FI76216B - BESTAEMNINGSFOERFARANDE FOR ANTIGEN. - Google Patents

Abstract

1. A method for determination of the antigen content of a sample by the antigen-antibody reaction, comprising diluting at least one of the two reaction components with a physiological saline solution, which contains polyethyleneglycol and a buffer substance, thereafter combining both reaction components and determining the antigen-antibody-reaction photometrically, characterized in that the buffer substance is a glycine, glycyl-glycine, acetate, citrate, N,N-bis-(2-hydroxyethyl)-glycine, histidine/HCl, 2-amino-2-(hydroxymethyl)- 1,3-propanol or 5,5-dialkyl-barbituric acid buffer.

Description

7621 6

Method for the determination of antigens

The invention relates to a method for determining the antigen content of a sample on the basis of an antigen-antibody reaction according to the preamble of claim 1. The invention also relates to a diluent for the antigen and antibody reaction components used in carrying out this method.

Determination of diagnostically important parameters by antigens in a sample, such as serum, plasma, or spinal fluid, based on an immunological or antigen-antibody reaction, which is interpreted photometrically by, for example, nephelometry or turbidometry, often requires specific preparation of both reaction components. In this case, the second reaction component containing the sample to be determined and the second component containing the antibody, which is thus usually formed by the action of the corresponding antiserum, is usually diluted with physiological saline or physiological saline solution further containing 4 X polyethylene niglycol (average ).

The purpose of the polyethylene glycol content is mainly to prevent the flocculent precipitation of the antigen-antibody complexes formed in the reaction, i. the formation of a precipitate in the test solution formed by combining the two reaction components during the antigen determination, i.e. the measurement.

This property of polyethylene glycol has long been known and is a basic prerequisite for these measurements, as otherwise not all antigen-diluent complexes can be included during the measurement.

When immunoglobulins were determined by laser turbidometry according to the following manual: "Arbeitsanleitung fur das 2 7621 6

Laeermed-Turbidometer <pm 4) zur Beetimmung der Immunglobu-line IgA, IgG, IgM mit ATAB-Antieeren, Firma AHS Deutschland GmbH, Bereich Merz und Dada, München ", 1982, it was found that various physiological saline solutions containing 4 wt. X polyethylene glycols, although prepared according to the same recipe, using the same chemicals and by the same person, gave different measurement signals on the laser turbidometer during the analysis, so that it was not possible to obtain reproducible results with sufficient accuracy, thereby disturbing the kinetics method according to the above instructions. In contrast to the so-called "Fix point Method" according to said protocol, i.e. for measuring the end point of a reaction lasting 30 minutes, the kinetic method thus does not, in particular, measure the rate of the antigen-antibody reaction after a pre-reaction of 10 seconds and then determine the antigen content. need to wait for the entire antigen-antibody reaction ion passage to the end.

The invention, the features of which are set out in the claims, solves the object of providing a method, i.e. a diluent, by means of which the antigen content in a sample can be determined accurately and reproducibly even according to a kinetic method.

The antibody molecule has a polyvalent and therefore very complex structural form, while polyethylene glycol is non-polar and therefore simple in structure.

When these two reaction components are combined, therefore, a certain homogeneous distribution takes place only after a long time and to some extent reproducible analytical results are obtained. The method of the invention is based on providing a rapid and homogeneous distribution of both reaction components by adding an additional 3 7 621 6 buffer to the polyethylene glycol-containing physiological saline solution, which removes the complex structure of the antibody molecule by leveling the charges.

Surprisingly, it has been found that the rate of the antigen-antibody reaction depends in particular on the chemical composition of the buffer, and even much more than on the concentration or pH of the buffer. In the context of the invention, it is particularly important not to select buffers which allow the fastest possible reaction rate, but above all buffer substances which lead to a constant initial reaction rate for a longer period of time. Namely, through this property of the wood curing agents to be added according to the invention, the reaction time is somewhat extended, so that a straightforward progression takes place for a relatively long time. This greatly improves the accuracy of the results of the kinetic method. For example, if 20 mmol / l of 5,5-diethylbarbituric acid, pH 7.5, is added as a buffer to a physiological saline solution containing 4% by weight of polyethylene glycol, then after combining both reaction components a pre-reaction of, for example, 10 seconds is expected, for example about 30 seconds. A period of 30 seconds is then a sufficient basis for even very accurate measurement results in an antigen assay.

Claims (7)

1. A method for testing the antigen content of beetame due to an antigen-antibody reaction or to exclude any of the reaction components from the anticoagulant cocaine release containing polyethylene glycol and buffer antigen, and the antigen antibody reaction component. that the buffer agent is 5,5-dialkyl barbiturate buffer. 2. A method according to claim 1, characterized in that the barbiturate is an 5,5-diatylbarbituric acid. 3. A process according to claim 1, characterized in that the buffer-mediated concentration in the coke solution is 5-200 mmol / l, preferably 20-50 mmol / l. 4. A process according to any of the claims above, characterized in that the concentration of polyethylene glycolene in the cocaine solution is 2-6 wt. X, preferably about 4 wt. 5. A process according to any of the claims above, characterized in that the pH value of the buffer medium containing the polyethylene glycol-containing physiological cocaine solution is 4.0-9.0 and preferably from 6.5-8. 6. A method according to any of the claims above, characterized in that the antigen-antibody reactions kinetically proceed with photometry. 7. Phyiologic cocoanalysis, which contains polyethylene glycol and buffer medals, as well as dilute ether monetone in other of two antigen-antibody reaction reaction components, which antigen content may be photomatriched, characterized in that the buffer agent is 5.