperature, the backing film was removed and the remaining liquid removed from the holes. Samples of 5 microliters of human serum were placed in the holes and the gel medium was placed in a moisture kairan. After 24 hours, the gel showed the desired precipitin rings around the holes, which showed the location of the reaction products of the serum and the reagent after diffusion.
Eksempel 5Example 5
En reagens-bindemiddel-film, der kan anvendes til de i eksempel 3 og 4 beskrevne fremgangsmåder, kan også fremstilles ved, at man blander 0,4 ml af antiseraene med 10 ml af en 0,5%'s vandig polyvinyl-pyrrolidonbindemiddelopløsning, frysetørrer den blandede opløsning 145286 8 og udspreder det frysetørrede faste stof på en plastbagklædning enten på et tyndt lag af våd polyvinylpyrrolidonfilm eller andet kontaktklæbemiddel.A reagent binder film which can be used for the methods described in Examples 3 and 4 can also be prepared by mixing 0.4 ml of the antisera with 10 ml of a 0.5% aqueous polyvinyl pyrrolidone binder solution. lyophilize the mixed solution and spread the lyophilized solid on a plastic backing either on a thin layer of wet polyvinylpyrrolidone film or other contact adhesive.
Eksempel 6Example 6
Man gik frem på samme måde som i eksempel 3/ 4 og 5, bortset fra at antigenet, normalt humant serum, anvendtes som reagenset i binde-middelfilmen, og antistoffet anvendtes som prøvestoffet.Proceed in the same manner as in Examples 3/4 and 5 except that the antigen, normally human serum, was used as the reagent in the binder film and the antibody was used as the test substance.
Eksempel 7Example 7
Man gik frem på samme måde som i eksempel 3, bortset fra at man i stedet for polyvinylpyrrolidonbindemiddel anvendte henholdsvis polyviiiylalkohol, polyethylenoxid, polyethylenglycol 20000, poly-acrylamid (vandopløseligt), natriumalginat, methylcelulose, klaret guargummi, vandopløseligt hydroxyethylcellulose, xanthangummi, opløselig stivelse og dextran som bindemiddel. Desuden anvendtes der i stedet for bagklædningsfilmen film af polyvinylidenchlorid ("Saran^ Wrap"), polycarbonat, polymethylmethacrylat og celluloseacetat, der alle er vanduopløselige. Selvbærende film af reagens-bindemiddel fremstilledes ud fra de ovennævnte blandinger ved anvendelse af polytetra-fluorethy lenf ilm ( "Tef lo'i^) som bagklædning og efterfølgende afrivning af den tørrede reagens-bindemiddel-film fra bagklædningen før anvendelse.Proceed in the same manner as in Example 3 except that polyvinylpyrrolidone binder was used instead of polyvinyl alcohol, polyethylene oxide, polyethylene glycol 20000, polyacrylamide (water soluble), sodium alginate, methyl cellulose, clarified guar gum, water soluble x dextran as a binder. In addition, instead of the backing film, films of polyvinylidene chloride ("Saran ^ Wrap"), polycarbonate, polymethyl methacrylate and cellulose acetate, all of which are water-insoluble, were used. Reagent binder self-supporting films were prepared from the aforementioned mixtures using polytetrafluoroethylene film ("Tef lO") as backing and subsequently tearing off the dried reagent binder film from the backing prior to use.
Eksempel 8Example 8
En mængde på 0,2 g af lipoprotein-farvestof "Fat Red 7B" (Sigma) opløstes i 0,2 ml polyethylenglycol 400 og blandingen indrørtes grundigt i 2 g smeltet polyethylenglycol 4000 , hvorefter man udspredte blandingen hurtigt og jævnt på en 81/4 x 10 cm bagklædningsfilm af hydrofiltpolyethylenterephthalat og lod den størkne. Filmen anvendtes uden yderligere ændring ved, at man anbragte reagens-bindemiddel-overfladen i kontakt med overfladen af et hydratiseret agarosegel-medium, hvori serumprotein på forhånd var blevet underkastet elektrofor ese. Efter 10 minutters kontakt ved stuetemperatur blev bagklædningsfilmen fjernet, hvorefter bånd af reageret lipoprotein var synlige i mediet.An amount of 0.2 g of lipoprotein dye "Fat Red 7B" (Sigma) was dissolved in 0.2 ml of polyethylene glycol 400 and the mixture was stirred thoroughly in 2 g of molten polyethylene glycol 4000, after which the mixture was spread rapidly and evenly on an 81/4 x 10 cm hydrophilic polyethylene terephthalate backing film and allowed to solidify. The film was used without further alteration by contacting the reagent-binder surface with a surface of a hydrated agarose gel medium in which serum protein had been electrophoresed beforehand. After 10 minutes of contact at room temperature, the backing film was removed, and bands of reacted lipoprotein were visible in the medium.
Ved en anden udførelsesform sattes der til reagens-bindemiddel-blandingen 0,01 g af et nonionisk overfladeaktivt middel, octylphe-noxypolyethoxy-(9yl0)-ethanol forhandlet under handelsbetegnelsen "Triton^i-100". Den således fremstillede reagens-bindemiddel-film 145286 9 udviste hurtigere diffusion ind i agarosegelmediet.In another embodiment, 0.01 g of a nonionic surfactant, octylphenoxypolyethoxy (9ylO) ethanol sold under the trade designation "Triton® i-100" was added to the reagent binder mixture. The reagent binder film thus prepared exhibited faster diffusion into the agarose gel medium.
Eksempel 9Example 9
Lige store rumfang af en barbitalpuffer med pH 8,2 og en ionstyrke på 0,08 og 1%'s vandig polyethylenglycol-4000-bindemiddel blandedes. 5 ml af denne blanding hældtes ud på overfladen af en 0,1 ran hydrofil polyethylenterephthalatbagklædningsfilm og tørredes i en ovn med tvungen luftcirkulation ved 40°C. Den opnåede, ca. 0,5 mm tykke reagens-bindemiddel-film anbragtes i kontakt med overfladen af et ca. 2 mm tykt hydratiseret agarosegelfilmmedium, idet man efterlod bagklædningsfilmen på plads på den side af reagens-bindemiddel-filmen, der vendte bort fra mediet. Efter 10 minutters forløb ved stuetemperatur fjernedes bagklaadningsfilmen, og gelmediet var parat til at modtage et prøvestof før en elektroforetisk proces.Equal volumes of a pH 8.2 barbital buffer and an ionic strength of 0.08 and 1% aqueous polyethylene glycol-4000 binder were mixed. 5 ml of this mixture was poured onto the surface of a 0.1 µm hydrophilic polyethylene terephthalate backing film and dried in a forced air oven at 40 ° C. The obtained, ca. 0.5 mm thick reagent binder films were placed in contact with the surface of a ca. 2 mm thick hydrated agarose gel film medium, leaving the backing film in place on the side of the reagent binder film facing away from the medium. After 10 minutes at room temperature, the backing film was removed and the gel medium prepared to receive a test substance prior to an electrophoretic process.
Eksempel 10Example 10
Dextran-sulfat, et specifikt fældningsmiddel for lipoprotéi-ner, kan anvendes som sådant, når det inkorporeres i en reagens-bindemiddel-f ilm. Ca. 0,5 g dextran-sulfat-reagens opløstes i 1 ml vand og udspredtes jævnt på overfladen af en forud fremstillet tørret film af polyethylenglycol-4000-bindemiddel af en tykkelse på ca.Dextran sulfate, a specific precipitant for lipoproteins, can be used as such when incorporated into a reagent binder film. Ca. 0.5 g of dextran sulfate reagent was dissolved in 1 ml of water and spread evenly on the surface of a pre-prepared dried film of polyethylene glycol-4000 binder of a thickness of approx.
0,25 mm og tørredes derefter i en ovn med tvungen luftcirkulation ved 40°C.0.25 mm and then dried in a forced air oven at 40 ° C.
Prøvemængder af humant serun underkastedes elektroforese i et ca. 1 mm tykt konventionelt hydratiseret agarosegelmedium båret på en bagklædning af hydrofil polyethylenterephthalatfilm.Efter fuldendelse af elektroforesen anbragte man overfladen af reagens-bindemiddel-f ilmen i kontakt med gelmediet, og lod det forblive der i 15 minutter ved stuetemperatur, hvorefter man vaskede mediets overflade med vand. Hvide bånd af de udfældede reaktionsprodukter af lipoproteiner med dextransulfatet var synlige i mediet, og måling af disse bånd muliggjorde en bestemmelse af mængden af lipoproteiner.Specimens of human sera were electrophoresed in a ca. 1 mm thick conventional hydrated agarose gel medium carried on a backing of hydrophilic polyethylene terephthalate film. Upon completion of the electrophoresis, the surface of the reagent binder film was contacted with the gel medium and left there for 15 minutes at room temperature, after which the medium was washed with water. White bands of the precipitated reaction products of lipoproteins with the dextran sulfate were visible in the medium, and measurement of these bands allowed a determination of the amount of lipoproteins.
Eksempel 11Example 11
En reagens-bindemiddel-film fremstilledes ved, at man fordelte 1 ml af et 1:5 alpha^-antitrypsin-antisera-reagens i 0,85% saltopløsning jævnt på overfladen af en forud fremstillet og tørret, ca.A reagent binder film was prepared by distributing 1 ml of a 1: 5 alpha 2 -antitrypsin antisera reagent in 0.85% saline evenly on the surface of a pre-prepared and dried, ca.
0,25 mm tyk film af polyethylenglycol-6000-bindemiddel. Det opnåede produkt tørredes hurtigt ved 20°C. Denne reagens-bindemiddel-film anvendtes på den i eksempel 10 beskrevne måde til humant serum af0.25 mm thick polyethylene glycol 6000 binder film. The product obtained was rapidly dried at 20 ° C. This reagent binder film was used in the manner described in Example 10 for human serum
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DK146176A DK145286C (en) | 1976-03-30 | 1976-03-30 | METHOD FOR APPLYING A MEASURED QUANTITY OF A WATER SOLUBLE COUNTER WATER DISPERSIBLE REAGENT TO A WATER SOLID MEDIUM AND RELATED PROCEDURE TO ANALYSIS OF A TEST |
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DK146176A DK145286C (en) | 1976-03-30 | 1976-03-30 | METHOD FOR APPLYING A MEASURED QUANTITY OF A WATER SOLUBLE COUNTER WATER DISPERSIBLE REAGENT TO A WATER SOLID MEDIUM AND RELATED PROCEDURE TO ANALYSIS OF A TEST |
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DK146176A DK146176A (en) | 1977-10-01 |
DK145286B true DK145286B (en) | 1982-10-25 |
DK145286C DK145286C (en) | 1983-04-05 |
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