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FIELD OF THE INVENTION
This invention relates to immunoassays generally and, more specifically, to
fluoroimmunoassays of biological species. Still more specifically, this
invention relates to an improved and modified polarization
fluoroimmunoassay method and apparatus.
BACKGROUND
Numerous biological substances are quantitatively or semiquantitatively
determined by immunological methods. Radioimmunoassay (RIA) opened a new
generation of trace determination techniques and permitted a degree of
sensitivity into the molecular range, not hitherto attainable. RIA
techniques are presently being displaced quite extensively, however,
nonradioactive determining methods are needed which permit an
approximately equal degree of sensitivity, but avoid the problems inherent
in handling radioactive materials. One such method is the widely used
fluoroimmunoassay, which is well known and is extensively described in the
published literature, See, for example, Davis et al, Microbiology, 2nd
edition, Harper & Row, 1973, pp. 397 et seq.; Cooper, THE TOOLS OF
BIOCHEMISTRY, Wiley-Interscience, New York, 1977; and Iesen, IMMUNOLOGY,
Harper & Row, 1974.
The literature has also reported that fluorescent molecules, when excited
by polarized light, emit luminous energy which, in its polarization value,
decisively depends upon the molecular size of the species which
fluoresces. The degree of polarization also depends upon other parameters,
such as, for example, the number of type of these molecules, the state of
the molecules, i.e., whether or not the molecules are bound at one
position or are unbonded to one another, etc. See the foregoing references
and Parker, C.W., in Weir, HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, third
edition, Volume I, pp. 18.1 et seq.
With the formation of the antigen-antibody complex, the molecular size of
the species is changed in all immunological reactions, As a result of this
change in size in the immunological reaction, a fluorescent tagged
immunological substance undergoes a change in its fluorescence
polarization characteristics, i.e., a fluorescent tagged antigen
fluoresces differently and, in particular, has a different degree of
rotation in its unbound condition as compared with its condition when
bound to an antibody. Similarly, a fluorescent tagged unbound antibody
behaves differently than the same antibody bound to an antigen.
One of the main disadvantages observed with conventional fluorometers is
lack of sufficient sensititivy to pick up very weak fluorescent radiation
from the samples, or they damage the samples--mainly biological samples
containing proteins through the radiation heat from a light source, and
they so reduce or destroy biological activity of the samples by heat
deterioration. Therefore, these instruments cannot be used for the
evaluating of very weak immuno-reactions.
SUMMARY OF THE INVENTION
The evaluation of the polarization fluorescence of a fluorescent tagged
species with respect to the intensity in the various polarization ranges
allows one to determine the presence and to semiquantitatively determine
the amount of an immunological reaction in a very simple manner, even in
cases in which the values obtained without polarized light are not taken
into consideration and; when this technique is combined according to the
present invention, with the use of a fiber optic light guide, and a
conventional high intensity beam producing source, great sensitivity is
attained. This is accomplished, according to the present invention,
without the sample being damaged, as occurs in conventional techniques
through the excessive accompaniment of heat evolution.
Accordingly, the present invention comprises, in one feature, apparatus for
directing polarized, high intensity beam of light which is guided through
a fiber optical element and into a sample containing fluorescent tagged
species; and measuring simultaneously the intensity of the fluorescent
emitted light seen by two photosensors through two differently (vertically
and horizontally) oriented polarizers, so determining directly and
quantitatively or semiquantitatively the amount of the bound versus the
unbound tagged immunopartner. Additional features of the invention
including the method are described hereinafter.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of the apparatus of this invention, and,
insofar as individual components are concerned, operates in the manner
described in my earlier U.S. Pat. No. 4,133,873, issued June 9, 1979,
entitled METHOD OF DETERMINING EXTRACELLULAR ANTIGENS AND ANTIBODIES.
FIG. 2 is a calibration curve typical of that used in the present method,
in semilogarithmic scale.
DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
A simple, tested embodiment which exemplifies, but does not limit, the
invention, is shown in FIG. 1. This type of apparatus has been used for
polarization fluorescence immunoassays according to the present invention.
Referring to FIG. 1, the excitation light for the apparatus of this
invention is provided by a conventional light source 1. The light is
monochromatized by an excitation filter 2, which, in the simplest case, is
simply an interference filter. Any monochromator commonly used in
photometry may, however, be used. The light is then polarized by the
polarization filter 3, which, again, is of conventional composition and
design. Polarizing filters are commonly used in photography and in many
scientific applications, and any good quality polarizing filters may be
used in this invention. The monochromatized, polarized pulse then passes
through a heat absorbing light guide 4 and enters the cell 5 in which the
sample containing the fluorescent tagges species is contained. For
identification in the drawing, the light beam used for exciting the sample
is identified as beam E.
The fluorescent tagged species, when excited by the beam E, emit
fluorescence radiation at all angles, and the intensity of such radiation
can be measured at any desired angle. However, it is preferred that the
measurements be made at 90.degree. to the incidence of the excitation beam
in order to minimize background resulting from the beam. Accordingly, as
shown in FIG. 1, two fluorescent beams identified as F-1 and F-2 at
90.degree. from the excitation beam and at 180.degree. from each other,
are selected. Beam F-1 is passed through a collimating slit 6, in one
direction, through a monochromator such as interference filter 7 to select
a desired emission wave length, then through a polarization filter 8 and
to a photomultiplier 9. The slit, monochromator and polarizer may be of
the type described with respect to the corresponding elements in the
excitation beam, and the photomultiplier is a conventional photomultiplier
used in photometry.
A similar arrangement is provided 180.degree. from fluorescent beam F-1
where fluorescent beam F-2 passes through a slit 10, monochromator
interference filter 11, which selects the same emission wave length, then
through polarization filter 12 and to a like photomultiplier 13.
The signals given off by the photomultipliers 9 and 13 are compared with
one another in an evaluation system 14, generally of the type described in
my aforesaid U.S. Pat. No. 4,133,873, and may be indicated by any
conventional device such as a meter 16, or may be recorded graphically as
is commonly done in photometry.
The switch 15 permits one to turn one of two photomultipliers out of the
circuit so that, by appropriate orientation of the polarization angle of
the polarizer filter 3, the apparatus can be used as a simple polarization
fluorometer and not as a difference fluorometer. Likewise, the
polarization filter 3 can be removed from the path F-1 and the apparatus
used as a nonpolarizing fluoroimmunoassay device.
In the evaluation system 14, the impedance of the pulse signals from the
photomultipliers 9 and 13 can be reduced and matched using conventional
impedance matching devices such as unity gain operational amplifiers. The
ultimate output is a voltage the magnitude of which is a function of the
difference between the signals taken by the two photomultipliers and,
consequently, the difference in the intensity of the fluorescence beams
F-1 and F-2. The polarization analysing filters 7 and 11 are, in a typical
application of the apparatus and of the method, rotated with respect to
one another by 90 .degree.. The polarization filter 3 in the excitation
beam is suitably affixed in a rotatable manner, e.g., simply mounted
loosely to permit rotation, in order to be able to optimiize the apparatus
in varying applications. Thus, the polarization orientation of the
polarizer 3 may be varied at the will of the operator to optimize the
desired polarization of the excitation beam E, merely by appropriate
filter adjustment.
EXEMPLARY APPLICATIONS OF THE INVENTION
Many potent modern medications have the disadvantage of displaying a
biologically effectiveness range of only a small order of magnitude, i.e.,
they must be administered and kept within any narrow range of
concentration, and the biological half life of the medication varies from
patient to patient. Thus, it is necessary to maintain nearly continuous
controls on the amount of the medication in the blood level of the patient
when these medications are used, in order to be able to correct the dosage
at the earliest possible time to protect against impending side effects
due to too high concentrations or inadequate medications due to too low
concentrations in the blood. Such substance include Digoxin, Norpace,
Amikacin, Kanamycin and Gentamicin.
An example of the method of this apparatus is given below using Gentamicin
as a typical medication which requires some monitoring.
The concentration of Gentamicin in the blood can be determined according to
the present invention in a simple manner, quickly, reliably and with the
highest degree of precision according to the method described below. A
plurality of samples, two ml each, of a solution adjusted to a pH of about
7.1 are treated, respectively, of fluorescein tagged Gentamicin, of any
desired or suitable concentration, in a 0.05 molar Tris buffered solution
adjusted to a pH of about 7.1 are treated, respectively, with 50 .mu.l of:
(A) patient serum
(B) Gentamicin free control serum
(C) Control serum with 1 .mu.g Gentamicin/ml
(D) Control serum with 2 .mu.g Gentamicin/ml
(E) Control serum with 4 .mu.g Gentamicin/ml
(F) Control serum with 8 .mu.g Gentamicin/ml
(G) Control serum with 16 .mu.g Gentamicin/ml
The samples are then incubated for 10 minutes at room temperature.
The samples are introduced, one after another, into the measuring cell 5
and the cells placed in the apparatus, and subjected to light excitation
energy. The difference in fluorescence as measured through the two
measuring systems, concluding with the two photo cells 9 and 13, is then
read off from the evaluating device 16. The fluorescence difference value,
i.e., the difference in the measured fluorescence according to this
method, of the patient's serum is compared with values shown in a standard
curve plotted from the data of the control series, samples (B) through
(G). A standard curve of this type is shown in FIG. 2. This immediately
gives the Gentamicin concentration in the patient's serum in .mu.g
Gentamicin/ml of serum.
In the case under discussion, the following values were obtained:
(A) Patient serum: 3.2 V
(B) Control serum 0 .mu.g/ml: 4.3 V
(C) Control serum 1 .mu.g/ml: 3.8 V
(D) Control serum 2 .mu.g/ml: 3.5 V
(E) Control serum 4 .mu.g/ml: 3.0 V
(F) Control serum 8 .mu.g/ml: 2.35 V
(G) Control serum 16 .mu.g/ml: 1.65 V
FIG. 2 shows the values graphically plotted on semi-logarithmic paper. The
graphic plotting produces the standard curve. From the standard curve it
becomes apparent that the Gentamicin content in the patient's serum is 3.2
.mu.g/ml.
The foregoing is given as an exemplary embodiment of the invention, and
does not limit, in any manner, the scope or application of the invention.
The present invention may be used, for example, and all immuno complexes
which have been labelled with a fluorescent tagged substance and which can
be evaluated by means of fluoroimmunoassay methods. This method can be
used for the competitive, as well as for the sandwiching immunoassay
techniques. Sandwiching or solid substrate fluorescent immunoassay
techniques are, of course, well known and are described in my aforesaid
U.S. Pat. No. 4,133,873, and also in the other references to herein before
and incorporating herein by reference. The present invention is,
therefore, of general applicability to fluorescent immunoassay techniques
in which there is a difference in polarization fluorescence according to
the state of the fluorescent tagged species, e.g., bound versus unbound.
The utilization of the polarization fluorescent immunoassay technique of
this invention can be used to identify and prove the existence of as well
as to quantitatively determine a concentration of immunologicalreaction
components of small and average sized molecules in cases where the highest
degree of sensitivity and precision is required and where great values
placed upon easy operation and rapid execution of determinations of this
general type.
The necessity of separating bound immuno reaction components from unbound
immuno reaction components is one of the greatest disadvantages of all
heterogenous immnoassay techniques. This great disadvantage is eliminated
in the present case by using the homogenous polarization fluorescence
immunoassay apparatus and method as described herein before, in which the
molecular size has an influence on the measured signal. This is based upon
the fact that, in liquid media, the immunological substances with small
molecular sizes are exposed to a much greater extent to the Brownian
molecular movement than those with larger molecular sizes. Accordingly,
small molecules change their spatial arrangement within the very short
time period between fluorescent excitation and emission of fluorescent
light as a result of that excitation and, therefore, appear to be less
polarized than large molecules which, during the same time period, change
their spatial arrangement only a minimal extent and consequently, change
their emission polarization orientation only to a miminal extent.
A solution containing small molecules of fluorescent tagged immunological
substances which, when viewed under nonpolarized light, shows exactly the
same fluorescence characteristics as the solution containing large
molecules of the same fluorescent immunological substances when viewed
under polarized light. However, the solution containing small molecules of
fluorescent tagged immonological substances displays a polarization which
is of a small, minimal order of magnitude, as compared with the
polarization magnitude of a solution containing large molecules with the
same fluorescent tagging. However, as the small molecules combined
themselves with other substances as, for example, through an immunological
reaction, to form an immuno complex, and consequently become larger, they
become far less subject to the Brownian molecular movement and,
accordingly, can be easily determined semiquantitatively by measurement of
the polarized light emitted therefrom.
The polarization fluoroimmunoassay according to the present invention has a
large number of advantages as compared with the conventional immunoassay
methods. The use of optical collimation means together with a
fiber-optical light guiding system allows it to take advantage of the
kinetic energy from a very bright conventional light source--for instance
from a tungsten light-source--and so to expose the sample to enough
excitation light for the generation of a significant amount of fluorescent
emission light from the fluorescently tagged molecules without
heat-deteriorating it. The fiber-optical light guide brings the collimated
light to the sample and simultaneously functions as a heat shield.
This feature overcomes one of the main disadvantages observed with
conventional fluorometers; those instruments are either not sensitive
enough to pick up very weak fluorescent radiation from the samples or they
damage the samples--mainly biological samples containing proteins--through
the radiation heat from the light source, and they so reduce or destroy
the biological activity of the samples by heat-deterioration. Therefore
those instruments cannot be used for the evaluation of very weak
immuno-reactions.
The polarization fluoroimmunoassay is not afflicted with the dangers and
disadvantages resulting from radioisotopes as in the more traditional RIA
techniques of the prior art.
The polarization fluoroimmunoassay can be carried out with extremely low
expenditures of time and, correspondingly, low monetary expenditures in a
quick and efficient manner with the highest degree of precision. The
polarization fluoroimmunoassay method of this invention can also be used
without difficulty for making kinetic determinations, thus making it
possible quickly and accurately to determine the reaction velocity of the
immunological reaction of interest.
One of the great advantages of the polarization fluoroimmunoassay of this
invention, as compared with most conventional immunological assays, is
that it can be used as a homogenous immunoassay.
It should be apparent from the foregoing that there are a number of
variations possible within the scope of the invention as disclosed herein.
For example, the exciting beam can be polarized and monochromatized and
directed to traverse the sample cell end to end, which typically is a
number of times the diameter of the cell, and the sensors can be arranged
at the sides of the cell and with a large field f vision, e.g. removing
the slits 6 and 10, measuring the polarization fluorescence over all or a
substantial area of the long side of the cell, increasing the amount of
incident fluorescent light on the photomultipliers several orders of
magnitude.
This invention can be used for many determinations by eliminating the time
consuming, and that time is rather costly and troublesome, and rather
critical separation of bound immunological reaction products from unbound
immunological reaction, a process which is conventionally required before
measuring can be accomplished.
INDUSTRIAL APPLICATION
This invention is useful in conventional diagnostic and scientific
immunlogical reaction measuring processes.
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