 |
Description  |
|
|
This invention relates to an imaging immunoassay detection system and
method.
BACKGROUND OF THE INVENTION
Highly sensitive instrumentation for immunoassay techniques has been
developed to enable measurement of reactions of extremely small quantities
of biological and chemical substances. For example, instruments for
radioimmunoassay are used which are sensitive, accurate and precise, but
require expensive gamma-counting equipment. Other disadvantages of such
systems include the short half-life of the radioisotopes, and the danger
of using and disposing of the radioactive compounds used in such assays.
Another prevalent technique is the colorimetric enzyme immunoassay which
utilises enzymes as labels. An enzyme-linked immunoreactant binds either
to an antigen or to an antibody, causing a reaction which yields a
quantitative measure of the antibody or antigen, which can be detected by
a colour change. Such an assay is usually slower than other conventional
techniques involving automated assays.
A third method that can be used is a fluorescence immunoassay, based on the
labelling of an antigen or antibody with fluorescent probes. U.S. Pat. No.
4320970 discloses a photon-counting fluorimeter that may be used in such
an assay. Disadvantages of such a system include the necessity of
processing only one sample at a time. Other systems attempt to use laser
beams as the external light source to excite the solution, as disclosed in
U.S. Pat. No. 3984533. Again, this system can process only one sample at a
time.
Instrumentation for luminescence assays advantageously involves a
self-exciting luminescing system, in direct contrast to fluorimeters which
utilise an external light source. In general, existing luminometers are
complex in operation and require the use of substantial quantities of the
reagent being sampled.
Further efforts to analyse more than one reagent sample simultaneously, in
a quantitative sense, have not been successful. Efforts toward this end
are illustrated by a system described by Schroeder et al in
"Immunochemiluminometric Assay for Hepatitis B Surface Antigen", Clinical
Chemistry, Vol. 27 No. 8 (1981), wherein a carrier is prepared containing
a plurality of reagents for analysis by a luminometer which measures light
production during reaction by photo-counting. However, this method and
apparatus have the disadvantage of requiring the reactions to be measured
sequentially, one at a time. A microprocessor was used to control fluid
and air valves for adding the desired chemicals to each well; the carrier
was moved in an x-y plan, to position the individual wells sequentially
over a phototube, in order to enable the photons emitted by the reaction
to be counted. The results were displayed by a printer. Even though
photons were counted for 2 seconds at 10 second intervals, obviously a
great deal of time would be required to analyse hundreds or thousands of
test specimens in sequential order, one at a time.
In addition, GB-A-2132347 discloses a chemiluminometer for simultaneously
handling multiple samples. The results obtained, however, are only
semi-quantitative.
It has heretofore not been possible to carry out luminescent assays on
multiple, small volume samples simultaneously in a short period of time,
i.e. seconds. Presently existing technology permits only one such assay at
a time and often requires large volume samples, i.e. of 200 .mu.l or more.
As used herein, the terms "luminescent" and "luminescence" mean all kinds
of light emission except incandescence and include chemiluminescence,
bioluminescence, prompt fluorescence, delayed fluorescence and
phosphorescence and the like.
Rees et al, J. Phys. E: Sci. Instrum. 14 (1981) 229-233, describe a
miniature imaging photon detector with a transparent photocathode. It is
proposed for use in astronomy and geophysics.
SUMMARY OF THE INVENTION
The present invention overcomes the given drawbacks and provides an imaging
immunoassay detection apparatus system and method capable of detecting and
quantifying multiple light-emitting reactions from small volume samples
simultaneously.
The present invention is very advantageous inasmuch as it is very rapid
because it analyses all samples simultaneously, is extremely accurate
because it requires no mechanical motion of components, has no
repositioning errors as in sequential resolution, can use an internal
standard such as a known sample to obtain comparative information, is
adaptable with a filter to handle any particular wavelength of light, and
is versatile in that it can detect assays requiring external light as well
as those that do not require external light, thus being able to operate
with immunoassays utilising luminescence and fluorescence and the like.
In brief, the present invention comprises an imaging system for detecting
photons generated by chemical reactions comprising sample carrier means
having a plurality of individual areas each containing individual chemical
reactant samples capable of emitting photons if a reaction takes place,
the plurality of reactant-containing areas being arranged in spaced
relationship with respect to each other; imaging means associated with
said carrier means for simultaneously receiving individual photons emitted
from each area sample where a reaction is taking place; and means coupled
to the photon-receiving imaging means for generating a signal representing
the x-y location of each area sample generating a photon, whereby the
reactants in each area sample having a reaction and the number of its
photon emissions over any predetermined period of time may be
simultaneously identified.
The invention also comprises a method of simultaneously detecting photons
generated by a plurality of chemical reactions, comprising the steps of
providing a plurality of individual chemical reactant samples each capable
of emitting photons when a reaction takes place, the samples being
arranged in spaced relationship with respect to each other, and
simultaneously detecting the presence and x-y location of each photon
emitted from any reacting samples, whereby the total number of photons
emitted from each reacting sample over a predetermined period of time may
be determined.
Thus, the present invention is not only extremely rapid and processes
simultaneously multiple assays, but also enables considerable economies to
be made in the use of often expensive reagents concerned because only
small quantities of samples are required.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagrammatic representation of the novel photon detection
system;
FIG. 2 is a diagrammatic representation of the imaging photon detector used
in the system of FIG. 1;
FIG. 3 is a representation of both background noise signals and signals
from discrete areas of reaction (whereby the background noise signals may
be cancelled, leaving only reaction signals);
FIG. 4 represents a carrier in which multiple antibody labels may be used
simultaneously or in which a standard reaction may be compared with other
reactions;
FIG. 5 is a diagrammatic representation of how a wavelength interference
filter may be used with the carrier of FIG. 4 to pass only a particular
light wavelength, thereby allowing only photons of a particular wavelength
corresponding to a labelled antibody to pass to the imaging detector;
FIG. 6 is a schematic representation of a circuit in a microprocessor for
subtracting background noise from the sample signals to obtain an output
signal representing substantially only pure photon emission from a
reactant sample; and
FIG. 7 is a diagrammatic representation of the use of invisible ultraviolet
radiation as a source of external light.
DETAILED DESCRIPTION
The apparatus of the present invention, which will be described in further
detail below, may be utilised with any number of different assay
techniques as stated earlier, but will be described with particular
reference to the use of luminescent immunoassays in the detection of
antigen-antibody reactions. More particularly, the invention can be
employed to detect the characteristic reactions of labelled monoclonal and
polyclonal antibodies with antigens found in samples such as urine,
faeces, blood, milk and water and the like.
Polyclonal antibodies are well-known. Monoclonal antibodies may be prepared
by the technique first described by Kohler and Milstein, Eur. J. Immunol.
6, 292 (1975). In order to detect the presence of particular antigens, the
monoclonal antibodies may be labelled with a multitude of different
labels, such as luminescent or fluorescent compounds. Further, the
particular labels utilised in the present invention must be capable of
emitting light once the antigen-antibody reaction occurs, and thus the
reactions are designated as "light-emitting reactions". The present
invention will be described in general with reference to a
luminescent-labelled monoclonal antibody, although fluorescent labels may
also be used as disclosed hereafter. As used herein, the term "reactants"
means the combination of (1) a monoclonal antibody labelled with a
luminescent or fluorescent compound, and (2) an antigen.
Luminescence is the emission of light by an atom or molecule as an electron
is transferred to the ground state from a higher energy state. In both
chemiluminescent and bioluminescent reactions, the free energy of a
chemical reaction provides the energy required to produce an intermediate
reaction or product in an electronically excited state. Subsequent decay
back to the ground state is accompanied by emission of light.
Bioluminescence is the name given to a special form of chemiluminescence
found in biological systems such as the firefly, in which a catalytic
protein or enzyme, such as luciferase, increases the efficiency of the
luminescent reaction. When this luciferase enzyme is combined with its
substrate, luciferin, in the presence of ATP (adenosine triphosphate),
magnesium and oxygen, a flash of light is produced, whose intensity is
proportional to the amount of ATP present in the sample. The firefly
luciferase/luciferin/ATP system is as follows:
##STR1##
where h.nu. is the energy of a photon, h is the Planck constant, and .nu.
is the frequency associated with the photon.
Assays of the invention can directly determine the number of live organisms
in a sample, either because the presence of ATP in a test sample indicated
live cells, or because of the presence of immunoglobulins labelled with a
luminescence-detectable enzyme (like peroxidase or luciferase).
Chemiluminescent substances such as luminol may also be utilised in a
horseradish peroxidase-catalysed oxidation, as follows:
##STR2##
In the present invention, the "light-emitting reactions" generate photons
which are coupled to an imaging device such as an imaging photon detector,
a charge-coupled device, or a vidicon tube (any of a variety of camera
tubes having a photoconductive target). In the preferred embodiment, an
imaging photon detector is used.
In particular, the reactions may be generated by reactants spatially
arranged in individual areas on a sample carrier in a single row or column
or by a two-dimensional array of reactants spatially arranged, for
instance, in rows and columns. For example, a carrier may have an array,
such as rows, of 1 mm outside diameter nylon tubes containing the labelled
monoclonal antibodies, to which is added the specimen or specimens being
tested for the presence of an antigen. The fluids involved are
self-contained and of a very small volume. Thus, an advantage of the
present invention is that the imaging photon detector can quantify (in 10
seconds or less) light emitted from multiple "light-emitting reactions",
in volumes of 3 .mu.l or less, i.e. much smaller than can be used in known
apparatus.
Another carrier suitable for use with an imaging photon detector is a
microtiter plate with multiple samples in rows and columns. A particular
plate may contain as many as 96 individual wells. Each well contains
different labelled monoclonal antibodies adsorbed on the surface of the
plate. A portion of the specimen is added to each well. The presence and
quantity of a particular antigen in an individual well is determined by
the number of photons generated by the antigen-antibody reaction.
A third carrier involves the principle of using immobilised antibodies on a
plurality of filaments; labelled monoclonal antibodies are immobilised in
individual areas on a plurality of filaments. Each filament may bear a
different labelled monoclonal antibody capable of emitting light upon the
detection of an antigen. Because the reactions on the individual filaments
generate light, the imaging photon detection system can quantitatively
determine the presence of particular antigens.
The present invention envisages the use of any number of different types of
multiple, chemically-produced, light-emitting reactions which can be
imaged by the image photon detector. As the samples emit light, the
present invention counts the individual photons impinging upon a light
sensitive photocathode of an imaging photon detector.
Each of the above means for containing a plurality of reactants can be used
in the apparatus system described below, in which the container is
identified as the specimen carrier means.
The apparatus system will be described with reference to FIGS. 1 and 2.
FIG. 1 is a system for quantitative assay analysis of multiple biochemical
images using an imaging photon detector. The system enables the detection
of very low concentrations of substances present in fluid samples or
specimens which, in the course of their reaction, emit light photons under
certain conditions. In particular, the system has demonstrated sensitivity
in the order of 10.sup.-16 and lower.
FIG. 1 shows a specimen carrier means 10 which may include a plurality of
fluid samples all capable of simultaneously undergoing a reaction. Samples
can be spaced in individual areas as a row or column or in a
two-dimensional array of rows and columns as shown in FIG. 3 and FIG. 4,
for example only. The reactions that produce light generate photons 12
which are focused by an optical system 14 to form the image of the light
outputs of each of the samples on a photoconductive target forming a
portion of an imaging photon detector (IPD) 16. The imaging photon
detector 16 will be disclosed in detail hereinafter but is known in the
art; it immediately converts incoming light into quantitative information
which can be stored and processed within a memory of any conventional
computing means such as a microprocessor 24.
The imaging detector 16 of the present invention takes simultaneous
readings of discrete sample areas such as the small darker shaded
orthogonal areas of FIG. 3 rather than averaging the readings of the
entire sample area (including the carrier area surrounding the sample).
Background noise, represented by the shading lines in FIG. 3, is caused by
non-specific binding antigens or antibodies to the solid surface of the
carrier, which are not washed away in the preparation of the carrier.
Conventional detectors read these signals generated by this undesirable
binding and, because these conventional detectors average the signal over
the entire sample area, they interpret these undesired false signals as a
positive reaction. Background noise effectively decreases the sensitivity
of the assay at relatively low levels of concentration, where the positive
reaction signal has nearly the same intensity level as the background
noise.
The present invention eliminates the background noise problem by
simultaneously reading the signal from the background environment and the
signal from the concentrated reaction area and comparing the two readings.
Because the present imaging immunoassay detection system can read signals
from numerous discrete reaction areas at the same instant, a real time
measurement of the signals from the discrete areas of reaction in a
two-dimensional array can be taken, averaged, and compared with the signal
representing the background noise caused by the non-specific binding. The
computer 24 can analyse and display the results by subtracting from the
signals representing the discrete areas of reactions the signals
representing the background noise, as shown in FIG. 6, thereby leaving the
pure reaction signals.
Further, since the present system can simultaneously evaluate a carrier,
such as a 96-well microtiter tray, without repositioning the carrier or
tray as in systems using sequential detection, resolution errors that
occur from imprecise mechanical repositioning of the samples between
measurements as required in the prior art are eliminated.
Since the present imaging immunoassay detection system can look at more
than one discrete reaction at a time, contrasting reactions can be
analysed relative to one another. By way of illustration, in a
two-dimensional array of samples as in a microtiter tray represented in
FIG. 4, reactions can be compared side-by-side simultaneously. The amount
of photons generated by each reaction can be read and analysed by computer
24 and the relative extent of reaction compared. In this way, a more
accurate comparison can be made between specific samples, thus providing
better test results. As an example, a negative reaction may be placed in a
discrete area A in FIG. 4, to serve as a control for purposes of
comparison with a positive reaction in a discrete area B. The negative
reaction in discrete area A may still generate spurious signals caused by
non-specific binding, as pointed out earlier. This background noise level
is potentially constant across a given carrier such as a microtiter tray
and is useful in setting up a base level of signal generation from which
more positive reaction can be compared.
Thus, the present imaging system is capable of rapid quantitative analysis
of samples. Because of its unique ability to simultaneously read and
analyse numerous samples, the time necessary to produce results is
dramatically reduced.
Furthermore, the sensitivity of the present imaging system allows for very
accurate measurements even at very low concentrations of the samples. For
instance, an imaging photon detector is capable of measuring individual
photons of light. By using amplifiers, the system is able to register very
low concentrations of materials and is therefore useful in areas such as
diagnosing for the presence of infectious organisms, as well as drug
monitoring and disease detection.
Because of its sensitivity, the imaging system can not only detect minute
quantities of a reaction samples, but can also read a very small area of
reaction. Thus, the amount of reagent and the area which are needed to
conduct the assay are less then before, thereby minimising the cost of
reagents and carrier materials.
The output of the imaging photon detector 16 on line 18 comprises analog
signals which represents the x-y spatial correspondence of each detected
photon, thus identifying electrically the x-y address of the sample or
specimen that produced the light. These analog signals are coupled to an
analog-to-digital (A/D) converter 20 which produces digital output signals
on lines 22 representing the spatial orientation of the specimen source
producing the photon received by the imaging photon detector 16 and thus
identifying the particular sample or specimen which produces the photon.
The digital signals 22 are coupled to a microprocessor 24 which stores and
analyses the image information and can be programmed to display it in any
desired format. The reactions continue to generate light throughout any
desired predetermined period of time, e.g. as little as 10 seconds or
less, and the number of photons produced by each reacting sample is
accumulated in the memory of microprocessor 24. Thus, microprocessor 24
produces signals on line 26 to video display 28 and printer 30 for visual
display and analysis of the light received, and accumulated, from samples
10.
A bar chart may be displayed on video terminal 28 which identifies each of
the samples and illustrates the relative amount of light or number of
photons being generated by each sample. Such a bar chart could also be
produced, for a permanent record, by printer 30.
Also, a two-dimensional image of the array of samples as they are
physically located (i.e. their x-y address) can be produced, the intensity
of the light generated by each sample being indicated either in colour or
by numerals, thus identifying which sample is generating the greatest
amount of light. Obviously, the microprocessor 24 can perform any
operation on the samples as desired to correct and calibrate the image and
to compensate for any inherent noise in the system such as by subtracting
the background noise, as explained earlier. Noise also may be reduced by
cooling the imaging photon detector 16 with a cooling unit 15 in any well
known manner such as by circulating a cooling liquid or a refrigerant
about the imaging photon detector 16. Cooling the detector 16 reduces the
tendency for free electrons to be emitted from elements of the detector
16, and which assist in generating the background noise.
FIG. 2 is a diagrammatic representation of the construction details of the
imaging photon detector used in the preferred embodiment herein and which
is known in the art. The detector may be type IPDG1 or type PIDF1
manufactured by Instrument Technology Limited in East Sussex, England. The
imaging photon detector 16 is a two-dimensional imaging sensor capable of
detecting extremely weak radiation, e.g, capable of detecting an ATP
content in the sample down to as low as 10.sup.-16 moles/sample. As
indicated earlier, that image is produced in analog form which is
converted through an analog-to-digital converter 20 to a digital form for
use by the microprocessor 24.
Light is composed of individual photons. Each individual photon has an
extremely small amount of energy associated therewith. In most common
images, the light contains fluxes of millions or billions of photons per
square centimeter and per second. Using the imaging photon detector 16,
each incoming photon has a high probability of detection by the
photocathode 32.
The photoconductive target 32 can thus be thought of as equivalent to a
photographic film except that it has a sensitivity of the order of 100
times greater. When a photon strikes the light sensitive photocathode 32,
a photoelectron is released from photocathode 32 and is immediately
accelerated into a series of microchannel plate intensifiers or amplifiers
34. As a result of the intensification created by microchannel plates 34,
a gain in the range of 3.times.10.sup.6 to 3.times.10.sup.7 electrons is
emitted from the rear of each microchannel plate for each incident
photoelectron and thus corresponds to each initially detected photon. The
combination of the microchannel plates 34 thus enables extremely small
amounts of light to be detected.
A resistive anode encoder 36 located immediately behind the microchannel
plates 34 translates the electron burst into signals which can be
processed easily into a two-dimensional x-y address of the detected photon
and thus the sample. Thus, the analog readout of the resistive anode 36 on
line 18 in FIG. 1 is used to present a linear x-y registration of each
photoelectron event. The read-out through four orthogonal electrodes 38 is
suitably processed to provide digital representation of the x-y position
of the incident photoelectron (and thus the sample) by analog-to-digital
converter 20 and microprocessor 24, both shown in FIG. 1. Thus, by the use
of the imaging photodetector 16, the full image of all of the samples 10
in two dimensions is created by integrating the image focused onto the
photocathode 32, photon-by-photon. Thus, the present system detects and
presents information relating to multiple imaging, presents the
information immediately upon the occurrence of the emitted light, detects
extremely small amounts of light down to and including a single photon,
and quantifies such information for each specific x-y sample or specimen
address.
Instead of an imaging photon detector (which is preferred), the system may
use a charge-coupled device (CCD) as the imaging device 16. CCD's are well
known in the art. They are used in conjunction with an optical lensing
system (such as optical system 14 i | | |
