Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~3ZO~
'I
--2--
The present invention is concerned wlth a process
for the determination of an opsonising, surface-binding
~2-glycoprotein, which is hereinafter referred to as
a~-SB-glycoprotein, and especially for the determination
thereof in body fluids.
a2-SB-Glycoprotein is a substance which is found
in blood and organ stroma. The serum protein has the
- electrophoretic motility of an ~2-globulin and a sediment-
ation coefficient of 12 to 14 S. Its molecular weight is
about ~00,000 to 440,000. It consists of t~o subunits,
which are joined together by disulphide bridges. ~he
concentration thereof in the plasma of healthy persons
is 300 to ~00 ~g./ml.
a2-SB-Glycoprotein is particularLy characterised
by speeial adhesion properties and, like an adhesive,
is able to bind other substances, for example cell debris.
a2-SB-Glycoprotein is a substrate of the acti-~ated
coagulation factor ~III, i.e. of factor ~IIIa (plasma
transglutaminase, fibrinoligase). It binds fibrinogen
and fibrin, especially in the cold. Since, during the
coagulation, it can react with fibrin, serum usually
contains smaller concentrations than plasma.
A physiological property of ~2-SB-glycoprotein is
its opsonising action. For example, the uptake of
particulate material is promoted by the reticuloendo-
thelial system (RES). Therefore, a direct correlation
is assumed between the ~2-SB-glycoprotein level in the
..
.~k
.~ .
,
~' ' ; '
~3;2Q~
~! -3-
plasma and the activity of the RES.
A decrease of the a2-SB-glycoprotein level occurs
in certain diseases. Increased values have been observed
in diseases of the connective tissue system and in
metastasing tumours in advanced stages.
It is assumecl that the removal of damaged auto-
logous tissue and of circulating colloidal components,
for example soluble fibrin, by non-immunoLogical opson-
ation is an important physiological process. For this
reason, a considerable decrease of a2-SB-glycoprotein
eould lead to organ failure in severely diseased
patients. Therefore, the determination of a2-SB-glyco-
protein is of diagnostic interest~ -
Processes are kno~n for the qualitative and
quantitative determination of this protein. Saba et al.
~J. Reticuloendothel~ Soc., 3, 398/1966) described a
radiological ~hagocytosis test for the semi~uantitative
determination of an opsonising serum factor which stim-
ulates the phagocytosis of particulate material in the
Kupffer cells of the liver. In this test system, the
uptake of radioactiv-labelled, gelatine-coated lipid
micelles in liver sections is measured. ~n immunological
detection process is described hy F.A. Blumenstock et al.
(J. Reticuloendothel. Soc., 23, 119/1978). In this ease,
the a2-SB-glycoprOtein is determined by means of an
electro-immune assay (roeket electrophoresis). In
~Ioppe-Seylers Z. Physiol. Chemie, 359, 247/lg78, there
.
is described a qu~ntit~tive method for the determin-
ation of CIG ~corresponds to ~2-SB-glycoprotein),
utilising the a,finity of CIG for 125-I-marked collagen,
the complex formed being precipitated out with anti-CIG
antiserum. In Int. J. Cancer, 20, 1/1977, there is
described an enzyme immuno assay in which plastic
test tubes are coated with gelatine or specific
antibodies against fibronectin and, after binding
fibronectin (corresponds to a2-SB-glycoprotein) on the
walls Oc the test tubes, there follows an incubation
with enzyme-labelled anti-fibronectin antibody (sandwich
principle). Furthermore, from J. Biol. Chem., 2~5,
5728/1970, there is known an immune-electrophoretic
method for carrying out a quantitative determination.
The known methods all suffer from the disadvantage
that, on the one hand, they require a relatively high
expenditure of labour and, on the other hand, the carry-
ing out of the test takes about 20 to 24 hours. Further-
more, several working steps are necessary.
Therefore, although it has already been possible
for quite a long time (1970) to determine ~2-SB-glyco-
protein ~uantitatively, it has previously not been
possible to develop for clinical practice a test which
is simple and ~uick to carry out. Because of the special
physiological properties and the diagnostic consequences
proceeding therefrom, a rapid carrying out of the test
would, however, be very desirable, a quick result being
- ' ' ' '
~13Z~
..
--5--
necessary, especially in acute cases, for the purpose
of clinical control and of continuous monitoring.
Therefore, it is an object of the present
invention to provide a rocess for the determination
of a2-S~-glycoprotein ~hich can be carried out within
a few minutes and in as simple a manner as possible
and thus, for the first time, to provide the pre-
requisite of detecting, ln a short time, changes in
the amount of this substance and to make it evaluable
for the purposes of diagnosis and treatment.
Thus, according to the present invention, there
is provided a process for the determination of an opson-
ising, surface-binding a2-glycoprotein, wherein a
buffer2d solution of antibodies against the opsonising,
surface-binding a2-glycoprotein is mi~ed with the sample
to be investigated and the turbidity formed is determined
turbidimetrically or nephelometrically at definite
intervals of time.
Surprisingly, we have found that it is possible
to determine a2-SB-glycoprotein turbidimetrically by an
immunological method although, having regard to the
adhesive properties of this substance, it wa~s to have
been expected that foreign substances would be entrained
and thus the proportionality bet~een the amount of the
protein to be determined and the resulting turbidity
could no longer be guaranteed and, furthermore, that
flocculation or agglomeration phenomena would occu-r
1~3-~:044
which would make a ~uantitative determination by turbidity
measurement im~ossible. This is also expressed by the
term "opsonising' which literally means "preparinq for
eating". This is to e~press the idea that this protein
serves, as it were, for the recognition of su~stances to
be removed and, by its adhesion to the cells of the
reticuloendothelial system, bring about the "eating up"
of these substances with the opsonising protein adhering
thereto.
The process according to the present invention is
preferably carried out at a pH value of from 5 to 9.5
and especially of from 6.5 to 8.5. Substances buffer-
ing in this range can be used. Buffers are preferred
with a low ion strength of up to about 100 mmolar.
However, buffer solutions with greater ion strength can
also be used.
In a preferred embodiment of the present invention,
a polyethylene glycol is added, the water-soluble types
of polyethylene glycol being preferred. An especially
preferred amount is from 1 to 5fi by weight of poly-
ethylene glycol, referred to the test solution.
The process is preferably carried out at a
definite temperature of from 15 to 35C. and more prefer-
ably of 20 to 30C In the case of lower temperatures,
precipitation takes place considerably more slowly and
at higher temperatures a dissociation of the immune
complex ta~ses place again.
1132~
The measurement ltself can he carried out at any
desired wavelength ~hich is known for turbidimetric
determinations, short wavelengths, for example 334, 360 and
405 nm., being preferr~d. A nephelometric determination
can aLso be used.
Measurement is carried out at definite intervals
of time, for exam~le after 1 minute and after 10 minutes,
the extinction di~ference of the measured values being
directly proportional to the content of a2-SB-glyco-
protein.
The antibody or antiserum can be ~roduced in the
usual manner by injecting the a2-SB-glyco~rotein serving
as antigen intradermally or intramuscularly, with re~eated
administrations, into suitable experimental animals, for
exam~le rabbits, sheep or goats. ~dministration can be
carried out with conventional immunological adjuvants,
for example Freund's adjuvant.
a2-S~-Glycoprotein is removed from human plasma
by affinity chromatography and the plasma then cross-
lin]~ed with glutardialdehyde to give a gel. The gel is
homogenised and, using the homogenate, the antisera
obtained from the experimental animals are absorbed up
to immunologica~ monospecificity (tested by immune
diffusion and lmmune electrophoresis). 'rhe antibodies
against the a2-SB-glycoprotein not absorbed in this
manner are used in th~ process.
The antiserum thus produced is diluted wlth the
.,
4~
above-descri'~ed ~uffer to the desired concen.tration,
optionally mixed with polyethylene glycol and stabilis-
ing agents and stored in liquid or lyophilised lorm.
.~xam~les oF sta'~ilising agents which can be used include,
for e~am~le, mannitol, alkali metal azides, serum albumin
and the ll~ie.
The process according to the present inventlon can
be used ~ot only for carrying out a manual test but also
in automatic analysers. This is e~plained in more
detail, with reference to the accompanying drawings, in
which:
~ig.l illustrates a standard curve l~roduced wit'n
a manual test;
Fig.2 illustrates a standard curve produced on an
automatic analyser (ABA-100); and
Fig.3 shows the correlation ~etween the determin-
ati~n o a2-SB-~lycoprotein in human plasma
according to the present invention, using a
mamlal test, with an immune electrophoretic
method (roc]cet electrophoresis).
r~he deterrnination is carried out by comparison with
a standard solution o F known a2-SB-glycoproteinconcentration.
The ~rocess according to the present invention
permits a rapid and e~act determination of a2-S~-glyco-
protein, which only re~uires a ew minutes and can be
carried out very simply with conventional photometric
devices. In the sc~me way, it can also be used for auto-
matic analysers.
1~32~
g
Tne following ~xample i5 given for the purpose
of illust~ating the pres2nt invention:
~3~ainl?1e .
A. ?roduction of the antiserum.
a2-S3-Glycoprotein was isolated by the method
descri"ed in 3iochem. 3iophys. Acta, 534, 210/197~ by
affinity chromatography on gelatine, bound on to cross-
linked agarose.
Rahbits, sheep and goats were i~munised according
to the following scheme:
, _ . .
day ¦ ~mount mode of administration ~ adjuvant
1 500 ~g.intradermally at several Freund
places
7 100 ~g.intramuscularly Freund
21 100 ~g.intramuscularly Freund
49 100 ~g.intramuscularly Freund
100 ~g.intramuscularly Freund
*) L~t monthly intervals.
The follo~7ing procedure was used for purification
of the antisera obtained from the experimental animals
100 ml. human plasma were freed, by affinity chromato-
graphy, from a2-SB-glycoprotein and then cross-linked
to give a gel using glutardialdehyde(by the process
described in Immunochemistry, 6, 53/1969)~ the gel
obtained then being homogenised. Antiserum obtained
~-
~32049~
--10--
from the experimental animals was filtered through the
gel. ~fter passage through the gel, the remaining anti-
serum displayed i~munological monospecificity.
- ~0 Car~yin~ out of the test
--
A standard solution of x2-SB-glycoprotein was
produced by dissolving definite amounts thereof in
0.15 mol/litre phosphate-buffered sodium chloride sol-
ution containing 1% by weight bovine serum albumin and
0.1~ by weight sodium azide.
Before use, the antiserum was brought to the
desired dilution with 0.66 ~ol/litre phosphate ouffer
(pH 7.4) ~hich contained 2.5,~, by ~eight polyethylene
glycol and O.lC' by weight sodium azide.
For carrying out the test, 500 ~1. of the diluted
antibody were introduced into a cuvette of 1 cm. layer
thickness and mixed with 10 ~l. of sample solution or
standard and then, at 20 to 25C., the extinction was
measured against air at T~g 334 nm in a commercially
available photometer 1 minute and 10 minutes after
mixing. The content of a2-SB-glycoprotein was obtained
by comparison of the extinction difference with a standard
curve obtained in the same manner.
'
