COUPLING OF ANTIGENS AND ANTIBODIES TO NON-FIXED ERYTHROCYTES

COUPLAGE D'ANTIGENES ET D'ANTICORPS A DES ERYTHROCYTES NON FIXES

English 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.

Claims

The embodiments of the invention in which an exclusive
property or privilege is claimed are defined as
follows:
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.

Description

2~6~~~~
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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.

- 2 -
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

- 3 -
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~
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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

- 5 -
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.

E
- 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

2090 39 2
- 10 -
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 ~'
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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.

2090392
- 12 --
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.

Representative Drawing