WO1986004684A1

WO1986004684A1 - Method for the determination of antibodies or antigens

Info

Publication number: WO1986004684A1
Application number: PCT/FI1986/000014
Authority: WO
Inventors: Juhani E.I. Luotola; Hannu Harjunmaa
Original assignee: Labsystems Oy
Priority date: 1985-02-06
Filing date: 1986-02-05
Publication date: 1986-08-14
Also published as: EP0214167A1; JPS62501647A; FI850481A0

Abstract

Method for the determination of antibodies, in which method fluorescent (6) and magnetic particles (5) are added to the sample, antigen of the antibody (3) to be determined being placed on the surface of each of the sorts of particles. In an immunological reaction, fluorescent particles adhere to the magnetic particles by the intermediate of the antibody. After the reaction the magnetic particles are pulled by means of a magnet into a separation layer (2), out of which the fluorescence is measured.

Description

Method for the determination of antibodies or antigens

The present invention is concerned with a fluoro etric or phosphorimetric iirununoassay method in which small polymer particles are used as the solid phase. The method in accordance with the invention can be used, besides for immunoassays in general, also for blood group determinations.

In prior art, methods are known which are based on immobilization of an antibody or antigen on an antigen or antibody in advance placed on a solid face as well as on the use of an antibody or antigen labelled with a tracer. Such methods are, e.g., RIA (Radioi muno- assay) and SP-FIA (Solid Phase Fluoroimmunoassay) . In all of these methods, the solid face on which the immuno- logical reaction has taken place and the reaction solu¬ tion must be separated from each other before the signal of the tracer is measured in order that the excess tracer present in the reaction solution should not cover the signal of the tracer present in the antibody or antigen immobilized on the solid phase. The signal concerned may be, e.g., radioactivity (RIA), fluores¬ cence signal (FIA) or even enzyme activity (EIA, i.e. Enzyme Immunoassay) . The separation of the solid phase from the reaction solution always includes washing of the solid phase, which, at present, requires operations whose automation is difficult. Thus, these operations are, as a rule, carried out manually. If small polymer particles are used, the operations include centrifuging or mag¬ netic deposition.

The principal objective of the present inven¬ tion is to provide such a method for the determination of antibodies or antigens in which inconvenient operations of separation are avoided and which is also suitable for being used in connection with such antibodies or anti¬ gens as are placed on the surface of cells or other particles of organic origin.

In the assay method in accordance with the invention, particles treated with a fluorescent (or phosphorescent) tracer are, together with particles treated with a magnetic material, immobilized on an antibody (or antigen) by means of an antigen-antibody bond. After the immobilization of the particles, the cells and the magnetic and fluorescent particles attached to them are pulled by means of a magnetic field from the reaction layer into the separation layer, whereupon the fluorescence is determined from the separation layer or from the reaction layer. Fluorescence is emitted only if the excitation light meets fluorescent particles. Mere magnetic particles alone do not emit fluorescent radiation. If the antibody does not become labelled with the polymer particles covered by the antigen concerned, only the magnetic particles arrive in the separation layer, and the fluorescent non-magnetic particles and the unlabelled cells remain behind the separation layer. The method is suitable for being used both as a direct method and as an indirect method. Thereat, for example, the determination of blood group can be carried out both from the cell side and from the serum side. The determination from the serum side takes place as indirect. The method in accordance with the invention is easy to carry out, because the solutions do not have to be removed from the vessel and because no separate washings have to be carried out.

Certain preferred embodiments of the invention will be illustrated by means of the attached schematical Figures 1 to 10.

Figures 1 to 4 illustrate the determination of dissolved antibody. Figures 5 to 8 illustrate the de¬ termination of an antigen placed on the surface of a cell, and Figures 9 and 10 illustrate simultaneous de¬ termination of several antibodies. Figures 1 to 4 illustrate the determination of dissolved antibody.

In the measurement vessel, there are two liquid layers (Fig. 1) : a reaction layer 1 and a so-called separation layer 2. In the reaction layer 1, there is the antibody 3 to be determined, in dissolved form. Moreover, in the reaction layer 1 , there are polymer particles covered with antigen 4 of the antibody 3, of which said polymer particles the particles 5 include a magnetic material and the particles 6 include a fluores¬ cent tracer. The polymer particles are pearls made of some suitable material, and their size is 0.1 to 10 μm.

The separation layer 2 is placed in the vessel below the reaction layer 1. It has been placed into the vessel before the reaction layer 1 , or it has been added afterwards to underneath the reaction layer.

The separation liquid 2 is preferably denser than the reaction solution 1 , and of such a colour that it prevents the fluorescence in the -reaction solution from being seen in the measurement. Suitable reaction solutions are, e.g., saccharose solution (concentration typically 10 to 60 %) and Ficoll-Paque density-gradient centrifuging solution, etc. If the separation solution is not of suitable colour by nature, a suitable colour is obtained by to it adding a colouring agent which ab¬ sorbs^" light strongly within the excitation or emission wavelength of fluorescence, or within both wavelengths. In particular, black is a suitable colour.

After the reaction layer and the sample layer have been measured into the measurement vessel, the conventional incubation stage follows, whereat the immunological reaction takes place (Fig. 2) . Thereby, the antibody 3 to be determined is bound with the antigen 4 placed on the surface both of the magnetic 5 and of the fluorescent 6 particle. The antibody 3 can now be both displaced magnetically and measured fluorometri- cally. If there is no antibody 3 in the sample, only the magnetic particles 5 can be displaced magnetically.

After the reaction has been completed, the magnetic particles 5 are pulled by means of a magnetic field 7 through the separation layer 2 onto the bottom of the measurement vessel (Fig. 3) . The fluorescent particles 6 , adhering to the magnetic particles by the intermediate of the antibody, follow along with them. At the same time, the separation layer 2 also acts as a physico-chemical washing layer. Into the flurometer used in the method, a particular magnetic separation device is built in. The more of the antibody 3 to be determined there is present in the sample, the more particles 6 covered with the fluorescent tracer are there now on the bottom of the vessel. The measurement takes place so that the exci¬ tation radiation 8 is passed to the bottom of the measure¬ ment vessel through its wall, and the emission radiation 9 is also collected along the same route for detection (Fig. 4) . The coloured separation layer 2 now acts as an optical shield layer against viewing of the fluorescent reaction layer 1. Thereby, no emitted fluorescent radiation has access from the reaction layer 1 into the detector system, and only the fluorescent particles placed on the bottom of the vessel are detected.

Figures 5 to 8 illustrate a case in which an antigen placed on the surface of a cell is to be de¬ termined.

In this case, in the measurement vessel there is also a reaction layer 1 at the top and a separation layer 2 underneath. In the reaction layer 1 , there are cells -10 on whose surface there is antigen 11. More¬ over, in the reaction layer, there are polymer particles covered with the antibody 12 of the said antigen, some of the said polymer particles being magnetic 5, and some of them being fluorescent 6. After the dosage stage (Fig. 5) , there are the incubation stage (Fig. 6) , the separation stage (Fig. 7) , and the measurement stage (Fig. 8) , in a way corres¬ ponding to the case described above, in which said case the antibody is present in the reaction solution in dis¬ solved form.

This method can be applied, e.g., to b ood group determinations as follows:

To among the blood cells to be studied, mag- netic and fluorescent particles covered with a known antibody are measured (the same antibody on the surface of each particle type) . It is assumed that in the mea¬ surement vessel A there are magnetic and fluorescent particles covered with anti-A antibody, and in the mea- surement vessel B there are magnetic and fluorescent particles covered with anti-B antibody. In connection with incubation, in a positive case, both of the particle types, magnetic and fluorescent, adhere to the same .cell. In the negative case, none of the particle types adheres to the cell surface, whereby only the magnetic particles respond to the magnetic field. The magnetized particles and the labelled cells, if any, are pulled against the bottom of the measurement vessel. When fluorescence is now measured through the bottom of the measurement vessel, only the fluorescent particles placed on the surface of the blood cells are noticed. If only the reaction vessel A gives a positive signal, the blood group is A from the cell side. If only the reaction vessel B gives a posi¬ tive signal, the blood group is B from the cell side. If both of the reaction vessels give positive signals, the blood group is AB from the cell side. If both of the reaction vessels give negative signals, the blood group is 0 from the cell side.

Correspondingly, from the serum side, the determination of the blood group is performed by in con¬ nection with the incubation adding a serum sample to be studied on the basis of known control cells (A and B cells) and corresponding magnetic anti-A and anti-B particles. If in the reaction vessel A the adherence of anti-A particles is prevented and the signal is negative, there has been anti-A antibody present in the serum, which has acted on the basis of competition and prevented the adhesion of anti-A particles. ^'If the reaction is posi¬ tive in respect of the anti-B particles present in the reaction vessel B, the blood group is B from the serum side. On the other hand, if there is a positive signal in the reaction vessel A,but a negative signal in the re¬ action vessel B, the blood group is A from the serum side. If the signal is positive both in the reaction vessel A and in the reaction vessel B, the blood group is AB from the serum side. On the other hand, if the signal is negative in both of the reaction vessels, the blood group is 0.

Of course, in the method, it is possible to use all known blood cell and serum types with their sub¬ groups. Program-technically, in the blood-group assays, it is possible to take into account the safe lower and _. upper limits. Moreover, from the serum side, the blood group can be determined by covering the particles with the corresponding blood cell antigen, which are also available commercially. In the determination of anti- bodies, when particles covered with red-cell antigens are used, no test cells are needed at all. When cells are used, by means of inhibition it is possible to establish, e.g., a person's secretor property.

The method can also be carried out by means of competition by using magnetic and fluorescent particles covered with the same antigen in the same reaction vessel. If there is the corresponding antibody (IgM class) in the serum, fluorescent pearls are also deposited in con¬ nection with the magnetic pearls, being linked by the antibody.

In the method, the Rh-factor can be established by incubating the cells to be studied with anti-D serum. whereby the antibody adheres to the face of the red blood cell. The antibody concerned is of the type IgG, which can be detected by means of anti-human-IgG. The antibody may be attached to fluorescent particles, or it may be present as its fluorescent conjugate. The methods may be the following: After red blood cells have been incubated in anti-D serum, magnetic pearls covered with anti-human-erythrocyte antibody are added to among the sensitized red blood cells, and the red blood cells are pulled down after the incubation time. Now it is possible to suck off any excess serum and unattached pearls out of the measurement vessel while keeping the magnetic field constantly on. When the magnetic field is switched off, the cells can be washed in a suitable medium, while finally depositing the cells down by means of the magnetic field. To among the washed sensitized cells, it is now possible to add fluorescent pearls covered with anti-human-IgG, and to underneath the mixture of cells and pearls, it is possible to add ^"a dense, coloured, suitable separating substance. Upon completion of the incubation, the cells are again pulled down by means of the magnetic field, whereby detection of the fluorescence is possible. Of course, it is also possible to add the coloured dense substance before the fluorescent particles, in which case the risk of fluo¬ rescence contamination is reduced. In place of the fluorescent particles, it is also possible to use a fluorescent anti-human-IgG conjugate. One mode of labelling the Rh-positive cells is to sensitize the cells by means of anti-D, whereupon the cells are pulled by means of magnetic anti-human-erythrocyte particles into the first separation-substance layer, wherein there is a very large quantity of fluorescent particles covered with anti-human-IgG or of fluorescent anti-human- IgG conjugate. The magnetic pulling is stopped at this layer for the time of the incubation, and upon completion of the incubation the cells are pulled through another coloured layer, of higher density, to the bottom of the measurement vessel, and the fluorescence is detected.

The techniques concerned can also be utilized in antibody screening and in a cross test. Fig. 9 illus- trates simultaneous determination of several antibodies in a case in which the antigens of all of the antibodies are attached onto the surfaces of magnetic particles of one sort.

In the reaction solution, there are magnetic particles 12, an antigen 4, 4' and 4" being attached to the surface of each of them. Moreover, in the reaction solution, there are fluorescent particles 6, 6' and 6", on whose surface there is only one antigen, either 4, 4' or 4", and each of which is provided with a characteristic fluorescent tracer of its own.

After the stages of incubation and separation have been carried out, measurement is carried out by using the specific excitation or emission wavelength of each of the fluorescent particles 6, 6', 6". In this way the desired antibodies 3, 3' and 3" can be determined.

Fig. 10 illustrates simultaneous determination of several antibodies in a case in which the antigen of each antibody is attached onto the surface of the par¬ ticles in its own group of particles. In the reaction solution, there are magnetic particles 5, 5' and 5", one antigen, either 4, 4' or 4", being attached to the surface of each of the said par- ticles. In the solution, there are also fluorescent particles 6, 6' and 6", each of which is provided with a typical tracer of its own.

After the stages of incubation and separation, fluorometric measurement is carried out at each of the wavelengths concerned in order to determine the anti¬ bodies 3, 3' and 3". The methods described above can, of course, also be applied by making use of phosphorescence. In the methods, in the solid phase, there may be an antibody equally well as an antigen.

If necessary, the separation layer may be placed on top of the reaction layer. In such a case, the measurement becomes less convenient, but thereby the background fluorescence caused by the vessel wall is avoided. If desired, the separation layer may also be formed in the vessel after the incubation.

Of course, if desired, the measurement may also be carried out from the reaction layer.

Claims

WHAT IS CLAIMED IS:

1. Fluorometric or phosphorimetric method for the determination of antibodies or antigens out of a sample by means of an immunological reaction, c h a r ¬ a c t e r i z e d in that to a sample placed in a measurement vessel in a layer (1) or reaction solution, particles (6) that include a fluorescent or phosphores¬ cent tracer as well as magnetic particles (5) are added, antigen of the antibody to be determined being placed on the surface of each of the said particles ; that in the measurement vessel, below or above the reaction layer, a separation solution layer (2) is formed that is not mixed with the reaction layer; that after the immuno- logical reaction, the magnetic particles with the fluorescent or phosphorescent particles linked to them by the intermediate of the antibody are pulled by means of a magnetic field into the separation -layer; whereupon the fluorescent or phosphorescent particles are deter- mined fluorometrically or phosphorimetrically out of the separation layer or out of the reaction layer.

2. Method as claimed in claim 1, c h a r ¬ a c t e r i z e d in that in the measurement vessel a separation layer (2) denser than the reaction layer (1) is formed below the reaction layer (1) and that the fluorescence or phosphorescence is measured out of the separation layer through the wall of the measurement vessel .

3. Method as claimed in claim 1, c h a r - a c t e r i z e d in that in the measurement vessel a separation layer (2) is formed, which absorbs strongly at the excitation or emission wavelength of the fluo¬ rescence or phosphorescence of the tracer, and that the magnetic particles (5) are pulled from the reaction layer (1) through the separation layer (2) to the opposite edge of the separation layer.

4. Method as claimed in claim 3 , c h a r ¬ a c t e r i z e d in that a separation layer (2) is formed which absorbs strongly both at the excitation wavelength and at the emission wavelength of the tracer.

5. Method as claimed in claim 4, c h a r ¬ a c t e r i z e d in that a black separation layer (2) is formed.

6. Method as claimed in claim 1 for the de¬ termination of several antibodies out of" one sample, c h a r a c t e r i z e d in that particles (6, 6', 6") that include a fluorescent or phosphorescent tracer are added to the sample, the surface of each particle being covered with the antigen (4, 4', 4") of one of the anti¬ bodies (3, 3' , 3") to be determined and each of the said particles having a tracer of its own, as well as that magnetic particles (5, 5', 5") are added to the sample, the surface of each of the magnetic particles being covered with the antigen of one of the antibodies to be determined, and that each of the fluorescent particles is determined separately at its specific wavelength.

7. Method as claimed in claim 1 for the de¬ termination of several antibodies out of one sample, c h a r a c t e r i z e d in that particles (6, 6', 6") that include a fluorescent tracer are added to the sample, the surface of each particle being covered with the antigen (4, 4', 4") of one of the antibodies (3, 3', 3") to be determined and each of the said particles having a tracer of its own, as well as that magnetic particles (12) are added to the sample, the surface of each of the magnetic particles being covered with antigens of all of the antibodies to be determined, and that each of the fluorescent particles is determined separately at its specific wavelength.