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Patent 1248873 Summary

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Claims and Abstract availability

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(12) Patent: (11) CA 1248873
(21) Application Number: 454853
(54) English Title: PARTICLES AND PROCEDURES FOR THE DETERMINATION OF ANTIGENS AND/OR ANTIBODIES USING THE PARTICLES
(54) French Title: PARTICULES ET METHODES POUR LE DOSAGE D'ANTIGENES ET (OU) D'ANTICORPS A L'AIDE DESDITES PARTICULES
Status: Expired
Bibliographic Data
(52) Canadian Patent Classification (CPC):
  • 167/37
(51) International Patent Classification (IPC):
  • G01N 33/533 (2006.01)
  • G01N 33/543 (2006.01)
  • G01N 33/58 (2006.01)
(72) Inventors :
  • TRIPATZIS, IOANNIS (Germany)
(73) Owners :
  • TRIPATZIS, IOANNIS (Not Available)
(71) Applicants :
(74) Agent: JOHNSON, DOUGLAS S. Q.C.
(74) Associate agent:
(45) Issued: 1989-01-17
(22) Filed Date: 1984-05-22
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data:
Application No. Country/Territory Date
P 33 22 373.4 Germany 1983-06-22
P 33 18 261.2 Germany 1983-05-19

Abstracts

English Abstract




- Abstract -




The invention concerns particles and a procedure for
the simultaneous determination of several antigens
or antibodies in a liquid using these particles. The
mixture of particles contains populations of particles,
which differ from each other in the following way:
Each population exhibits a specific combination of
the following characteristics: 1) fluorescent substances
with differing emission spectra, 2) quantity of these
fluorescent substances, 3) particle size. Each particle
population is loaded furthermore with a different
type of antibody or antigen. Using this mixture of
particles, the simultaneous investigation of several
types of antigen or antibody can be carried out a
time and effort saving manner. The particle mixture
is mixed with the liquid containing the antibodies
or antigens to be determined. The subsequent reaction
steps correspond to the steps of a conventional immuno-
fluorescence procedure. Finally, each particle is
measured using a measuring device (e.g. flow cytometer)
for its fluorescence (emission spectrum and intensity)
and size. One the basis of the measured data, a computer
identifies the particle and correlates the measured
immunofluorescence with a defined specificity.


Claims

Note: Claims are shown in the official language in which they were submitted.


THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:


1. 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 antibodies or antigens differing from
one another for determining antigens and/or antibodies.

2. A test agent according to claim 1, characterized in that
said particles are approximately spherical and of approximately
the same size.

3. A test agent according to claim 2, characterized in that
said particles are of a size in the range of approximately 1 µm to
approximately 100 µm.

4. A test agent according to claim 1, characterized in that
said particles are applied in several discrete sizes, whereby they
can be instrumentally categorized with great certainty, according
to their size, into particle populations which can be
distinguished one from another, whereby the size of the particles
serves as an additional labelling (distinguishing)
characteristic.

5. A test agent according to claim 1, characterized in that
said particles consist of a material selected from the group
consisting of plastic, rubber, polysaccharide polymer (e.g. agar)
or another polymer or glass or that said particles are cells, e.g.
erythrocytes.


6. A test agent according to claim 2, characterized in that
said particles consist of a material selected from the group
consisting of plastic, rubber, polysaccharide polymer (e.g. agar)
or another polymer or glass or that said particles are cells, e.g.
erythrocytes.

7. A test agent according to claim 3, characterized in that
said particles consist of a material selected from the group
consisting of plastic, rubber, polysaccharide polymer (e.g. agar)
or another polymer or glass or that said particles are cells, e.g.
erythrocytes.

8. A test agent according to claim 4, characterized in that
said particles consist of a material selected from the group
consisting of plastic, rubber, polysaccharide polymer (e.g. agar)
or another polymer or glass or that said particles are cells, e.g.
erythrocytes.

9. A test agent according to claim 1, 2 or 3, characterized
in that said particles are loaded by direct binding of the antigen
to the particle or characterized in that the particles are
initially loaded with the specific antibody and then the antigen
is bound to this antibody by immunological reaction (particle-
antibody-antigen).

10. A test agent according to claim 4, 5 or 6, characterized
in that said particles are loaded by direct binding of the antigen
to the particle or characterized in that the particles are
initially loaded with the specific antibody and then the antigen
is bound to this antibody by immunological reaction (particle-
antibody-antigen).





11. A test agent according to claim 7 or 8, characterized in
that said particles are loaded by direct binding of the antigen to
the particle or characterized in that the particles are initially
loaded with the specific antibody and then the antigen is bound to
this antibody by immunological reaction (particle-
antibody-antigen).

12. A process for the determination of antigens or
antibodies using particles variously labelled by labelling
substances selected from the group of fluorescing substances,
colouring agents or pigments and/or their size, characterized in
that said particles are loaded with antigens or antibodies
differing from one another, 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 to 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.

13. A process according to claim 12, wherein said labelling
substances exhibit different but defined emission spectra.

14. A process according to claim 13, wherein said labelling
substance is one or more fluorescing substances with predetermined
differing concentrations.

15. A process according to claim 14, wherein said labelling
substances are positioned on the surface of said particles,
embedded within said particles or bound to the molecules of the
material of said particles.

16. A process according to claim 15, wherein said particles
are approximately spherical and of approximately the same size.


7. A process according to claim 16, wherein the size of
said particles is in the range of approximately 1 µm to
approximately 100 µm.

18. A process according to claim 17, wherein said particles
are applied in several discrete sizes, whereby they can be
instrumentally categorized with great certainty, according to
their size, into particle populations which can be distinguished
one from another, whereby the size of the particle served as an
additional labelling (distinguishing) characteristic.

19. A process according to claim 18, wherein said particles
consist of a material suitable for loading with antigen or
antibody.

20. A process according to claim 19, wherein said material
is plastic, rubber, polysaccharide polymer (e.g. agar) or another
polymer or glass or wherein said particles are cells, e.g.
erythrocytes.

21. A process according to claim 20, wherein the said
surfaces of said particles are loaded respectively with different
antigens or antibodies.

22. A process according to claim 21, wherein said loading of
said particles with said antigen is either carried out by direct
binding of the antigen to the particle or such that said particles
are initially loaded with the specific antibody and then said
antigen is bound to said antibody by immunological reaction
(particle-antibody-antigen).



11

3. A process according to claim 19, characterized in that
the following procedural steps are carried out for the
identification of the bound antibodies:
(a) washing of the particles after completion of the
reaction time, for the removal of non-bound substances;
(b) in the case of the investigation of antibodies, addition
of a liquid with labelled antibodies, which react species-
specifically with the antibodies to be determined or investigated
(e.g. anti-human globulin in the investigation of human
material);
(c) washing for the removal of non-bound substances; and
(d) analysis of the labelling of the individual particles.

24. A process according to claim 23, wherein fluorescence-
labelled antigens are also employed for the determination of
antibodies.

25. A process according to claim 19, characterized such
that, for the identification of the bound antigens, and before the
addition of the liquid with labelled antibodies, a liquid
containing a mixture of non-labelled antibodies is added, which
reacts specifically with the antigens to be determined, followed
by a washing step after the completion of a preset reaction time,
and that the labelled antibodies which are used react species
specifically with the non-labelled antibodies which are used.

26. A process according to claim 25, wherein fluorescence-
labelled antigens are also employed for the determination of
antibodies.

27. A process according to claim 24, characterized in that
the antibodies or antigens are labelled with fluorescent
substances.

12

. A process according to claim 26, characterized in that
the antibodies or antigens are labelled with fluorescent
substances.

29. A process according to claim 27, characterized in that
the fluorescein-labelled antibodies or antigens are added
simultaneously with the liquid to be investigated to the
particles, or after a certain reaction time following addition of
the liquid to be investigated, without prior washing of the
particles.

30. A process according to claim 28, characterized in that
the fluorescein-labelled antibodies or antigens are added
simultaneously with the liquid to be investigated to the
particles, or after a certain reaction time following addition of
the liquid to be investigated, without prior washing of the
particles.

31. A process according to claim 29, characterized in that
the washing of the particles after the reaction time with the
liquid to be investigated and the fluorescein-labelled antibodies
is deleted.

32. A process according to claim 30, characterized in that
the washing of the particles after the reaction time with the
liquid to be investigated and the fluorescein-labelled antibodies
is deleted.

33. A process according to claim 31, characterized in that
the analysis is carried out using fluorescence photometry or
photometry such that the fluorescence data of each particle are
determined (identification fluorescence of the particles and
fluorescence of the labelled antibodies) and evaluated.

13

?. A process according to claim 32, characterized in that
the analysis is carried out using fluorescence photometry or
photometry such that the fluorescence data of each particle are
determined (identification fluorescence of the particles and
fluorescence of the labelled antibodies) and evaluated.

35. A process according to claim 33, characterized in that
the emission spectrum is excited by radiation with a broad
spectrum.

36. A process according to claim 33, characterized in that a
certain line of the emission spectrum is excited by radiation of a
defined wavelength, or that several discrete lines are excited by
radiation with several defined wavelengths.

37. A process according to claim 34, characterized in that
the emission spectrum is excited by radiation with a broad
spectrum.

38. A process according to claim 34, characterized in that a
certain line of the emission spectrum is excited by radiation of a
defined wavelength, or that several discrete lines are excited by
radiation with several defined wavelengths.

39. A process according to claim 12, characterized in that,
for the identification of the particles, the fluorescence spectrum
or spectra are measured and evaluated on the basis of wavelength
and/or intensity of the fluorescence radiation of the labelling
substance or substances and/or the particle size or that the
labels of colouring agents or pigments are evaluated on the basis
of the reflected light.

14

?. A process according to claim 18, characterized in that,
for the identification of the particles, the fluorescence spectrum
or spectra are measured and evaluated on the basis of wavelength
and/or intensity of the fluorescence radiation of the labelling
substance or substances and/or the particle size or that the
labels of colouring agents or pigments are evaluated on the basis
of the reflected light.

41. A process according to claim 33 or 39, characterized in
that the evaluation is carried out on the basis of the
categorization of the immunological reactions which have taken
place in one or several antigen/antibody systems.

42. A process according to claim 34 or 40, characterized in
that the evaluation is carried out on the basis of the
categorization of the immunological reactions which have taken
place in one or several antigen/antibody systems.

43. A process according to claim 31, characterized in that
the particles are directed through a thin tube in the form of a
dispersion (sol or suspension) following the procedural steps in
claim 31 in a concentration which permits individual
determination, and are measured (flow-cytometer).

44. A process according to claim 32, characterized in that
the particles are directed through a thin tube in the form of a
dispersion (sol or suspension) following the procedural steps in
claim 32 in a concentration which permits individual
determinations, and are measured (flow-cytometer).

45. A process according to claim 12, characterized in that a
mixture of the labelled and loaded particles is fixed to a
carrier.


6. A process according to claim 45, characterized in that
said carrier is of plastic or glass.

47. A process according to claim 13 or 45, characterized in
that the liquid is added after fixing of the particles.

16

Description

Note: Descriptions are shown in the official language in which they were submitted.


73
\


2 --



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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.

-- 3 --

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.


`~E3

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~ .
- 3a -


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

. . _ _ . . . . .. .

': ` ' ' .~5; ' ' ' ` `
. .

3B~



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




'
. ,

7;3

-- 6

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. ~ ~



,~



~' ,

-- 7 --

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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Representative Drawing

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Administrative Status

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee  and Payment History  should be consulted.

Administrative Status

Title Date
Forecasted Issue Date 1989-01-17
(22) Filed 1984-05-22
(45) Issued 1989-01-17
Expired 2006-01-17

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $0.00 1984-05-22
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
TRIPATZIS, IOANNIS
Past Owners on Record
None
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Drawings 1993-09-14 1 16
Claims 1993-09-14 9 280
Abstract 1993-09-14 1 34
Cover Page 1993-09-14 1 24
Description 1993-09-14 7 300