METHOD FOR THE DETERMINATION OF ANTIGENS

METHODE DE DETERMINATION DE LA TENEUR EN ANTIGENES

English Abstract

- 1 -
Abstract
In the photometric determination of the antigen content by
the antigen-antibody-reaction at least one of the two
reaction components is diluted with a physiological salt
solution, which contains, beside polyethylenglycol a buffer
substance, before the two reaction components are combined.

Claims

Claims:
1. A method for the determination of the antigen content
of a sample by an antigen-antibody reaction involving the
mixing of a reaction component containing the antigen and
a reaction component containing an antibody, comprising
first diluting at least one of the two reaction compon-
ents with a physiological saline solution which contains
polyethyleneglycol and a buffer substance, thereafter
combining both reaction components and determining the
antigen-antibody reaction photometeically in the presence
of a buffer substance, wherein the buffer substance is
selected from the group consisting of a glycin, glycyl-
glycin, acetate, citrate, bicine, histidin/HCl, tris or
5.5-dialkylbarbituric acid buffer.
2. A method according to claim 1, wherein the 5.5-dialkyl-
barbituric acid is 5.5-diethylbarbituric acid.
3. A method according to claim 1 wherein the concentration
of buffer substance in the salt solution is 5 to 200 mmol/l.
4. A method according to any one of claims 1 to 3, wherein
the concentration of the polyethyleneglycol in the salt
solution is 2 to 6% by weight, and the average molecular
weight of the polyethyleneglycol is 4000 to 12000.
5. A method according to any one of claims 1 to 3, wherein
the pH-value of the physiological salt solution containing
the buffer substance and the polyethylenelgycol is 4,0 to 9,0.
6. A method according to any one of claims 1 to 3,
wherein the photometrical determination of the kinetic
progression of the antigen-antibody-reaction is performed.
7. A polyethylene containing, physiological salt solution
which can be used as a diluent of at least one of two
reaction components of an antigen-antibody-reaction for
the photometrical determination of the antigen content,
wherein said solution contains a buffer substance selected
from the group consisting of a glycin, glycylglycin,
acetate, citrate, bicine, histidin/HCl, tris or 5.5-
dialkylbarbituric acid buffer.

Description

- ~Z08548
-- 1 --
The invention relates to a method for the determination
of the antigen content in a sample by the antigen-antibody-
reaction. It also relates to a diluent for an antigen
and antibody component, respectively, for preforming this
method.
The determination of diagnostic relevant parameters with
antigens in samples, as serum, plasma or liquor, by the
immunological or antigen-antibody-reaction, which is
evaluated photometrically for instance by nephelometry
or turbidimetry, makes frequently a special preparation
of both reaction components necessary. Usually the re-
action component which contains the sample which antigen
content has to be determined, and the other component
which contains the antibody, i.e. which is normally the
corresponding antiserum, are diluted with physiological
salt solution or with physiological salt solution which
contains in addition 4~ polyethylenglycol (average
molecular weight 6000).
The content of polyethylenglycol serves particularly to
avoid a precipitation of the antigen-antibody-complexes
formed during the reaction, i.e. to avoid the formation
of a precipitation in the test solution formed by the com-
bination of both reaction components during the antigen
determination, i.e. during the measurement. This property
of polyethylenglycol is known for a certain period of
time and is a necessity for these measurements, because
otherwlse not all formed antigen-antibody-comple~es are
subjected to the measurement.
l'he determination of immune globulins by laser turbidimetry
according to the "Arbeitsanleitung fur das Lasermed-
Turbidometer (pm 4) zur Bestimmung der Immunglobuline IgA,
IgG, IgM mit ATAB-Antiseren (Instructions for the Lasermed-
turbidometer (pm 4) for determining the immune globulins
'~

208S~8
-- 2 --
IgA, IgG, IgM with ATAB-anitsera), AHS Deutschland GmbH,
Bereich ~lerz und Dade, Munich, 1982", showed that with
different physiological salt solutions with 4% by weight
polyethylenglycol different measurement signals were
obtained with the laser turbidometer during the assay,
although they (the salt solutions) were prepared according
to the same formulation and with the same reagents by the
same person. Accordingly no sufficiently reproducible
results were obtained. The so called kinetic method
shows a particular interference when according to said
instructions after a prereaction of 10 sec. the rate of
the antigen-antibody-reaction is measured and the antigen
content is determined. In contrast to the so called "Fix
point"-method according to said instructions, i.e. the
final poin~ measurement after a reaction period of for
instance 30 min, it is not necessary with said kinetic
method to wait until the end of the completion of the
antigen-antibody-reaction.
According to one aspect of the invention there is provided
a method for the determination of the antigen content of
a sample by an antigen-antibody reaction involving the
mixing of a reaction component containing the antigen and
a reaction component containing an antibody, comprising
first diluting at least one of the two reaction compon-
ents with a physiological saline solution which containspolyethyleneglycol and a buffer substance, thereafter
combining both reaction components and determining the
antigen-antibody reaction photometrically in the pre.sence
of a buffer sub~tance, wherein the buffer substance is
selected from the group consisting of a glycin, glycyl-
glycin, acetate, citrate, bicine, histidin/HCl, tcis or
5.5-dialkylbarbituric acid buffer~
According to another aspect of the invention there is
provided a polyethylene containing, physiological salt

` ~2~)85~18
- 2a -
solution which can be used as a diluent of at least one
of two reaction components of an antigen-antibody-reaction
for the photometrical determination of the antigen content,
wherein said solution contains a buffer substance selected
from the group consisting of a glycin, glycylglycin,
acetate, citrate, bicine, histidin/HCl, tris or 5.5-
dialkylbarbituric acid buffer.
The invention solves the object to provide a method and a
diluent, respectively, with which the antigen content of
sample is exactly and reproducibly determinable even with
the kinetic method.
The antibody molecule has a polyvalent and therefore a
strongly structured form whereas the polyethyleneglycol
is non-polar and therefore not structured. When koth
reaction components are combined a fairly homogenous
partition, which gives fairly reproducible results, is
obtained not before a certain period has lapsed. The
method according to the invetion is based on the fact
that a fast and homogenous partition of both reaction
components is obtained by adding a buffer substance to
the polyethyleneglycol containing, physiological salt
solution, which eliminates the structured form of the
antibody molecule by balancing the charges.
~5

- ~20B54B
It is surprising that theirate of the antigen-antibody-
reaction is particularly dependent on the chemical composition
of the buffer substance, even far more than the concentration
of the buffer substance or the pH-value~ Also important is
in connection with the invention that not such buffer
substances are selected which provide the fastest reaction
rate, but such buffer substances are prefered, with which
a constant reaction rate in the beginning is obtained which
_ lasts for a certain period of time. This property of the
buffer substances used according to the invention stra1ghtens
the reaction to a certain degree, so that a linear progress
over a relatively long period is obtained. This improves the
accuracy of the results of the kinetic method considerably.
If for instance 20 mmol/l 5.5-diethylbarbituric acid, pH 7,5,
are added as buffer to the physlological salt solution
containing 4 % by weight polyethylenglycol a pre reaction
time of for instance 10 sec is followed by a measurement
time of for instance 30 sec. The period of 30 sec is a
measurement basis for the antigen determination which is
sufficient even for very accurate results.