Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Particles and proc~dures for the determination of
antigens and/or antibodies using the particles
The invention concerns a particle and a procedure
for the determination of antigens and/or antibodies
using these particles.
There are various procedures f~r the determination
of antigens or antibodies, bas~d on the principles
of agglugination, precipitation, complement binding
reaction, immunofluorescence, radioimmunoreaction,
enzyme immunoreaction etc. All of these procedures
have in common, that only one type of antigen or antibody
can be determined at a t~me~
However, it is commonly the case that a search is
for several types of antigen or antibody, e.g. for
the differential diagnosis of infectious or other
diseases, in screening tests in healthy subjects,
e.g. in serological diagnosis of tumors (multiple
tumor antigens), or in allergy tests (several allergens)
or in the determination of the immunological status
of the organism. Rccording to this state-of-the-art
of the techniques, particular~ investigations have
to be performed for each type of antigen or antib`ody
which is sought. This represents a~multiple investment
of time and materiàl. ~ ~
The object underlying the discovery is to reduce~ the
expenditure of time and material in the investigation
and determination of several types~of antigen or antibody.
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According to the present invention, the solution of this problem
is possible using a mixture of particles characterized by one or
more distinguishable labeling substances. Thus the particles in
the mixture can be distînguished on the basis of their different
labels. Also on the basis of this fact, the fluorimetrically
measured immunoreaction between the antigen or antibody with the
particle, loaded with antigens or antibodies, and the antibody or
antigen in the liquid to be inYestigated, can be categorized into
a particular specificity, which represents the combination of
labeling signals of the particles.
Accordingly, in the present invention, there is provided a test
agent with labelling for analyzing a plurality of different
antigens and/or antibodies in a liquid sample, characterized by
neutral carrier particles which are labelled individually or in
groups with a fluorescent labelling substance and which are
loadable with different antibodies or antigens for determining
antigens and/or antibodies.
A further aspect of the present invention is a procedure for the
determination of antigens or antibodies l~sing particles variously
labeled by labeling substances sele~ted from the group of
fluorescing substances, colouring agents or pigments and/or their
size, characterized in that said particles are loaded with
different antigens or antibodiesa that a mixture of such loaded
particles is mixed with a liquid containing the antibodies or
antigens to be investigated or determined, and that, after a
reaction period in which the antibodies or antigens to be
determined are bound ~o the antibodies or antigens fixed to the
particles, with which the particles are loaded, the antibodies or
antigens are identified by subsequent reaction steps and
measurements. Using this procedure, it is possible, through the
categori~ation of the reacting antigens or antibodies towards the
distinguishable particles of the mixture, to determine the
antigens or antibodies by the measurement of immunofluorescence
and by probing and analysis of the labels.
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The invention is described below in more detail U5i ng the
following example, whereby reference is made to the accompanyin9
drawings which illustrate embodiments of the invention as follows:
Figure 1 depicts a particle adapted to the determination of bound
antibodies according to the present invention; and
Figure 2 depicts a particle adapted to the determination of bound
antigens according to the presen~ invention.
A serum is to be investigated for antibody types 2 and 16, e.g.
Akl, Ak2, ..., Akj, ..., Akn. Groups (populations of
small particles 4, preferably in the form
'I`qi,
of spheres with a diameter of approximately 10 ~m
and of a suitable carrier material (plastic or poly-
saccharide polymer, e.g. agar), are loaded each with
a t~pe of antigen, 6 or 12, Agl, Ag2 ~ A~ ,....
Aqn ~ach type of antigen ist
bound to a particular type of particle, which were
previously labeled in the following way.
The particles ~ are labeled with a combination of
substances 8, whose fluorescence spectrum is defined
and can be determined fluorimetrically. The labeling
can also be carried out in a simple manner, such that
the individual particle populations are only labeled
with one fluorescing labeling substance with a particular
concentration, differing however from particle population
to particle population, or with several labeling substan-
ces with a particular concentration, differing however
`from particle population to particle population. This
type of labeling (labeling with distinguishable concent-
rations of the same fluorescing substance) can be
combined with labeling with labeling using a combination
of fluorescing substances with differing emission
spectra. The evaluation is then per~ormed on the basis
of the spectrum and/or intensity of the emitted fluores-
cence. In this way, using one labeling substance,
applied in two distinguishable concentrations (0%
and 100% of a particular concentration), 21 = 2 particle
populations can be distinguished (particles with the
substance and particles without the substance). If
during the labeling, the substance is applied in three
distinguishable concentrations, e.g. 0%, 50% and 100
of a given concentration, then 31 = 3 particle populations
can be distinguished. With n labeling substances with
differing emission spectra, each applied in ~ ~istin-
guishable concentration, the~ number of labeling possibi-
lities amounts to m , e.g. with three labelin~ substances
each in ten distinguishable concentrations, 103
1000 different particle populations can be characterized
and identified. The labeling of the particles occurs
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during or after their production. For each type of
antigen ~Agj ), a particle population (Pj ) definable
fluorescence spectrophotometrically or by its size
is applied. The antigen or antibody is chemically
or physically bound to the particle. This is done
separately for each type of antigen or antibody. There-
after, all the particles are mixed in the desired
combination. A mixture of particles Pl , P2 ~ ~ Pj
..., Pn ~ loaded with the respective antigens Ayl ,
Ag2,..., Agj,..., Agn is thus produced.
This mixture of particles is mixed with the liquid
(e.g. blood serum), containing the antibodies to be
determined. After a reaction time, the the antibodies
to be determined, 16 and 2, bind specifically to the
corresponding antigens 12 and 6. After a washing step
for the particles using a washing fluid for the removal
of non-bound substances, the particles are mixed with
a solution o fluorescein-labeled antibodies lO, which
react species specifically with the antibodies to
be determined. These fluorescein-label~d antibodies
react with al~l antibodies (every antigen specificity)
o the animal species, from which the antibodies to
be determined originate. After the reaction time,
in which the antibodies lO are bound to the antibodies
16 and 2, the particles are again washed to remove
non-bound fluorescein-labeled antibodies. Then, not
only the fluorescence of each individual particle-
bound antibody 10 (immunofluorescence as a parameter
for the immune reaction which has taken place) is
measured, but also the fluorescence label which identifies
the particle, the size of the particle using a suitable
measuring device, are also measured. A flow~cytometer
which measures the fluorescence data (and also the
size) of each individual particle is suitable for
this. The data are processed by ~ computer, whereby
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the immunofluorescence is appointed to the appropriate
particle population. In this way, a profile of the
various antigen-antibody reactions Ag 1 Ak1 ,..~Agn Akn
is presented.
The procedure described above is equally applicable
to the determination of antigens 12 in the liquid
to be investigated. In this case, after the first
reaction and washing step, a mixture of antibodies
16 towards all the antigens to be investigated is
added. These antibodies originate preferably from
another animal species than antibodies 14. After renewed
reaction and washing step, the fluorescein-labeled
antibodies 10, which react species specifically with
the antibodies 16, are added. The measurement follows
as in the investigations for antibodies.
The procedure can also be carried out in the following
way, which deviates from the flow-cytometry:
The particle mixture is fixed to a slide, e.g. to
the base of a microtitre plate. The serum to be investi-
gated is then applied to this slide (particle mosaic).
After a reaction time, the antibodies present in the
serum bind to the corresponding antigens on the particles.
The non-bound substances are removed by subsequent
washing.
In a second step, a fluorescence-labeled globulin
antibody 10 is applied, which is speclfic for the
animal species from which the antibodies to be investi-
gated 2 or 16 originate. In this second reaction,
the fluorescence-labeled globulin antibody~ 10 binds
to the antibodies (globulins) 2 and 16, which were
bound to the particles 4 ln the first reaction step.
The non-bound material is removed in a further washing
step. ~ ~
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The preparation is the(h investigated photometrically
using a fluorescence photomicroscope, which is fitted
out with filters etc, so that it can measure the spectrum
and intensity of the fluorescence radiation emitted by~
a single particle. For the investigation, it is expedient
to guide the detector of a fluorescence microscope
in a predetermined course, e.g. columnwise, over the
particle mixture on the slide which is to be investigat~d.
This can also be performed automatically with a suitable
appliance, where required.
The data are evaluated using a comFuter. The investigated
particle is indentified on the basis of the fluorescence
spctrum of the labels containéd in the particle, e.g.
as particle Pj . Thus, the measured fluorescence, which
derives from the globulin antibody (immunofluorescence)
can be attributed to the immunological reaction
Agj Akj . In this way, the emission data of a large
number of particles can be gathered one after the
other automatically, and evaluated by the computer.
The data of all the particle populations can be assimi-
lated and processed according to the statistical distri-
bution of the particles. Thus, a profile of the different
antigen-antibody reactions Ag 1 Akl , Ag 2 Ak2 ,...,AgnAkn
can be presented.
The emission spectrum or the emission spectra of the
labels of the particles can be excited by a radiation
of a wide spectrum; however, a ~efined line of ~he
emission spectrum can also be excited by radiation
of a defined wavelength or several defined lines through
excitation by several defined wavelngths.
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