Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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One-Way Reaction Vessel for the Solid-Phase Immunological ~nalysis
of, and a Method of Measuring Constituents which can be determined
via Immune Reactions
Description
The present invention concerns a disposable reaction vessel for
the solid-phase immune analysis, and a method for its application.
Qffinity chromatography is a technology which is broadly applied
in preparative purification of bio-molecules. Therein, advantage
is taken of a specific interaction between the molecule to be
determined and a complementary binding partner. In the general
practice of such methods a sample containing the bio-molecule to
be purified is applied to a chromatographic column, which contains
one of' the mutually complementary binding partners bound to a
solid substrate. Examples of pairs of complementary binding
partners are enzymes and their substrates, antibodies and hapten
resp. antigen, and mutually complementary DN~ or RNQ single
chains.
In immunoaffinity chromatography, for quantification use is made
of the interaction between an antigen resp. a hapten and the
complementary antibody.
Up to now, in immunoassays using affinity chromatography reusable
columns are generally used which are packed with the antibody or
antigen (named ligand in the following) bound to a solid
substrate. The sample to be analyzed is pressed through the
chromatographic column by high pressure to guarantee a rapid
chromatographic processing. The application of high-pressure
liquid chromatography (HPLO) for immunoassays is described e.g. by
de Elvis, W.U., and Wilson, G.S., in "~nalytical Chemistry", 1985,
Vol. 57, pp. 2754-2756, and Sportsman7 3.R. et al. in "Qnalytical
Chemistry", 1983, Vol. ~5, pp. 771-775. However, the use of HPLC
for immunoassays has several drawbacks On the one hand, the use
of costly instruments is necessary for this technique, and on the
other hand, the chromatographic columns for the analysis must meet
high technical demands. The columns must withstand the high
pressures applied in HPLC as well as be reusable to allow a
rational application of this technology. However, the reuse of the
columns re~uires additional measures before a new sample can be
analyzed on the same column. ~s pubtished in DE-OS 37 11 894,
reusable columns are regenerated prior to every analysis to
prevent effects of displaced and incompletely eluted substances of
the previrus test.
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In the DE-OS 24 48 411 a reaction vessel is described for solid-
phase immune analysis However, prior to a quantitative
determination, this vessel for multiple use requires a calibration
with standards of the component to be determined. Besides, long
incubation times are necessary in this described reaction vessel,
during which the flow through the column is stopped. 6 hours to 2
days are recommended to guarantee sufficient binding of the
component to be determined to the immunologically complementarY
binding partner loaded onto the substrate However, the binding
reaction is always terminated before the binding equilibrium is
reached, which would take 1-2 days, to guarantee a rational
performance The requirement of calibration with standard
solutions is a consequence as we~l of this early termination of
the binding reaction.
Rccordihg to the preference in practice for disposable columns,
the DE-PS 26 26 8~3 describes a procedure for the preparation of a
prefabricated disposable adsorption column especially for radio
immunoassays using analytical instruments applying centrifugal
forces. However, this column is not a reaction column, and is
useful for the separation a~ter an immune reaction only, because
it is not loaded with a specific immunoreactive component. To
prevent disturbing effects during the separation via gel bed, the
flow resistance of the porous retaining dèvices is kept as low as
possible.
The object of the present invention is to provide a disposable
reaction vessel for the solid-phase immune analysis which allows
the rapid direct performance of immunoassays, which is easy to
handle, and which is ready to immediate use without prior
calibration or regeneration, and a method for the application of
this vessel.
With respect to the device, this is accomplished in the present ,~ r
invention by a disposable reaction vessel for the solid-phase
immune analysis which is open at the upper and lower ends, in
which at least one immunologically reactive component is bound to
a substrate, characterized by a substrate bed volume of up to 600
ul, and by the determination of the liquid flow velocity by the
permeability of the substrate. The substrate is either a solid
frit, membran or the like, or it is contained between an upper and
a lower separating device.
With respect to the method, this is accomplished by a procedure
for the determination of components which can be determined Vi3
immune reactions, which is characterized by the application of the
sample to be analyzed to d disposable reaction vessel according to
any one of the claims 1-20, which is further characterized by the
retention of the sample component, which has to be determined, by
the complementary immunologically reactive component in the
disposable reaction vessel during the direct flow-through, and in
which the component to be determined is
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a) eluted and determined, or
b) marked in the disposable reaction vessel via amplification
reactions with common immunological markers, and then
determined, or
c) determined via a marked compound in reactive competition to
the component to be determined.
The invention is described in detail with relation to figures 1
and 2 which show:
Fig. la a longitudinal section of a disposable reaction vessel 1
according to the invention containing two separating
devices 2a and 2b, between which the substrate with the
' immunologically reactive component 3 is located,
Fig. lb a top view of the disposable reaction vessel 1, and
Fig. 2 a scheme of an online-system for antibody production
control.
The dimensions given in figures la and lb are for example only.
The described disposable reaction vessel 1 and the described
method, in which the disposable reaction vessel 1 is used, exhibit
several considerable advantages compared to known reaction vessels
resp. methods.
The described disposable reaction vessel 1 allows a rapid
performance of the quantitative determination of components which
can be determined by immunoreactions. The handling of the reaction
vessel 1 is very simple and does not require the use of costly
instruments such as HPLC apparatus. Besides excellent results are
obtained with the aforesaid disposable reaction vessel 1 at
extremely low consumption of substrate 3 with bound
immunologically reactive component (named substrate bed in the
following). R disposable reaction vessel 1 with a substrate bed
volume 8, i.e. the volume between the upper and lower separating
device 2a and 2b, of 50 ul is preferably used. The small substrate
bed volume allows the use of small quantities of elution liquid
with the consequence that the dilutio.- effect of the elution
remains small, and thus the detection limit is not decreased in
contrast to other methods in which larger elution volumes are
necessary.
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Besides, the aforesaid reaction vessel 1 allows the quantitative
determination at binding equilibrium (end-point determination)
between the component to be determined and the immunologically
reactive component. The binding equilibrium is reached within an
incubation time of 1 minute already in the aforesaid disposable
reaction vessel 1.
The rapid e~uilibration is rendered possible by the small
substrate bed volume 8, its flow resistance, and the high loading
of the substrate with the immunologically reactive component. The
consequence of the controlled flow and the high loading of the
substrate with the reactive component is that the component to be
determined is bound quannatitatively to the reactive component
within about 1 minute, i.e. before the flowing liqu;d leaves the
substra,te bed volume. Therefore, a comparative analysis with
standard references is spared.
Besides, a rapid analysis of samples is possible, because the
disposable reaction vessel 1 allows immediate use, because the
substrate 3 with the immunologically reactive component can be
standardi~ed and ready-made, and the disposable reaction vessel 1
does not need to be calibrated before use.
The substrate 3 does not need to be stable against pressure,
because the disposable reaction vessel 1 allows the rapid analysis
of samples without application of HPLC. For the substrate 3, all
common materials useful for affinity chromatography can be
considered. Prefered substrate materials are polymeric sugars,
plastics, plastic-modified substrates, metal oxides, or silicates.
Substrate materials in the form of frits are especially prefered.
For the determination of very large components such as viruses,
which do not have access to the pores of such substrate materials,
graft copolymers with side chains with active groups are prefered.
The immunologically reactive component loaded onto the substrate
material 3 can bs bound to the substrate material covalently as
well as adsorptively. The immunologically reactive component can
be selected from the group containing haptens, antigens,
antibodies, and immuno-affine proteins. Polyclonal as well as
monoclonal antibodies can be used.
.. . . .
The vessel material 4 does not need to be stable against pressure,
because the disposable reaction vessel 1 does not need to be
subjected to high pressures in the present procedure. Therefore,
besides pressure-resistant materials such as metals, other not
pressure-resistant materials can be considered for the production
of the disposable reaction vessel 1 such as glass, natural
materials, and plastics. Prefered plastics are polyethylene~
polypropylene, and/or polystyrene, and prefered metals are
aluminum and stainless steel.
For practi~cal purposes, the disposable reaction vessel 1 has a
swelling 5 at one end to allow adaption of the reaction vessel
into a holding and moving device for automatic transport. This is
necessary for the use in automated sample processing devices.
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Besides, for practical purposes, the disposable reaction vessel 1
has a smaller-diameter joininy at the end opposite to the
swelling, preferentially a joining ~or a socket-switch connection
with another disposable reaction vessel~ This facilitates e.g.
joining in series of several disposable reaction vessels 1,
whereby the outlet 6 of the first vessel is,connected with the
inlet of the second vessel by a easy simple male-female
connection.
Besides, the disposable reaction vessel 1 is closable by caps 7
and 9.
The present procedùre using said disposable reaction vessel 1 is
superior due to, among others, a strongly simplified procedure
compared to common procedures, and because it allows a rapid
quantitative and qualitative determination. This is enabled partly
because the determination of the component to be determined does
not require a preceding calibration or regeneration of the used
substrate in the disposable reaction vessel 1, and because no
series reference measurements with standard solutions are ',
necessary. The easy handling is a consequence of the possibility
to apply samples and other solutions by usual laboratory equipment
or by a pipetting automate. ~-
Besides manual procedures, automated procedures are improved by
application of the invented reaction vessel 1. In such procedures,
the application of the sample and the further steps of the
procedure ars performed by an automated sample processing device.
Such a sample processing device is used for example in the online
production process control of antibodies produced in a fermenter.
In such procedures the samples are automatically applied in time
intervalls of about 6 to 8 hours, and the antibody concentration
in the fermentation medium can be continuously determined rapidly
and easily over a long time period using the disposable reaction
vessel 1. Q system for antibody production control using the
invented disposable reaction vessel 1 is schematically described ~ ;
in fig. 2. Therein, the disposàble reaction vessel 1 is included
in the automated sample preocessing device.
The present procedure can be performed rapidly without application
of high pressures to the disposable reaction vessel 1 after sample
application. But low suction or pressure forces can be used for
quicker performance, e.g. by centrifugation or pumping.
If necessary, several disposable reaction vessels 1 can be
connected in series or parallel in the present procedure. This
allows e.g. the simultaneous determination of several parameters
in one single sample resp. the simultaneous analysis of several
samples.
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The connection of several disposable reaction vessels in series is
advantageous e.g. for the performance of allergy tests. Therein, a
different antigen (allergen) is applied to each different column,
then the columns are connected in series, and the sample
containing the antibody of the class IgE which mediates the
allergic reaction is applied to the first reaction vessel.
The sample flows successively through the different disposable
reaction vessels 1 containing the different allergens. The IgE of
the sample will be bound in the reaction vessel which contains the
allergy-evoking antigen, and can be detected e.g. via fluorescence
detection. In this way, one single sample can be tested
simultaneously with many different potential allergens.
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