PROCEDURE AND DIAGNOSTIC AID TO THE QUANTITATIVE IMMUNOCHEMI EXAMINATION OF ANTIGENS

The invention concerns a procedure for the quantitative immunochemischen examination of antigens as well as a diagnostic aid to the Durehführung of the procedure. The immunochemischen procedures for the quantitative regulation of antigens, used so far, are based on Tmmunodiffusion, e.g. after their inventors e.g. admitted IVIancinioder Ouchterlony procedure, or on Immunoelektrophorese, like the Elektroimmunoprüfung after Laurell. These procedures and their modifications accomplish the necessary reciprocal effect between antigen and antibody by a Diffusionsvorgang and/or a elektrophoretischen procedure. On the other hand the erfindungsgeraäße procedure is based to that on the utilization cape larkräfte between a porous substrate, are bound at which antibody, and which sample which can be examined. the le procedure according to invention consists of that the antigens containing sample with a diagnostic mechanism, those from a strip from a porous, kapi! larhaltigen substrate also to it bound antibodies exists, in contact is brought, by this absorb is let and the antigenhaltigen ranges of the mechanism then by addition by antibodies in an aqueous carrier, which are bound to a suitable water-soluble color indicator, e.g. to a fluorescent connection or to a Einzym, which catalyzes a color development reaction, be determined. The diagnostic aid to the execution of the procedure is characterized thereby that it consists of a strip of a porous, kapillarhaltigen substrate also to it by adsorption or kovalente connection bound antibodies. At the time of the execution of the procedure the kapillarhaltiffe diagnostic aid is immersed into the sample, in which the quantity of an interesting antigen is to be examined. It enters then a capillary migration, according to which the antigenhaltigen ranges of the diagnostic aid are indicated by addition by antibodies, which are bound to a suitable color indicator, e.g. to a fluorescent connection or an enzyme, which catalyzes a color reaction. For the use with the invention suitable antibody is won in well-known procedures by Immunisieren of an animal, which is kind different from that one, whose antigen best (mrùt will is. For example antibodies are won against human proteins (albumin, Gammoglobulin, Fibrinogen, Transfe1 Jrin) easily from hares, goats, sheep, horses or guinea pigs. Such antibodies are contained in the blood of immunisierten individual. In particular the antibodies contain the G T-maglobulin-Fraktien (Irùrùunoglobulin). Suitable according to invention kapillarhaltige materials are e.g. different kinds by cellulose fibers containing materials such as filter paper, chromatography paper, ion exchange paper, Zelluloseacetatfflm, cellulose acetate disks, ZeUuloseseheiben for the Dünnschichtchromatographie as well as films from materials such as strength, e.g. a three-dimensional network from Dextranketten, transverseinterlaced with epichlorohydrin, films from plastic material such as polyvinyl chloride, keramisehes material and combinations of such materials, e.g. silicon dioxide containing polyvinyl chloride. The connection of the antibodies to the slow-acting material is preferably managed by bromine cyanogen or Glutaraldehyd. Suitable ones water-soluble color indicators are in particular Fluoresceinisothiocyanat, Rhodamin or Dansylchlorid. The quantitative determination of the antigens takes place for example in the following way: The material which can be examined is preferably applied in well-known quantity, in lVlengen between 1 pl and 3 pl, on an aid according to invention from a kapillarhaltigen substrate. The same volume of a standard solution with well-known content of the antigen which can be examined is applied in Parallelversuchen in different Verdüunungen. 43 0.075 M Natrinmbarbituratpuffer with an additive of 0,34 albumin can be used e.g. as mobile phase for the relief of the capillary migration. The Kapiltarwanderung can be made both in open and in closed attempt containers. In addition kanh the mobile phase upward or downward walks to be left, whereby the combination of a closed attempt container and a rising mobile phase is preferred at any time between approximately 10 and 75 min after beginning of the capillary wall the damp substrate brought into an anti-body solution, which contains either at Fluoreszeinisothiocyanat or at Yälchsäuredehydrogenase bound antibodies. The incubation period in each of these indicator solutions amounts to preferably CH of the Inkubation the substrate under Nr.347601 flowing water with + 37°C 5 min is long washed and then preferably 2 times 5 min into 20 ml sodium bar bit urate buffers, which contains 0.2% albumin and 0.15 M NaC1, inkubiert. If for the Identiflzierung fluorescent antibodies are used, the migration distance of the antigens in ultraviolet light is measured with 340 Nm. If LDH bound antibody becomes used s, the substrate in a coloring material bath is treated with the following composition: mg Nitroblautetrazolium (2.2 ' - Di-p-nitrophenyl-5,5' diphenyl-3,3' (3.3 ' - dimethoxy-4,4' diphenylen) - ditetrazoliumchlorid) and 40 mg NAD are solved in 56 ml 0.05 M TRIS buffers with a pH value from 7,4. For mixture 1.60 ml 3.6 M Lithiumlactat solution, and some grains Methylphenazoninmmetosulfat, solved in 2 ml distilled water, are given to 5 ml 0.06 M calcium cyanide solution. The I0 kapJ] larhaltige material is inkubiert in the coloring material bath with + 37°C, until a clear colouring is recognizable. The Farbenentwicldung been based on it, the LDH the reaction Lactat + NAD Pyruvat + NADH2 catalyzed and the NADH2 quantitatively the Nifroblautetrazolinm to an insoluble fliederfarbenen Formazanverbindung reduces. Indicated marks of the diagnostic mechanism covered with antigen are all the larger, the content of antigen in the examined sample is the larger. It assumed that the bound anti-body molecules the migration of the antigen molecules by Austauschreaktionerl during that cape {]] arwanderung retard. The more highly the concentration at antigen, the smaller the delay effect, because the average time, which remains bound at an antibody an antigen molecule, with rising concentration of the Aatigens sinks. Another method consists of it, strips of the Antikörper haltigen, porous substrate in accordance with the invention with a certain depth, e.g. 3 mm dive into a small volume of a solution of the antigen material which can be examined. The capillary migration caused thereby is left long about 3 to min to run off and interrupted then, as the strip is removed from the solution. The migration distance of the antigen is then measured, how described above, and that result as erreehnet above. The diagnostic aid according to invention is manufactured in form of a test meeting on the porous substrate. The strip can have the thickness of a Fllterpapiers or if required a somewhat larger thickness possess or in form of a film or than disk be trained. The strip can be trained also like a thin flat Stäbchen or a Stäbchen with small diameter. In case of of micro-porous plastic sheets, where finely divided Siliziumdloxyd is present, 3S amounts to the middle pore diameters 0.1 to 1 p and over it. In the case of a measurement of the overall porosity (by mercury) this results between approximately 1.1 and 1.8 ml/g. In the following the invention is described by examples of the production of diagnostic aids according to invention: B e i s p i e 1 1: Connection of antibodies on kapillarhaltigem material with CNBr 10 g filter paper as zellulosehaltiges material 5 min is immersed long into distilled water. Afterwards a solution is added to water distilled by 8 g CNBr in 300 ml and the pH value with i M NaOH solution is stopped to 10,5 and maintained by addition of suitable quantities of a means for the maintenance of the pH value. The reaction is left to 20 min long to run off and decanted then the supernatant solution. The cellulose material treated with CNBr is washed with 2 l 0.005 M Nai_iCO3 _Lösung. After removing the Waschlösuug the material becomes over night with + 4°C in a solution of 500 mg Gammaglobulin from hares against human Cammaglobulin (IgG Fc-Fragraent) in i0 mi 0.1 M NaHCO3 - solution inkubiert and then recently 3 h long with i00 ml more aqueous 0.005 M ethanol amine solution inkubiert. Afterwards becomes with 500 ml 0.5 M NaHCO3 - solution washed and 200 ml 0, I M acetate buffer with a pH value of 4,0 and 500 ml 0.075 M sodium bar bit urate buffers with 0,34 is added to albumin in such a way with + 4°C. B e J s p i e l 2: Connection of antibodies to kapillarhaltiges material with Glutaraldehyd 0.6 g of a cellulose acetate film with a solution by I00 mg Gammaglobulin are long inkubiert by hares against mensahliehes Gammaglobulin (IgG Fc-fragment) in 10 nll 0.01 M phosphate buffers with a Nr.347601 pH value of 6,8 30 min. After Abdekantieren of the solution the film 30 min is inkubiert long in ml casually Glutaraldehydlösung in water, on which a Inkubation in 10 ml 1.0 M of aqueous methyl amine solution for 15 min follows. Afterwards the film is washed with 100 ml 0.075 M Natrinmbarbituratpuffer and kept in the same buffer, were caused to which 0.3% albumin. B e i s p i e 1 3: Connection of antibodies at kapillarhaltlgem substrate by adsorption or absorption g polyvinyl chloride with a content of approximately 1 silicon dioxide is inkubiert over night with + 4°C with a solution by 500 mg Gammaglobulin by hares against menschliehes Gammaglobulin (IgG Fe-fragment) in 100 ml 0.1 M phosphate buffers at a pH value of 5,8. After complete Inkubation lo the material is washed with 1 1 physiologiseher saline solution buffered with phosphate with a pH value of 7,0. In such a way received material is dried between filter papers. B e i s p i e 1 4: Production of Konjugaten from fluorescent lugs and antibodies. a) 720 mg Gammaglobulin of hares against menschHches Gammaglobulin (IgG Fe-fragment), 0.57 g NaC1, 0.259 g NaHCO3 and 0.049 g Na2 CO3 .10H2 0 are solved in 72 ml distilled water. The developing test solution is cooled on an ice bath and shifted with 36 mg Fluoreszeinisothiocyanat under agitating. The solution is left long then 18 h under agitating in cooling. Afterwards the mixture is dialysiert against a physiological saline solution with a pH value of 7,0, buffered with phosphate, until the Dialysierungsflüssigkeit stops fluoreszieren. The developing Antikörper Konjugat is frozen. b) 720 mg Gammaglobulin of hares against human Gammaglobulin (IgG Fc-fragment), 0.57 g NaC1, 0.259 g NaHCOs and 0.049 g Na2 CO3 .10H2 0 are solved in 72 ml distilled water. The developing test solution is cooled solved in an ice bath and shifted with 9 mg Rhodamin, in 2 mI acetone, under agitating. The solution ward 18 h long under agitating in cooling held. Afterwards the laschung becomes against a physiological saline solution with a pH value of 7,0, buffered with phosphate, dialysiert' to the dialysierende liquid to fluoreszieren stops. The developing Antikörper Konjugat is frozen. c) 720 mg Gammaglobulin of hares against human Gammaglobulin (IgG Fc-fragment), 0.57 g NaC1, 0.259 g NaHCO3 and 0.049 g Na2 CO3 .10H2 0 are solved in 72 ml distilled water. The developing test solution is cooled for acetone solved in an ice bath and shifted under agitating with 20 mg Dansylchlorid, in 7 ml. The solution is kept long under agitating 18 h in cooling. Afterwards the mixture is dialysiert against a physiological saline solution buffered with phosphate at a pH value of 7,0, until the dialysierende liquid stops fluoreszieren. The developing Antikörper Konjugat is frozen. B e i s p i e 1 5: Production of a Konjugats from LDH Isoenzym H4 and antibody a suspension from 4,5 mg Milchsäuredehydrogenase (LDH) - ISO enzyme H4 in ammonium sulphate is dialysiert against 0,1 M phosphate buffers with a pH value of 6,8. Under agitating 2.25 mg Gammaglobulin are added by hares against menschHches Gammaglobulin (IgG Fc-fragment), and with phosphate buffer on a total volume of 1,35 ml one brings. After dissolving the entire Gammaglobulins 45 is drop by drop added pl l ge Glutaraldehydlösung, and the mixture is let long stand to 2 h at ambient temperature. The mixture is then dialysiert against sodium bar bit urate buffers and the Konjugat with + 4°C is kept. Before the use it is diluted with 10 ml bar bit urate buffers, which 2° albumin is added. B e i s p i e 1 6: Examination of the anti-body connection to the carrier 2 strips from the porous material, which was treated in accordance with the preceding Beispieien with antibodies, and 2 strips of the same porous material without treatment are used for the examination. A strip of each kind (i.e. treats and untreatedly) is inkubiert in a solution by 1 mg human G.mmaglobulin (IgG) per ml sodium bar bit urate buffer. All strips are then washed to 0.15 M NaCl, for altogether ten times, with 20 ml Natriumbarbimratpuffer, containing according to which they are long inkubiert in a solution of a Konjugats from Fluoreszeinisothiocyanat and antibody 10 min. After repetitive washing as in the preceding step the strips are dried and examined in ultraviolet light with a wavelength by 340 Nm, in order to guarantee that only those strips fluoreszieren, which were inkubiert bound antibodies contained and in the IgG solution. With the examination of the strips of the example 3 as control strips are used, which were treated with cattle albumin.