CA2090392C

CA2090392C - Coupling of antigens and antibodies to non-fixed erythrocytes

Info

Publication number: CA2090392C
Application number: CA002090392A
Authority: CA
Inventors: Yves Lapierre; Dieter Josef; Jean-R. Adam; Susanne Greber-Widmer
Original assignee: Stiftung fur Diagnostische Forschung
Current assignee: Stiftung fur Diagnostische Forschung
Priority date: 1992-02-25
Filing date: 1993-02-25
Publication date: 2000-01-11
Anticipated expiration: 2013-02-25
Also published as: EP0557546A1; HK1002126A1; EP0557546B1; DE59208460D1; JPH0688822A; ATE152835T1; GR3023970T3; ES2101763T3; DK0557546T3; CA2090392A1

Abstract

1. In order to determine an analyte in an aqueous sample solution by agglutination of non-fixed erythrocytes, one proceeds according to the present invention in such a way that (a) erythrocytes are incubated with one or several antibodies or/and antibody derivatives which do not lead to premature agglutination whereby the antibodies or/and antibody derivatives are directed towards one or several antigens on tie erythrocyte membrane and whereby before or after incubation with the erythrocytes the antibodies or/and antibody derivatives are coupled to a first binding partner of a high affinity binding pair, (b) a second binding partner of the high affinity binding pair is incubated with the treated erythrocytes from (a) whereby the second binding partner is chosen such that it has more than one binding site per molecule for the first binding partner, (c) the first binding partner of the high affinity binding pair which is coupled with the analyte or/and an antigen-active determinant of the analyte is incubated with the treated erythrocytes from (b), (d) a sample solution which has been treated with an antibody or antibody derivative directed towards the analyte is incubated with the treated erythrocytes from (c) and (e) the absence or presence of the analyte in the sample solution is determined on the basis of the occurrence or absence of an agglutination reaction.

Description

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D a s c r i p t i o n The present invention concerns a method for the determination of an analyte in an aqueous sample solution by agglutination of non-fixed erythrocytes.
Agglutination tests in which antigens or antibodies are bound to fixed erythrocytes have been known for a long time. In this process the cell membrane of the erythrocyte is stabilized by the fixation so that the cells can no longer haemolyze. When such coated cells are then brought into contact with the corresponding antibody or antigen an agglut9_nation takes place.
This test procedure in itself is a very simple and rapid determination method for analytes in an aqueous sample solution. However, in recent years it has become much less important since it is susceptible to interference, it is difficult to interpret i.n borderline cases and is not sensitive enough compared to more recent methods.
Lapierre et al. (Transfusion 30 (1990), 109; CH-A
85 02010) have reported a new method in blood group serology in which the agglutination reactions are carried out in microtubes which are filled with a gel suspension. After sedimentation the agglutinates are either on the gel or in the case of weak reactions distributed within the gel. In the absence of agglutination the cells collect at the bottom of the tube. This procedure is simple, very sensitive and less subject to interference. The results are easy to read and easy to interpret.

It is, however, not possible to apply this above-mentioned gel method to reactions in which antigens or antibodies are bound to fixed erythrocytes. The reason for this is that the erythrocyte membrane can no longer be deformed after the fixation process and the fixed erythrocytes can no longer pass through the gaps between the individual gel particles as a consequence of which it is not possible to differentiate in practice between a positive and a negative reaction.
This problem could only be partially alleviated by a weaker fixation of the erythrocytes (EP-A 0 305 337) since the reaction can only be controlled with difficulty. In addition it turned out that even after a short time there is a change in the treated cells in that they either lyse or become non-deformable. A pure absorption or pre-treatment of cells with chromium (III) chloride also has no or only very little success.
The object of the present invention was therefore to provide a method for the determination of an analyte i.e. an antigen or antibody by agglutination of erythrocytes in which the disadvantages of the state of the art are at least partially eliminated.
The object according to the present invention is achieved by a method for the determination of an analyte in an aqueous sample solution by agglutination of non-fixed erythrocytes, which is characterized in that (a) erythrocytes are incubated with one or several antibodies or/and antibody derivatives which do not lead to premature agglutination whereby the antibodies or/and antibody derivatives are directed towards one or several antigens on the erythrocyte membrane and whereby before or after incubation with the erythrocytes the antibodies or/and antibody derivatives are coupled to a first binding partner of a high affinity binding pair with, (b) a second binding partner of the high affinity binding pair is incubated with the treated erythrocytes from (a) whereby the second binding partner is chosen such that it has more than one binding site per molecule for the first binding partner, (c) the first binding partner of the high affinity binding pair which is coupled with the analyte or/and an antigen-active determinant of the analyte is incubated with the treated erythrocytes from (b), (d) a sample solution which has been treated with an antibody or antibody derivative directed towards the analyte is incubated with the treated erythrocytes from (c) and (e) the absence or presence of the analyte in the sample solution is determined on the basis of the occurrence or absence of an agglutination reaction.
It surprisingly turned out that antibodies or/and antibody derivatives which do not lead to a premature agglutination of erythrocytes are suitable for linking non-fixed erythrocytes. In principle a multitude of different antibody types are suitable for this but not those antibodies which have a constant IgM domain.
Antibodies of the IgG class or antibody derivatives such as Fab or F(ab)2 fragments are preferably used.
The above-mentioned antibodies are directed towards antigens on the erythrocyte membrane. Examples of these are anti-D, anti-C, anti-E, anti-c, anti-e, anti-M or anti-N antibodies. Anti-D antibodies are preferably used. In order to amplify the reaction it is also ~t~p~

possible to use several of these antibodies at the same time. The use of human antibodies (e. g. of antibodies with a human constant domain) has proven to be particularly advantageous since when antibodies with a non-human constant domain are used it can lead to false species-specific reactions.
Antibodies against erythrocytes are commercially available or can be produced in a simple manner. A
purification can if necessary be carried out according to known methods. IgG antibodies are e.g. purified by ammonium sulfate precipitation and column chromatography. It is particularly preferable to use monoclonal antibodies since these are present in a substantially purified form. Such antibodies can for example be obtained from Bioscott UK, Monocarb Sweden, Diagast France etc.
The antibody directed towards the antigen on the erythrocyte membrane is coupled either before or after incubation with the erythrocytes to a first binding partner of a high affinity binding pair. The term "high affinity binding pair" denotes two substances, i.e. the first and the second binding partner, which are capable of forming a non-covalent bond with each other with a binding constant of at least 10-$ mol/1. The use of the streptavidin- or avidin-biotin system has proven to be particularly advantageous whereby one can also use an appropriate biotin derivative instead of biotin. A
further example of a suitable binding pair is a hapten, i.e. a low molecular antigen such as fluorescein, and a specific antibody directed towards it.
The use of the biotin/streptavidin system is particularly preferred since streptavidin has four binding sites for biotin so that the number of coupled antigens or antibodies can be increased three-fold and in addition the production of the reagents is very simple since all reaction steps can be carried out on a solid phase. Instead of streptavidin or avidin, derivatives thereof can also be used such as avidin-succinyl.
Step (a) of the method according to the present invention can be carried out :in two different ways;
according to a first embodiment the erythrocytes are firstly incubated with antibodies or/and antibody derivatives which have already previously been coupled to the first binding partner of the high affinity binding pair. According to a second and particularly preferred embodiment the erythrocytes are firstly incubated with non-coupled antibodies or/and antibody derivatives and only then are the antibodies or/and antibody derivatives coupled to the first binding partner of the high affinity binding pair.
The addition of the second binding partner in step (b) of the method according to the present invention is preferably carried out in such a way that the first binding partner and the second binding partner are present in a molar ratio of about 1 . 1. The addition of the analyte-coupled first binding partner in step (c) of the method according to the present invention is preferably carried out in such. a way that all free binding sites of the second binding partner are occupied. In the case of the streptavidin/biotin system this means a molar ratio of ca. 1 . 3 (streptavidin .
biotinylated analyte).

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The method according to the present invention serves to determine an analyte in an aqueous sample solution. The term analyte includes all substances which can be determined by an immunologica:L antigen-antibody reaction. A preferred application for the method according to the present invention is, however, to carry out a pregnancy test in which for example HCG is determined as the analyte. It is, however, obvious that other analytes which play a role in the detection of diseases, in particular hepatitis, AIDS, syphilis or hormone disorders, can also be determined. Moreover it is also for example possible to test for medicines.
The method according to the present invention can also be used to determine several different analytes simultaneously so that screening tests for infectious diseases can be greatly simplified.
It is intended to elucidate the method in more detail in the following using a pregnancy test as an example. The sequence of the individual steps in the method is also shown schematically in Figure 1. Firstly a biotinylated antibody (anti-D) of the IgG type which is directed towards the surface D antigen of erythrocytes is added to human blood (e. g. blood group 0, Rh (D) positive).
The amount of antibody which is necessary in each case depends on the affinity of the antibody to the antigen.
After incubation and a washing step, streptavidin or avidin is added and thereupon binds with one of its four binding sites to the biotinylated antibody. The addition is preferably carried out such that the molar ratio of streptavidin to biotin is 1:1. After a further washing, the free binding sites of the streptavidin or avidin are occupied with the biotinylated antigen, the pregnancy hormone HCG, which is thus bound via the biotin-avidin 2~~Q~~~
complex to the antibody. The molar ratio of avidin to biotinylated analyte is particularly preferably 1:3. The antibody in turn mediates binding to the erythrocytes.
After washing and diluting, one obtains an erythrocyte reagent which is ready to use.
On the other hand, it is also possible to firstly allow the antibody to react with the erythrocytes and, after washing the cells, to biotinylate antibody bound to the erythrocytes with the aid of ~~ reactive biotin derivative (e. g. biotin-N-hydroxysuccinimide ester).
When patient serum and anti-HCG antibodies are added to the erythrocyte reagent one observes an agglutination provided that the serum contains no HCG. The agglutination is inhibited by sera from pregnant women which contain HCG. The gel test system described in CH-A
85 02010 has proven to be particularly advantageous for evaluating this test. Using this system it is possible to achieve a 25-fold higher sensitivity compared to conventional tests. A person skilled in the art can easily determine the amount of antibody required to neutralize the analyte by simple experiments (in a corresponding way to known agglutination tests).
The present invention also concerns a reagent for the determination of an analyte in an aqueous sample solution by agglutination of non-fixed erythrocytes which is characterized in that it contains erythrocytes on the membrane of which one or several antibodies or/and antibody derivatives which do not lead to premature agglutination are bound and whereby a first binding partner of a high affinity binding pair is covalently bound to the antibodies or/and antibody derivatives.

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- g -The reagent according to the present invention preferably additionally contains the second binding partner of the high affinity binding pair bound to the first binding partner whereby the second binding partner has more than one binding site and particularly preferably four binding sites for the first binding partner. In this case it is preferable that the binding sites of the first binding partner are essentially quantitatively occupied. In addition it is preferred that the first binding partner- of the high affinity binding pair which is coupled to the analyte or/and an antigen-active determinant of the analyte is in turn bound to the second binding partner.
The reagent according to the present invention is stable for approximately 2 months at 4°C in a ready-to-use form, in particular when it is present in the form of a suspension.
In addition the present invention also concerns a reagent kit which contains a reagent according to the present invention and separate from this an antibody or a corresponding antibody derivative which is directed towards the analyte to be determined. The reagent kit according to the present invention also preferably contains test vessels filled with a gel suspension (e. g.
tubes suitable for centrifugation in a microcentrifuge).
It is intended to further elucidate the invention by the following examples in conjunction with Figure 1.
Figure 1 shows: the agglutination of non-fixed erythrocytes as exemplified by a pregnancy test.

._ 2090392 _ g -Example 1 Indirect pregnancy test 1. Production of biotinylated anti-D
12 ~,1 biotin-N-hydroxysuccinimide ester (1 mg/ml in DMSO, SPA Milano) is added to 1 ml anti-D (IgG, 100 ~Cg/ml) in 0.1 mol/1 NaHC0,3, pH 8.5. The mixture is allowed to stand for 72 hours at 4°C and subsequently dialysed against PBS (phosphai~e-buffered saline solution).
2. Production of biotinylated HCG
12 ~,1 biotin-N-hydroxysuccinimide ester (1 mg/ml in DMSO) is added to 1 ml HCG (0"1 mg/ml 3000 U/mg) in 0.1 mol/1 NaHC03, pH 8.5. The mixture is allowed to stand for 72 hours at 4°C and subsequently dialysed against PBS.
3. Coatinct of erythrocytes with biotinylated anti-D
antibody Fresh human erythrocytes of blood group 0, Rh (D) positive are diluted to a concentration of 10 % with 0.9 % NaCl. 0.5 ml biotinylated anti-D is added to 5 ml of this suspension. After incubating for one hour at room temperature they are washed three times with 0.9 NaCl and the cells are suspended in 5 ml ID CellStab (stabilizing solution, Dialled Murten).
4, Reaction of the biotinylate:d cells with avidin 1 ml avidin-succinyl (6.39 U/mg; SPA Milano) is dissolved in 70 ~,1 PBS. 60 ~,1 of this is added to 5 ml of the erythrocyte suspension described under (3). After incubating for one hour at room temperature they are washed three times with 0.9 % NaCl and the sediment is suspended in ID-CellStab~:
5. Reaction of the avidinated cells with HCG
5 ml of the erythrocyte suspension described under (4) is reacted with 2.5 ml biotinylated HCG (0.1 mg/ml).
After incubating for one hour at room temperature they are washed three times with 0.9 o NaCl and a ca. 0.8 suspension is prepared with ID-CellStab~ The reagent prepared in this way is ready for use and is stable for about two months at 4°C.
6. Test procedure 50 ~,1 of the erythrocytes coated with HCG are applied to prefabricated microtubes already filled with a gel suspension (ID-Karte, Dialled Murten). Subsequently 25 ~,1 patient serum and 25 ~1 anti-HCG (the correct dilution of the antibody is determined beforehand) are added. (In order to prevent cross-reactions with LH or FSH it is expedient to use anti-B-HCG as; the antibody).
After incubating for 10 to 15 minutes at room temperature it is centrifuged for 10 minutes at ca.
910 r.e.m. in a centrifuge suitable for the microtubes in the ID-Karte (ID centrifuge, Dialled Murten).

7. Interpretation of the results a) positive result The antibody reacted with the HCG in the serum and has thus been neutralized. It can therefore not react with the HCG on the erythrocytes. The non-agglutinated cells collect at the bottom of the microtube after centrifugation (sedimentation) and are visible as a red button.

2090 39 2 ~' b) negative result The antibody reacts with HCG on the erythrocytes.
Depending on the intensity of the reaction the agglutinates are present either on the gel or in the gel after centrifugation (sedimentation).

8. Sensitivity of the test A dilution series in HCG-negative serum was carried out using the second international HCG standard of the WHO.
The sensitivity was 25 to 50 U/1 and therefore corresponds to the latest generation of commercially available ELISA tests.
g' Accuracy of the test 500 sera of pregnant and non-pregnant women were examined using a commercially available pregnancy test (Hoffmann LaRoche, sensitivity 50 U/1). The results were 100 o in agreement.
Example 2 Biotinylation of anti-D on a solid phase (i.e. on the erythrocyte surface) 1, Coating the erythrocytes with anti D
Fresh erythrocytes of the blood group 0, Rh (D) positive are diluted to 10 % with 0.9 =NaCl. 0.5 ml anti-D serum (100 ~g/ml) is added to 5 ml of this suspension. After incubating for one hour at room temperature they are washed three times with 0.9 o NaCl and the cells are suspended in 5 ml of an isotonic solution of 0.1 mol/1 NaHC03, pH 8.5.

2. Cout~linct of biotin to the <~nti-D
1 mg biotin-N-hydroxysuccinimide ester (SPA Milano) is dissolved in 1 ml DMSO. 6 ~,1 of this are added to 0.5 ml of the erythrocyte suspension described under (1). After incubating for 24 hours at 4°C they are washed three times with 0.9 % NaCl and the cells are resuspended with 5 ml ID-CellStabM (Dialled MurtEan) .
3. Further reactions are carried out as described in example 1.

Claims

1. Method for the determination of an analyte in an aqueous sample solution by agglutination of non-fixed erythrocytes, wherein (a) erythrocytes are incubated with one or several antibodies or antibody derivatives which do not lead to premature agglutination whereby the antibodies or antibody derivatives are directed towards one or several antigens on the erythrocyte membrane and whereby before or after incubation with the erythrocytes the antibodies or antibody derivatives are coupled to a first binding partner of a high affinity binding pair, (b) a second binding partner of the high affinity binding pair is incubated with the treated erythrocytes from (a) whereby the second binding partner is chosen such that it has more than one binding site per molecule for the first binding partner, (c) the first binding partner of the high affinity binding pair which is coupled with the analyte or an antigen-active determinant of the analyte is incubated with the treated erythrocytes from (b), (d) a sample solution which has been treated with an antibody or antibody derivative directed towards the analyte is incubated with the treated erythrocytes from (c) and (e) the absence or presence of the analyte in the sample solution is determined on the basis of the occurrence or absence of an agglutination reaction.

2. Method as claimed in claim 1, wherein in step (a) erythrocytes are incubated with antibodies or antibody derivatives which have been previously coupled to the first binding partner of a high affinity binding pair.

3. Method as claimed in claim 1, wherein in step (a) the erythrocytes are firstly incubated with non-coupled antibodies or antibody derivatives and then the coupling of the antibodies or antibody derivatives to the first binding partner of a high affinity binding pair is carried out on a solid phase.

4. Method as claimed in claim 1, 2 or 3, wherein antibodies of the IgG class which are directed towards antigens on the erythrocyte membrane are used as antibodies.

5. Method as claimed in claim 1, 2 or 3, wherein antibodies of human origin which are directed towards antigens on the erythrocyte membrane are used as antibodies.

6. Method as claimed in claim 4, wherein antibodies of human origin which are directed towards antigens on the erythrocyte membrane are used as antibodies.

7. Method as claimed in claim 2, 3 or 6, wherein biotin or a biotin derivative is used as the first binding partner and avidin or streptavidin or a derivative thereof is used as the second binding partner.

8. Method as claimed in claim 4, wherein biotin or a biotin derivative is used as the first binding partner and avidin or streptavidin or a derivative thereof is used as the second binding partner.

9. Method as claimed in claim 5, wherein biotin or a biotin derivative is used as the first binding partner and avidin or streptavidin or a derivative thereof is used as the second binding partner.

10. Method as claimed in claim 1, 2, 3, 6, 8 or 9, wherein the occurrence or absence of an agglutination reaction is determined in a gel sedimentation test.

11. Method as claimed in claim 4, wherein the occurrence or absence of an agglutination reaction is determined in a gel sedimentation test.

12. Method as claimed in claim 5, wherein the occurrence or absence of an agglutination reaction is determined in a gel sedimentation test.

13. Method as claimed in claim 7, wherein the occurrence or absence of an agglutination reaction is determined in a gel sedimentation test.

14. Method as claimed in claim 1, 2, 3, 6, 8, 9, 11, 12 or 13, wherein anti-D antibodies which are directed towards antigens on the erythrocyte membrane are used as antibodies.

15. Method as claimed in claim 4, wherein anti-D
antibodies which are directed towards antigens on the erythrocyte membrane are used as antibodies.

16. Method as claimed in claim 5, wherein anti-D
antibodies which are directed towards antigens on the erythrocyte membrane are used as antibodies.

17. Method as claimed in claim 7, wherein anti-D
antibodies which are directed towards antigens on the erythrocyte membrane are used as antibodies.

18. Method as claimed in claim 10, wherein anti-D
antibodies which are directed towards antigens on the erythrocyte membrane are used as antibodies.

19. Method as claimed in claim 1, 2, 3, 6, 8, 9, 11, 12, 13, 15, 16, 17 or 18, wherein a pregnancy test is carried out.

20. Method as claimed in claim 4, wherein a pregnancy test is carried out.

21. Method as claimed in claim 5, wherein a pregnancy test is carried out.

22. Method as claimed in claim 7, wherein a pregnancy test is carried out.

23. Method as claimed in claim 10, wherein a pregnancy test is carried out.

24. Method as claimed in claim 14, wherein a pregnancy test is carried out.

25. Method as claimed in claim 19, wherein HCG is determined as the analyte.

26. Method as claimed in claim 20, 21 or 22, wherein HCG is determined as the analyte.

27. Method as claimed in claim 1, 2, 3, 6, 8, 9, 11, 12, 13, 14, 15, 16, 17 or 18, wherein a test is carried out for analytes which play a role in the detection of diseases.

28. Method as claimed in claim 4, wherein a test is carried out for analytes which play a role in the detection of diseases.

29. Method as claimed in claim 5, wherein a test is carried out for analytes which play a role in the detection of diseases.

30. Method as claimed in claim 7, wherein a test is carried out for analytes which play a role in the detection of diseases.

31. Method as claimed in claim 10, wherein a test is carried out for analytes which play a role in the detection of diseases.

32. Method as claimed in claim 27, 28, 29, 30 or 31, wherein the diseases are selected from the group consisting of hepatitis, AIDS, syphilis and hormone disorders.

33. Reagent for the determination of an analyte in an aqueous sample solution by agglutination of non-fixed erythrocytes, wherein it contains erythrocytes on the membrane of which one or several antibodies or antibody derivatives which do not lead to a premature agglutination are bound and whereby a first binding partner of a high affinity binding pair is coupled covalently to the antibodies or antibody derivatives.

34. Reagent as claimed in claim 33, wherein in addition a second binding partner of the high affinity binding pair is bound to the first binding partner whereby the second binding partner has more than one binding site per molecule for the first binding partner.

35. Reagent as claimed in claim 33 or 34, wherein in addition the first binding partner of the high affinity binding pair is bound to the second binding partner which is coupled to the analyte or an antigen-active determinant of the analyte.

36. Reagent as claimed in claim 33 or 34, wherein it is present in the form of a suspension.

37. Reagent as claimed in claim 35, wherein it is present in the form of a suspension.

38. Reagent kit, wherein it contains a reagent as claimed in claim 33, 34 or 37 and an antibody or a corresponding antibody derivative which is directed towards the analyte to be determined.

39. Reagent kit, wherein it contains a reagent as claimed in claim 35 and an antibody or a corresponding antibody derivative which is directed towards the analyte to be determined.

40. Reagent kit, wherein it contains a reagent as claimed in claim 36 and an antibody or a corresponding antibody derivative which is directed towards the analyte to be determined.

41. Reagent kit as claimed in claim 38, wherein in addition it contains a test vessel which is filled with a gel suspension.

42. Reagent kit as claimed in claim 39 or 40, wherein in addition it contains a test vessel which is filled with a gel suspension.