Note: Descriptions are shown in the official language in which they were submitted.
CA 02291458 1999-12-02
CENTEON PHARMA GMBH 1998/Z020 - Ma 1195 - C30
Process for the specific detection of glycosylated proteins
The invention relates to a process for the determination of glycosylated
proteins which are bound to a solid phase and, after reaction, are detected
photometrically using a labeled substrate.
The glycosylation of proteins is a complex process which takes place
subsequent to protein synthesis in the cell interior. As a result of
glycosylation, proteins are stabilized and in some cases also even
equipped with their specific properties. The sugars linked to amino acids by
means of O- or N-glycosylation have considerable physiological functions,
for example as part of the protein structures recognized by the cell
receptors, which in turn can play a role in cell activation processes or the
half-life of proteins. Although monosaccharides are ubiquitous and not
characteristic of a certain species, it is not unusual for complex sugar
structures, which are constructed from the known sugars in different ways,
to be found for certain living beings.
Glycosylation plays a particular role in recombinantly prepared proteins. A
protein prepared, for example, in yeast cells, which completely resembles
the amino acid sequence of the human protein, as a rule has a markedly
different glycosylation pattern if appropriate glycosylation sites are
present.
This can lead to a modified half-life of the recombinant protein, for example
in the plasma, or to an induction of antibodies which can provoke allergic
reactions on repeated administration of the protein. This is particularly
significant if appropriately high doses of the protein are administered.
CA 02291458 1999-12-02
- 2 -
In the expression of recombinant proteins, it is not unusually observed that
only a part of the protein is glycosylated which is to be detected and, if
appropriate, to be removed from the product. It is therefore important to be
able to detect even traces of glycosylated proteins. Although the person
skilled in the art is familiar with some chemical methods for the
determination of glycosylated proteins, these methods are often
complicated and do not make the simultaneous quantification of the
specific protein in a more complex solution possible. The ELISA technique
also offers a possibility for the detection of glycosylated proteins, specific
antibodies being directed against the glycoside structures and, by means
of a second antibody, make the detection of another part of the primary
structure possible. However, polyclonal and monoclonal antibodies are
often produced in rodents such as rabbits or mice, which often do not have
the ability to produce antibodies which are directed against glycoside
structures. In these cases, the ELISA technique does not lead to utilizable
results.
One possibility of replacing the antibodies recognizing a glycoside
structure in a detection process consists in the use of lectins. These are
specific proteins which very specifically recognize and bind saccharides
even in lipid- or protein-bound form. They are widespread in all living
organisms. The longest-known lectins are concanavalin A, abrin, ricin and
phasin. Depending on their nature, they can bind different glycoside
radicals. It was therefore to be suspected that the binding of glycosylated
proteins to immobilized lectin and their subsequent detection by means of
a specific, labeled antibody against the nonglycosylated region should be
possible. It was likewise to be expected that it should be possible to
immobilize an antibody against the specific protein on the solid phase and
to carry out the detection of the glycosidic structures using a labeled
lectin.
However, it has been shown that the production of a standard curve using
appropriate test proteins does not lead to any clear dependence on
concentration and measured signal. Surprisingly, however, this is possible
CA 02291458 1999-12-02
- 3 -
if instead of the complete, monoclonal antibody (mAb) a corresponding
labeled Flab) fragment is used.
The invention therefore relates to a process for the specific detection of
glycosylated proteins, in which '
a) incubating the sample to be investigated with a solid phase
which is coated with a substrate which immobilizes the
glycosidic groups bonded to the protein,
b) removing the remains of the sample not bound to the solid
phase by washing,
c) allowing an enzyme-labeled Flab) fragment or F(ab')2 fragment
of a monoclonal antibody directed against the specific protein to
act on the immobilized, glycosylated protein, and
d) after washing out the unbound, labeled Flab) fragment or
F(ab')2 fragment by addition of a substance which is
chromogenic or induces chemiluminescence, detecting the
bound, glycosylated protein photometrically.
The substrate employed for coating the solid phase can be a lectin.
Moreover, it is also possible for coating the solid phase to use a
monoclonal antibody directed against the specific protein or a fragment of
an antibody of this type. The detection of the bound, glycosylated protein
can then be carried out using a labeled lectin.
The surprising observation that the complete, monoclonal antibody (mAb)
cannot be employed for the detection process according to the invention
but the corresponding labeled Flab) fragment, in particular the F(ab')2
fragment, can be used, was explainable by the observation that the
complete monoclonal antibody is bound to over 90% to a ConA matrix
CA 02291458 1999-12-02
- 4 -
(concanavalin A matrix), while over 99% of the F(ab')2 fragment was to be
found in the column eluate. The reason for this observation could be seen
in the fact that the complete monoclonal antibody has glycosylated Fc
portions which are bound to the ConA matrix so that the monoclonal
antibody is no longer adequately available for the detection of the
immobilized, glycosylated protein.
For the binding of the glycosylated proteins to the solid phase, the
abovementioned lectins or their derivatives, which are known to the person
skilled in the art, can thus be used. In this case, particular attention is to
be
paid to the fact that divalent cations are present, especially magnesium,
calcium and/ or manganese ions in the form of their water-soluble salts, for
example as chlorides. The presence of one or more of these ions is
necessary in order to maintain the complex lectin structure which is
necessary for the binding of the glycoside groups. The use of lectins as
agents for the immobilization of glycosylated proteins can be carried out,
for example, using microtiter plates which are coated with concanavalin A.
Attention is to be paid here to the presence of divalent cations for the
binding of the glycosylated protein by the lectin. This also applies to the
subsequent washing processes, the incubation with the sample to be
investigated and the subsequent detection reaction. The presence of the
divalent cations mentioned is also important if the labeled lectin is used as
a detector in a corresponding test.
A preferred detection process for glycosylated proteins is thus carried out
by first incubating a sample containing glycosylated protein with a lectin
which is adsorbed on or bound to a solid phase; the solid phase is then
washed and incubated with an antigen-binding fragment of an antibody
specific for the protein, preferentially an F(ab')2 fragment which, for
example, is labeled with an enzyme. Detection with the aid of peroxidase
has proven particularly suitable. Moreover, however, detection is also
possible by means of a labeled antigen-binding fragment of a second
antibody. Conversely, the F(ab')2 fragment can also be immobilized on the
CA 02291458 1999-12-02
- 5 -
solid phase, it then being possible to detect the glycosidic groups of the
glycosylated protein using a labeled lectin as a detector reagent after
incubation with the sample to be investigated.
The invention is illustrated by the following example:
CA 02291458 1999-12-02
- 6 -
Example 1:
Albumin was used in this example. To a certain amount, this contained
glycosylated molecules which were to be detected and quantified.
In order to obtain a reference substance, glycosylated albumin was
obtained preparatively by means of a ConA-Sepharose~ (Pharmacia AB,
Sweden) and quantified by conventional protein determination methods.
Owing to the removal of the saccharified molecules from the albumin
starting solution and the abovementioned quantification, the content of
saccharified albumin in the starting solution could also be calculated. Both
solutions, i.e. albumin having a known content of glycosylated albumin and
the "pure" saccharified albumin were used for plotting standard curves in
the test system described.
Microtiter plates were incubated at room temperature for 16 hours with 150
~I per well of a concanavalin A (type IV-S, Sigma, Germany) containing 10
~.g/ml. The coating buffer contained 20 mM Na acetate, 4 mM MgCl2, 4
mM CaCl2, 4mM MnCl2, pH 5.5. After washing the plates, the various
dilutions of the standard or the samples were applied. These had been
diluted beforehand with sample buffer, consisting of 50 mM tris, 150 mM
NaCI, 10 mM MgCl2, 0.5% Tween 20, pH 7.2. After incubation at 37°C
for
one hour, the wells were washed three times with sample buffer and then
incubated at 37°C for a further hour with 100 p,l each of a peroxidase-
labeled monoclonal antibody against human albumin or its F(ab')2
fragments. After washing three times, a POD substrate was pipetted into
the wells and incubated at room temperature (in the dark) for 5 minutes,
the reaction was stopped and the OD450 was determined photometrically.
Samples which contained a known amount of glycosylated albumin were
included and quantified in each case.
CA 02291458 1999-12-02
Result:
The standard curves were determined on four successive days. The
results obtained by means of.the complete mAb's were poorly reproducible
and in some cases revealed rio dependence or an unsatisfactory
dependence of the measured signal on the concentration employed (not
shown). The mean values and standard deviations of the measured
values, however, which were obtained with the aid of the labeled F(ab')2
fragments, are shown in Fig. 1. In this figure:
- triangles signify pure glycosylated albumin
- squares signify albumin with a corresponding content of
glycosylated albumin.
The parallel and almost identical course in the case of the standard curve
shows that the reaction or the measured signals are specific and only the
saccharified albumin was shown.
The measured values obtained with known total contents of glycosylated
albumin are shown in the following table:
Sample No. Content of glycosylated Measured content of
albumin (%) glycosylated albumin
(%)
1 0.66 0.61
2 0.55 0.59
3 0.72 0.73
4 0.59 0.54
0.60 0.68
