Note: Descriptions are shown in the official language in which they were submitted.
L~ S 3
The invention relates to the detection and measure-
ment of predetermined immunochemical substances, using a novel
method and apparatus therefor which provide for rapid
qualitative and quantitative determinations in an advantageous
manner.
$ Various meth~ s have ~2en developed in the last two
or three decades for the determination of a variety of immuno-
chemical substances, including antigens, antibodies, haptens,
and certain low molecular weight substances. Examples of these
methods are:
1. Radio assay techniques
a. Competitive protein binding assays
b. Radioimmunoassay (RIA)
c. Immunoradiometric assays
d. Sandwich or 2-site immunoradiometric assays
2. Fluoroimmunoassays (FIA)
3. Enzime immunoassay (EIA)
4. Lysis-initiating immuno~ssays (LIA)
5. Latex-particle agglutination ~LPA)
6. Charcoal-particle agglutination (CPA)
7. Hemagglutination and Hemagglutination
Inhibition Assays (HA), (~IA)
8. Complement Fixation (CF)
9. Counter-immunoelectrophoresis (CIEP)
l0. ~adial Immunodiffusion and Double
diffusion ~RID)
11. Viroimmunoassay ~VIA), and
12. Spin immunoassay (SIA) among others.
l3. Turbidity (physical assay~
One type of immunochemical test system involves the
use of labeLs. Within these, there are many types of labels
*~
useful in assays for the detection and measurement in serum or
other media of biolo~ically important or interesting compounds
or substances. The administration of most of these tests are
hampered by one or m~re of the ~ollowing limitations: (1) lack
of sensitivity, (2) complexity of the test procedure, (3~
instability of reagents, (4) hazardous reagents, (5) impure
reagents, and (6) expensive equipment required to perform
quantitative and qualitative analysis of the amount of label
involved in an immunochemical reaction. For a review of the
development and evaluation of immunological methods and their
uses as diagnostic tools, reference is made to "In~unology as a
Laboratory Tool", by FRANZ PEETOOME, 37 American Journal of
Technoloqy (2) 445-469 (1971).
There are other immunochemical test systems which do
not use labels for a means of detection, some o~ these are the
so-called "agglutination" tests, wherein the analysis depends
on the measurement of certain electromagnetic radiation proper-
ties of the liquid samples containing immunochemical constitu-
ents, with the measured properties depending on whether or not
an immunochemical reaction has taken place. A pioneed reference
in this area of technology is Schuurs, U.S. Patent No. 3,551,555
(1970). See al50 Price et al, U.S. Paten~ No. 4,066,744 (1978).
Examples of these "agglutination" tests include the so-called
"NOSTICON"*latex tests by Organon Incorporated (West Orange,
~ew Jersey3, including P~NOS~ICON ~ R~EUMANOSTICON ~, and
GONOSTICON~ latex agglutination or latex agglutination
inhibition Slide Tests (distinguishable from the "NOSTICO~"*
erythrocyte ag~lutination inhibition tests).
It must be emphasized that both labeled and unla~eled
immunochemical testing may employ various devices to separate
immunochemical constituents which have reacted from non-reacted
* ~r ade ~lark
l~ 'Y3
immunocllemical collstituents and from substances irrelevant to
the test. ~or example, some EIA patents require separation
through the use of one component in the antigen-antibody reac-
tion being in an "insolubilized" phase for separation - see
Schuurs and co-workers in U.S. Patent Nos. 3,654,090, 3,791,932;
3,~50,752, 3,839,153, 3,879,262, 4,016,043 and Reissue 29,169.
~nother method does not require separation of free
and ~ound label because the assay depends on the inhibition or
- activation of the enzyme label by antibody binding ~e.g., the
EMIT ~-type system of Syva Corporation of Palo Alto, California,
for EIA and FRAT, or "free radical assay technique", for SIA) -
see V.S. Patent Nos. 3,880,715, 3,852,157, 3,875,011, 3,935,074,
and 3,905,871, and an article by Kenneth S. Rubenstein et al in
"Homogeneous Enzyme Immunoassay, a New Immunochemical Technique",
Biochemical_and Bio~hysical Research Co~mnunications 47, No. 4,
846-851 (1972). These are examples wherein an insolubili~ed
` phase is not employed and the assay depends on inhibition or
activation of the enzyme label by antibody binding. See also
G. Brian Wisdom, "Enzyme Immunoassay", Clinical Chemistrv 22/8,
20 1243 (1976).
Radioimmunoassay (RIA) is now considered a classical
and well-known techni~ue for detectin~ antigens at very low
concentrations. It is based upon the competition between radio-
labeled and unlabeled antigen for a fixed, limited amount of
antibody, a5 described by R. Yalow and S. Berson in J. Clin.
Invest., 39 1157 (1960). The amount of unlabeled antigen
in1uences the distribution of the labeled antigen in antibody~
bound ~B3 and antibody-free (F3 labeled antigen, i.e., the more
that unlabeled antigen is present, the le3s the labeled antigen
is able to combine with the antibody. In order to obtain con-
clusive results froo the distrihution, a ~ood separat~on between
1~ '. 3
B and F must ~ made. Methods used for this purpose are, for
instance, chromatoelectrophoresis, described by S. Berson and
R. Yalow in The Hormones, edited by G. Pincus et al, Academic
Press, New York (1964), vol. IV, 557, or i~solubilization of the
antibodies. This insolubilization can be achieved by chemical
means ~cross-lin~ing or covalent binding to an insoluble
carrier) or by physical methods (adsorption to an insoluble
1~
carrier).
Of the limitations cited above, a most serious
limitation until recently has been lack of adequate sensitivity
to detect some antigens. In general, three levels of sensi-
tivity are recognizable. Low sensitivity techniques, where
materials detected and measured exist in microgram~milliliter
quantities, include RID, CF, CEP, CPA, and LPA. Intermediate
sensitivity techniques, where microgram/milliliter to nanogram/
milliliter quantities of materials may be measured, include
HIA, ~A, CF, FIA, SIA, VIA, and EIA. Until recently only RIA
was able to measure with ultrasensitivity the picogram/
milliliter to femtogram/milliliter region.
Many of the techniques listed above require that some
form of physically or chemically identifiable label be attached
to reagents in the assay system in order that the result of a
test can ~- detected. RIA, FIA, EIA, LIA, VIA, and SIA all
fall into this category. Radioactivity, fluorescent moieties,
enzymes, complement, viruses, and electro,n-spin la~,els are
used respectively to generate some form of end-point signal.
The sensitivity with which these labels can ~e detected
directly and fund2mentally affects the useful ranges of the
test systems using them.
The sensitivity with which a lahelin~ moiety can be
measured depends upc,n the nature of the signal that it ~enerates,
the ability to detect that signal, and the intensity of signal
available per unit amount of marker molecule, i.e., its
specific activity. With radioactive labels, heretofore the
most popular label in use, the signal is decay radiation.
Because of the penetrating properties of the emissions
generated, radioactive decay can be detected easily. Modern
counting equipment very efficiently measures the radio~ctive
emissions from even a small amount of radioactive material.
Furthermore, there is a range of specific activities offered
by isotopes currently used for tagging.
As noted, up to the present time, the radioimmunoas-
say (RIA) method in its various forms has been the most
sensitive system available. The RIA method, unfortunately, has
several serious disadvantages, including the requirement o~
special equipment, trained staff, the recited need for extra
safety measures to protect against harmful radiation, special
licensing, controlled radioactive wastes disposal and the con-
tinuous disappearance of labeled compound by radioactive decay.
The possibility of replacing the radioactive 7abel with an
enzyme label was proposed in 1968 in an article by L. E. M.
Miles and C. N. E~ales, entitled "Tabeled Antibodies and I~muno-
logical Assay Systems", Lancet, II, 492 (1968), and Nature 219,
168 ~1968). No procedural details were provided, the article
offered on1y the general idea, leaving it to future wor~ers to
determine the basic step and to perform the extensive experi-
mentation needed to establish a practical operative enzymatic
immunoassay method.
More recently, methods for detecting and measuring
immunochemical substances have been developed in which, in lieu
of a radioact ive isotope, immunochemical substances have ~er
labeled ~;ith otner materials which can be detected by various
t~c}~ es, e.g., optical and electronic instrument methods.
One useful group of materials are enzymes which, because of the
great nurnber of analytical permutations, has created a whole
family of techniques known collectively as enzyme immunoassay
(EIA) techniques.
Amol2g the more recent patents issued that are repre-
sentative of the state of the art in the detection and measure-
ment of immunochemical substances are, as recited, U.S. Patent
Nos. 3,654,090, issucd April 4, 1972; 3,666,421, issued May 30,
1972; 3,791,932, issued February 12, 1974, 3,839,153, issued
October 1, 1974, 3,850,752, issued November 26, 1974, 3,879,262,
issued April 22, 1975, 4,016,043, issued April 5, 1977, and
Reissue Patent 29,169, reissued April 5, 1977.
A specific example of a recent latex agglutination
- inhibition method is a qualitative in vitro test for determin-
ing the present of human chorionic gonadotropin (HCG). HCG is
a hormone that is characteristic of pregnancy and may he found
in the urine of a pregnant human. An antiserum specific to
HCG can be prepared from rabbits immunized with HCG to produce
the antibody.
According to the PREGNOSTICON ~ Slide Test, if the
antiserum is mixed with latex that has heen sensitized with
HCG, agglutination of the latex occurs. If, on the other hand,
the antiserum is mixed with a sample of urine containing I~CG,
i.e., from a pregnant person, the antiserum is neutralized,
and upon subse~uent mixing of the antise~m-urine mixture with
the HC~-sensitized latex, the agglutination of the latex is
inhibited. The latex appears as a milky homogenous suspension,
its agglutination having been inhibited. This is a positive
test for pregnancy.
Although usually a positi~e or ncgativc rcsult can
'f''~
be determined by a lac~ of agglutination or agglutination of
the latex, respectively, a maximum inhibition of agglutination
may not OCCUl- in the early stages of pregnancy when the con-
centration of IICG in the urine has not increased above a certain
threshold level ~hich can be detected by this method. The
sensitivity of the described pregnancy test is normally such
that the concentration levels of ~CG are usually sufficiently
elevated by the twelfth day after menstruation fails to occur
that the HCG can be detected with the test. If the result of
the test is inconclusive, the test must be conducted again in
another week or two, to allow sufficient time for- an increase
in the l-lCG concentration in the urine to detectable levels. Of
course, this is very undesirable from a diagnostic viewpoint,
since it is often important to be able to determine the
existence of pregnancy at the earliest stages.
The above-described test is qualitative in nature,
giving either a positive or a negative test for some threshold
concentration of HCG. If HCG is detected in a urine sample, a
more quantitative determination of the concentration of HCG
2Q in the sample can be made by conducting a series of tests on
a series of systematic serial dilutions (commonly referred to
in the art as a "dilution series"~ of the urine samples. Of
course, the necessity of conducting a series of tests to deter-
mine the concentration o~ ~CG in a single urine sample is time-
consuming and costly.
In an available embodiment, the foregoing technique
for qualitatively detecting the HCG antigen characteristic of
pregnancy is known as the PREGNOSTICON ~ -Slide Test {Or~anon
Inc., West Orange, New Jersey~.
The same general agglutination process principles
underlying th(' PREGNOSTICOM ~ -Slide Test (and erythrocyte
'^ 3
test) can aLso ~? applied to the determination of other immuno-
chemical substances which can be specifically bound, such as
antigells i.e., those associated with gonorrhea, rheumatoid
factor, etc., and antibodies such as those specific to GC, IgG,
and so forth. It would be desirable to provide a test for
determining immunochemical substances which would both detect
and quantitatively measure the presence of such specifically-
~indable immunochemical substances rapidly and at low concen-
trations, to thereby insure ~arly diagnosis,
- 10 T~le use of light-scattering photometers in analyzing
the electromagnetic properties of various substances is well
known and photometric methods can be used in the analysis of
immunochemical substances. There are many different embodi-
ments in which light-scattering photometers have been used, but
in most cases, such instrumentation is very sophisticated and
expensive. One reason for this is that such instruments
typically include a number of lens systems and complicated
mechanisms for positioning the cuvette containing the subs-
tance being determined, for example, the BRICE-PHOENIX Model
OM-2000 Light Scattering Photometer (Virtis Co., Gardiner, New
York), SCIENCE SPECTRUM Differential Light-Scattering ~ Photo-
meter (Santa Barbara, Cal.). For example, U~S. Patent No.
3,036,~92 issued May 25, 1962, describes a complex adiustable
specimen chamber for determining the light transmittance
properties of a sample at varying angles. ~nother example
is U.S. Patent No. 3,9l8,817 issued ~ovem~er 11, 1975, which
describes a turbidimeter, a particular type of photometer,
including a special thermal insulating housing and using a
glass test tube of rectangular cross-section. Buffone,
"Improved Nephelometric Inst~umentation", LaboratorY Manac~e-
ment, ~pril, 1977, desc3-ibes at page l9 a nephelomet~r, a
similar type of photomoter, llsinc3 an incandescent lamp with
filters to p~oduce a band of radiation between 450 and 650 nm.
In general, much of the existing literature has been
concerned primarily with textile quality control techniques.
For example, in other variations involving the use of light-
scattering photometers, textile color analyzers involving
instrument heads using a plurality of fiber-optic bundles
positioned to receive diffuse light reflected from the textile
samples have been devised, as described in U.S. Patent No.
3,986,778 issued October 19, 1976, and U.S. Patent No.
3,999,860 issued December 28, 1976.
It is interesting to note that light-scattering
photometric methods for determining particular substances have
: in the past typically required measurement at a particular
wavelength, that is, essentially monochromatic light.
For instance, in Blume and Greenberg, "Application
of Differential Light Scattering to ~he Latex Agglutination
Assay for Rheumatoid Factor" Clinical ChemistrY, Vol. 21, No.
9, 1975, page 1234 et seq., it is disclosed that in the tech-
nique of differential light scattering it is essential that
the lic~ht source be highly monochromatic, such as, e.g., that
produced by a helium/neon-laser (632.8 nm). The requirement of
essentially monochromatic light in photometric determinations
is again described by Lichenbelt, Pallmarnanobaran, and
Wiersema, "Rapid Coagulation of Polystyrene Latex in a Stopped
Flow Spectrophotometer", Journal of Colloid & ~nterface Science,
Vol. 49, ~o. 2, 1974, page 2~1 et seq., and Dininno and
McCandless, "Agarose Medium Turbidimetric Assay for Cross-
Reacting Antigens", Journa~ of Immunoloqical Methods, Vol. 17,
1977, pages 73-79. See also Fluorometry Reviews, March 1969
(monthly bulletin of Turner Inc., Palo Alto, California).
Surprisingly, it has now been found tha~ in the
instant invention that for the detection and measurement of
insolubilized particles in suspension, i.e., non-agglutinated
or ag~lutinated latex particles, turbid liquid samples, chemical
precipitates, etc., the requirement for a monochromatic, incident
light source is illusory. It has also been found in the instant
invention that the widest spectral band oE incident light
available, whose upper wavelength limit being equal to or less
than the mean diameter of the insolubilized particles in the
suspension Or interest (the value herein which may be expressed
in nanometers or microns), is preferred for optimum detection
and measurement sensitivity. The use of such wide-band spectral
~ilters, commonly known as low-pass optical filters, in asso-
ciation with an appropriate light source, is indeed unique
and novel for the aforementioned applications.
The use of such low-pass optical filters are suitable
for forward-scattering measurements at the 0 optical-axis
mode as well as for light-scattering measurement at any off
axis detection angle mode from 1 to gn to the optical axis
o~ the test system. The 90 detection mode is more commonly
known as the nephelometric mode. Exàmples of present art for
the 0 detection mode is the Klett-Surnmerson Model 900-3
colorimeter ~Klett Mfg. Co.), for off-axis detection modes,
the Brice-Phoenix Models 2000D and 2000DM Light-Scattering
Photometers (Phoenix Precision Instrument Company), and ~or
the 90 detection mode, the Volu-Sol Models 300 and 299 Nephelo-
meters ~Volu-Sol Medical Ind., Inc.).
The preceding discussion illustrates that a need
exists for (a) new improved methods and ~b) low cost, simple
to use apparatus therefor for making rapid, accurate, and
economical measurements of immunochemical su~stances in
agglutination and colorimetric medical-diagnostic laboratory
1()--
tests employin~ inexpensive disposable cuvettes (having
scratches and small defects) normally manufactured for the
science laboratory.
The invention relates to the detection and measure-
ment of predetermined, specifically-bindable, immunochemical
substances in ag~lutination-type immunochemical test systems
or labeled immunochemical systems (as are known to those
skilled in the art, such as EIA, or FIA, subject to spectro-
photometric, colorimetric, or nephelometric analysis, using
a novel method and novel apparatus therefor that provide for
rapid and accurate quantitative determinations in an efficient
and economical manner). It must also be understood that the
invention relates to the detection and measurement of particles
by turbidimetry, and to the demonstration and determination,
i.e., qualitative and quantitative analysis, of clarity of
various non-opaque liquids, whether or not containing suspen-
sions of particles therein.
The devices, i.e., novel features of the invention,
may have direct applications in other analytical instrumentation
ZO for studying molecular and micellar weights of compounds (from
about 3 x 102 to about 109), particle size and size distribu-
tions shapes and orientations of macromolecules, interactions
in solutions, ~inetics of reactions, and polarization of ~luo-
rescence, as well as the optical properties of liquids by
measuring (as the case might be~ turbidity, forward light
scattering, off-axis light scattering, transmitted light,
optical density, depolarization or fluorescence. As will be
appreciated by those in the art, it must be understood th~t
qualitative analysis is inclusive in the phrase l'detection and
measurement" or the equivalent, and that the user, of course,
need not at his option utilize the data provided by the method
for a quantitative analysis; i.e., "detection and measurement"
-Ll~
L~7 3
may ~? read "detection and measurement, or, if desired, only
detection."
In a preferred embodiment of the invention there is
provided an optical method for detecting and measuring in a
liquid sample, comprising a suspension of coated (preferably
latex) particles, the presence and concentration of a pre-
determined immunochemical substance in the sample. By
measuring the electromagnetic radiation transmission properties
of the sample using a calibrated radiation-measuring apparatus
according to the novel apparatus of the invention, the presence
and concentration of the immunochemical substance can be
determined .
The novel method for detecting and measuring a pre-
determined specifically bindable immunochemical substance in a
liquid sample, comprises the steps of
ta) providing, in an immunoassay technique for a
liquid sample, a component comprising a suspension of particles
which may be agglutinated or insolubilized in relationship to
the presence and concentration of the immunochemical substance
in the sample, and
(b) determining the presence and concentration of the
immunochemical substance by measuring the electromagnetic
radiation transmission properties of the sample using a cali-
brated radiation-measuring apparatus, said apparatus comprising:
(1) a suitab~e electromagnetic radiation source
capahle of providing radiation at wavelengths equal to
or less than the mean diameter of said paxtic~es,
(2~ means for concentrating and collimating
radiation from the electxomagnetic radiation source to
form a beam,
(3) means for filtering the beam to (i) eliminate
-12-
73
radiatiorl having wavelengths greater than the mean
diameter of the particles and (ii) transmit
radiati.on of a wavelength equal to or below the mean
diameter of the particles over a wavelength range
of at least 100 nm,
(4) means for (i) positioning a sample-containing
cuvette and for (ii) allowing the filtered beam
incident on the cuvette to be transmitted through the
cuvette and sample, and for (iii) receiving a portion
of the filtered beam transmitted through the sample
at two or more predetermined angles with respect to
the beam, and
(5) means for detecting and measuring the portion
of the beam transmitted at a predetermined angle.
The novel apparatus of the invention for detecting
and measuring electromagnetic radiation transmitted at pre-
determined angles through a liquid sample having a suspension
of particles comprises-
(a) a suitahle electromagnetic radiation source
capable of providing radiation at waveiengths equal to or lessthan the mean diameter of the particles,
(b) means for concentrating and collimating radiation
from said electromagnetic radiation source to form a beam;
(c~ means ~or filtering the beam to (i~ eliminate
radiation having wavelengths greater than the mean diameter of
the particles and (ii) transmit radiation of a wave7ength below
the mean diameter of the particles over a wavelength range of
at least 100 nm;
(d) means ~or (i) positioning a sample-containin~
cuvette and for (ii) allowing the filtered beam incident on
the cuvette to ~e transmitted through the cuvette and sample,
and for (iii) receiving a portion of the filtered beam
transmitted through the sample at two or more predetermined
angles with respect to the beam, comprising:
a cuvette fixture comprising a tubular incident
radiation beam aperture, a tubular cuvette opening, and at
least two tubular radiation-receiving apertures, wherein the
axes of all the apertures are coplanar and intersect along
the axis of the cuvette opening and are perpendicular to the
cuvette opening, and wherein the axes of the radiation-receiving
apertures are fixed at predetermined angles with respect to the
axes of (1) the incident radiation beam aperture and t2) the
tubular cuvette opening, and attached thereto: and
a receptor-conveyor means comprising a tubular fiber-
optic bundle terminated at one end by a substantially coaxial
tubular detector cone, wherein the detector cone is (1)
inserted in sealing engagement with one of said radiation-beam
receiving apertures, (2) has a substantially coaxial orifice at
the end of the detector cone remote from the bundle at a
distance such that electromagnetic radiation is transmitted at
about a 6 admittance angle from th~ orifice to said fiber-optic
bundle, the ori~ice being at the intersectjon of the receiving
aperture and the cuvette opening' and
(e) means with the non-cone-te~minated end of the
fiber-optic bundle and with the cuvette-positioning means for
detecting and measuring substantially only a portion of the
transmitted beam. Preferably, the means with the non-terminated
encl of the fiber-optic hundle comprises a signal processing
circuit with a photodiode.
The mean diameter of the particles may ~enerally be
from about 0~20~ m to about 1.~ m, preferably from 0.40 ~ m
tG about 0.65~ m, and most preferably about O.'~ m, SG 101-~
_ 1 ~
'73
as the light source provides radiation of a wavelength below
said mean diameter over a wavelength range of at least about
100 nm.
- The apparatus o~ the invention can be operated at
ambient conditions of temperature, pressure and humi.dity in an
ordinary light-filled room, and has the advantage of no moving
~arts alld mec}~ar~ical adiustmeJlts that encumbered the prior art.
In the drawings which illustrate the invention,
Figure 1 represents an overall schematic diagram of
an apparatus according to the invention;
Figure 2 shows an enlarged and more detailed view
of the cuvette fixture depicted in the schematic diagram of
Figure 1,
-; Figure 3 shows a vertical sectional view of the cuvette
- ~ixture of the apparatus,
- Figure 4 shows a vertical sectional view of the plane
taken along lines 4-4 of Figure 3,
Figure 5 shows a horizontal section view taken along
lines 5-5 of Figure 4;
Figure 6 shows an axial sectional view of the detector
cone portion o~ the receptor-conveyor means,
Figure 7 represents a schematic diagrarn of the signal
processing circuit,
Figure B is a graph illustrating the linear relation-
ship ~etween the concentration of the immunochemical substance
and the meter reading, for use in Example III.
Figure 9 is a graph illustrating the re~ponse of a
preferred ern~odiment device to varying levels of HCG (see
example I), and
Figure 10 representg a schematic diagram of a
modification of the circuit of Figure 7 ~Example ~
i3
It will ~c noted that Figure 7 is on the same sheet
as Figure 1, Figure 4 is with Figure 6 and Figure 5 appears
with Figure 8.
While applicable to any labeled system subject to
photometric analysis for detection and measurement o~ the
immunochemical substa~ce to be determined, the instant invention
is preferably applied to any of the insolubilized agglutination
and EIA tests commercially available. The latex agglutination
tests may be utilized in the methods of Schuurs in U.S. Patent
No. 3,551,555 (1970) and Price et al, U.S. Patent No. 4,066,744
(1978). The aforementioned EIA diagnostics tests are represent-
ed by Schuurs U.S. Patent No. 3,654,090 and its progeny as
mentioned above.
Enzyme-marked compounds for use in enzyme-immunoassays
(EIA) now possess, formost antigens and antibodies to be
detected, all of the advantageous properties that were formerly
achieved only by radioimmunoassay (RIA~: e.g., high specific
activity (enzymatic or radioactive), chemical stability, immuno-
logic similarity to the substance to be measured, and chemical
purity.
If an EIA technique is employed, the choice of the
enzyme which is taken up in the coupling product is determined
by a number of properties of that enzyme. It is, of course,
essential that the catalytic property of the enzyme shou~d be
resistant to the coupling with another molecule. Also of great
importance is the specific activity of the enzyme. As less
enzyme conjugate is needed to be added to reach a measurahle
enzyme effect, the sensitivity of an immunoassay system can be
increased. With a specified enzyme whose rate of conversion is
fixed an~ whose purity is high, the specific activity o~ a
labeled compound is proportional to the de(~ree o incorporation
of enzyme molecules per molecule of marked substance, and a
higher specific enzymatic activity results. See German Patent
No. 2,430,356 (1975)i German Patent No. 2,557,419 (1976); U.S.
Patent No. ~,85~,987 (1974), Michel F. Aubert, "Critical Study
of the ~adioimmunological Assay for the Dosage of the Polypeptidc
Hormones in Plasma", J. Nuclear and Biological Medicine 13, 1-19
~970), Robert Roberts and A. Painter, "Radioimmunoassay for
Carrier Creatine Kinase Isoenzymes", 480 Biochimica BioPhvsica
Aeta 521-526 (1977), Michael G. Grattain, J.M. Puttman, and
T.G. Pretlow in "The Use of Glutaraldehyde-Conjugated Horse-
radish Peroxidase-Bovine Serum Albumin in the Visualization of
Concanavalin A Binding to Tissue Sections of Human Colonic
Tumor", Laboratory Investiqation 35/6, 537-541 ~1976).
Those enzymes can be determined colorimetrically that
catalyze a reaction in which a colored substance appears or dis-
appears.
Also, the enzyme should be stable when stored for a
period of at least three months, and preferably at least six
months at temperatures which are convenient for storage in the
laboratory, normally about 4C or below.
A product should be either formed or destroyed as a
- result of the enzyme reaction, which product absorbs light in
the ultra-violet region or the visible region, that is in the
range o a~out 250-750 nm, preferably 300-600 nm.
The enzyme should have a satisfactory turnover rate at
or near the pH optimum for immunoassay conditions, this is
normally at about pH 6-10, and most typically from about 6.0 to
about 8Ø Pre~erably, the enzyme will ha~e the pH optimum
for the turnover rate at Gr near the pH optimum fol^ binding
of the anti~ody to the liquid.
The enzyme which is employe~ or other enzymes with
-17- ,
1~2~
like activity wili not be present in the fluid to be measured,
or can be easily removed or deactivated prior to the addition
of the assay reagents. Also, one must insure that naturally
occurring inhibitors for the enzyme present in fluids to be
assayed are not pre.sent in concentrations at which they will
interfere.
; Also, although enzymes of up to 600,000 molecular
weight can be employed, usually relatively low molecular weight
enzymes will be employed of from 10,000 to 300,000 molecular
weight, moxe usually from about 10,000 to 150,000 molecular
weight, and frequently from 10,000 to 100,000 molecular weight.
Where an enzyme has a plurality of subunits the molecular weight
limitations refer to the enzyme and not to the subunits.
A summary of properties of enzymes useful for enzyme
labels is given below:
1. Available and inexpensive in high purity.
2. High enzymatic specific activity.
3. Soluble under labeling and assay conditions.
4. Chemically and functionally stable under
labeling and assay condi~ions.
5. Enzymatic activity detected simply, sensitively
inexpensively, rapidly and with standard
laboratory equipment.
6. Missing or in negligible concentration in
analyte.
7. Interfering factors missing in analysis.
Enzymes currently used as labeling moieties in enzyme
immunoassay (from G.B. Wisdom, Clinical Chemistry 22, No. 6,
1243-1255 (1976)) are sho~n in Table I.
-1~-
15~3,'~3
,,
TABLE I
Enzymes Currently Used as Labeling Moieties in
Enzyme Immunoassay
Enzyme Source Enzyme
; Commission
: Malate dehydrogenase Pig Heart 1 37
. mitochondria 1.1. .
Glucose-6-phosphate Leuconostoc
dehydrogenase mesenteroides 1.1.1.49
Glucose oxidase Fungal 1.1.3.4
. 10 Peroxidase Horse-radish 1.11.1.7
Acetylcholinesterase Bovine erythrocytes 3.1.1.7
Alkaline phosphatase Calf intestinal 3.1.3.1
mucosa and E. coli
- Glucoamylase Rhizopus nivens 3.2.1.3
Lysozyme Egg wh.ite 3.2.1.17
-Galactosidase E. coli 3.2.1.23
-1'3- 1
Preferable enzymes generally include catalase,
peroxidases, ~-glucosidase, ~ -D-galatosidase, ~'-D-glucosidase,
urease, glucose oxiclase, galatose oxidase, and alkaline
s phosphatase; In general the glucuronidases, galatosidases
ureases and the oxidoreductases. An extremely preferable
enzyme is horseradish ~roxidase (HRP) which can be obtained
relatively inexpensively for pure material, has a high conversi~
i of substrate, and has a substantially flat, fixed rate of con-
version.
Use of an enzyme immunoassay system offers attractive
advantages: elimination of radioactive substances and their
associated hazards and license requirements, common, inexpensive
laboratory equipment used, amplification of results through
repeated use of enzyme catalysis (a radio-isotope atom decays
only once) and ready availability commercially of the enzymes.
Unlike radioactively labeled compounds where high specific
radioactivities lead to increased auto-radiolytic destruction
these high specific enzymatic activity enzyme systems are stable
chemically, there being no radioactive emissions present to
cause destruction. Hence, preferablé markers for the invention
are suitable enzymes, with HRP being most preferable.
As known to those s~illed in the art, in some ins-
tances a "cofactor" or coenzyrne, which is a small nonprotein
prosthetic group ~i.e., compound), is required before an enzyme
can exert its catalytic effect on a substrate. An exemple of
such an enxyme is malate dehydrogenase.
The novel method and novel apparatus are also
especially suited for agglutination-type immunochemical tests
i.e., the so-called latex agglutination tests such as the
"NOSTICON"*tests mentioned above.
The term "antibody" or "antibodies" as employed
* Tr ade Mar}~
-- 2Q
'73
herein means a g~oup of serum proteins, also referred to as
gamma globulins or immunoglobulins, that will specifically react
with an antigen. Most of these antibodies belong to the Ig
class, while the other classes are termed IgA, IgM, IgD, and IgE.
It is also used herein to include certain naturally occurring
binding proteins which recognize certain humoral constituents,
for example, such proteins for testosterone, cortisol and
thyroxine.
The term "antigen" is employed herein to mean a
substance that will react with an antibody. Antigens are often
characterized as capable of inducing the formation of an anti-
body and o~ reacting with that antibody. However, as will be
recognized by those in the art, in the case of "haptens",
defined infra, it is necessary to be coupled to a carrier such
as, for example, inert adsorbing particles, synthetic peptides,
or natural protein molecules, in order to induce antibody
formation. Materials commonly employed as carriers include,
for example, the albumins (human, bovine,or xabbit), synthetic
polypeptides (for example, polylysine), inert adsorbing
particles (for example, charcoal particles) and polymers (for
example dextrans). It is noted that haptens will, in the
absence of a carrier, still react with antibodies and can be
employed in the antigen-antibody reaction assays of the present
invention either with or without carriers.
The term "pure protein" or simply "protein" as
employed herein is intended to include proteins and poly-
peptides that are ~ree of contamination, and it is good
practice to use such pure material to avoid unnecessary inter-
fering factors.
The following Table II lists a partial representation
of diseases, causative organisms, antigens, and antibodies
within the scope o the invention:
-21-
'Y3
TABLE II
REPRESENTATIVE ~NTIGENS AND ~NTIBODIES
Disease States and Antigen Derived From
the Causative Organism or Other Specific
Antigens Used in the Diagnosis of Certain
Disease States
I. Infectious Diseases
A. Parasites
Disease Orqanism _ Antigen
A~oebiasis Entamoeba histolytica Organism sonicate
of strain HK-9
Toxoplasmosis Toxoplasma qondii Whole organism or
their sonicate de-
rived from tissue
culture or mouse
peritoneal fluid
Chagas Trypanosoma cruzi Organism sonicate
derived from
tissue culture
Schistoso- Schistosoma Haematobium
miasis Schistosoma iaponicum
Schistosoma mansoni Culture filtrates
- B. Bacteria
Infectious Neisseria Capsular polysac-
meningitis mënin~itidis ~ charide
Gonorrhea Neisseria qonorrhoeae Pili isolated
from the bacterial
cells
Typhoid fever Salmonella ~ Bacterial cells or
their extracts
Pneumonia Diplococcus Capsular poly-
pneumoniae saccharide
C. Fun~i
Histoplas- HistopLasma Culture filtrate
mosis capsulatum
Blastomy- Blastom~ Culture filtrate
cosis de_matitidis
Coccidiodo- Coccidioides Culture filtrate
mycosis immitis
-22-
TABLE II
-
REPRESENTATIVE ANTIGENS AND ANTIBODIES
(continued)
Disease Oraanism Antigen
D. Viruses
Rubella Rubella virus Virus particles
Measles Measles virus Virus particles
(Rubeola)
Rabies Rabies virus Virus particles
E. Allerqie
Ragweed pollen Pollen extract
Tomatoes Tomato extract
Bermuda grass seed Seed extract
Cat dander Fur extract
House dirt Dust extract
F. Disease _tates
Lupus Erythem- DNA molecules
atosis
RNA molecules
Rheumatoid Human IgG
arthritis
Colon cancer CEA antigen
Hepatoma Alpha l-feto protein
-
The novel apparatus and method of the invention can
also be employed for the determination of haptens, which may be
regarded as a special group of low molecular compounds, and
their anti-substances. These substances mostly occur in low
concentrations. As will be recognized by those in the art,
and according to t~le original definition of K. Landsteiner,
haptens are protein-free substances whose chemical configuration
is such that they can react with specific antibodiesr but not
such that they are capable of causing the formation of anti-
bodies. In order to be able yet to make antibodies againsthaptens, the haptens must be coupled to polypeptides, inert
adsorbing particles, or natural protein molecules before being
injected into a test animal. In the determination of a low
molecular weight compound by classical enzyme immunoassay (EIA),
the substance to be determined and its enzyme conjugate enter
into competition for a given quantity of the antibody. The
more unlabeled compound the sàmple contains, the less the
soluble enzyme conjugate of that compound is able to combine
with the speci~ic binding protein and the more of the conjugate
will remain unbound in the liquid phase. Following a separa-
tion of bound and free phases (frequently but not always nec-
essary), the enzyme activity can be measured in a simple manner.
As examples of haptens are mentioned: steroids, such
as esterone, estradiol, estriol, cortisol, corti70ne,
testosterone, pregnanediol, and progesterone' vitamins, such as
- vitamin ~12 and folic acid, l-thyroxine, triiodo-l-thyronine,
histamine, serotonin, digoxin, prostaglandin, adrenalin; nor-
adrenalin morphine vegetable hormones such as auxin, kinetin,
and gibberellic acid, and antibiotics, such as penicillin.
~ence, the compound or substance to be labeled is a
conventional diagnostic material such as a hapten, a drug, a
-24- !
hormone, a protein, nucleic acid or other biologically or
immunologically useful or interesting molecule, or viruses or
bacteria. If an enzyme marker is chosen and a preference is
made to use an insolubilized phase in the reaction scheme, one
may adapt the novel process and method for a simple "competitive"
method (as taught in U.S. Patent No. ~,654,090), or for a
"sandwich" method (as taught in U.S. Patent No. 4,016,043 or
U.S. Reissue Patent 29,169, for example), or for a double anti-
body solid phase "DASP" method (as taught in U.S. Patent No.
3,839,15~). The instantly claimed method and apparatus of
the invention can be used with conventional test kits, for ex-
ample, those kits also set forth in detail in U.S. Patent Nos.
3,654,090, 3,850,752, 3,838,153, 3,879,262 and 4,016,043. The
term "kit" is employed herein to mean a collection of all or some
of the chemicals, including the assay tubes or cuvettes, and
instructions necessary to do a enzyme immunoassay.
The practice of the novel method of the invention
involves in each case the determination of the electromagnetic
radiation transmission properties of a sample in a cuvette using
the novel apparatus of the invention;
The apparatus is most conveniently discussed by
reference to the drawings, although it is to be understood that
the drawings are referred to only for purposes of illustration
and example, and the scope of the invention is not limited
thereto.
~n Figure L, the apparatus of the invention is shown
schematically. As depicted, mirror 1 (Rolyn 61,200) and lens
assembly 5 act to concentrate and collimate the electromagnetic
radiation emitted by electromagnetic radiation source 3 (here a
Syl~ania 6.6~/T2.50/Cl quartz-halogen lamp) to form a beam 7.
The lens assembly 5 usually comprises two condenser lens (Rolyn
-25-
L~
10,0140) ancl a collimator lens (Rolyn 10,0050). It is prefer-
able to surround the beam 7 by a black barrel (not shown)
slightly greater than the diameter of the incident radiation
beam aperture 13 so as not to lose appreciable energy. A low-
pass optical filter 9 is interposed in the path of the beam to
modify certain characteristics of the beam, as discussed herein-
after, before the beam reaches cuvette fixture 11 at tubular
incident radiation beam aperture 13. Preferably, a standard
Leitz-Wetzlar BG12 filter is employed.
A cuvette fixture 11 provides a means for positioning
a sample-containing cuvette 17 and allowing the filtered beam
incident to the cuvette to be transmitted through the cuvette
and sample. The cuvette fixture is preferably machined from
a DELRIN ~ (du Pont) black resin material (machined by Organon).
A receptor-conveyor means 25 is snugly positioned in one of
alternate radiation receiving apertures 19, 21 and 23 originat-
ing through cuvette opening 15 and providing a means ~or
receiving a portion of the filtered beam transmitted through
the sample at a predetermined angle with respect to the axis of
incident radiation beam aperture 13.
A suitable light transmission means such as a fiber
optic bundle 27 ~here a Ski~ bundle, LG-093-008, not shown in
Figure 1) comprising a part of a receptor-conveyor means 25
(or receptor assembly) conveys the received radiation from
cuvette fixture 11 to signal processin~ circuit 33, which trans-
forms the signal received into a needle displacement in meter
43, indicative of the electroma~netic radiation transmission
properties of the sample.
The electromagnetic radiation source 3 suitable
according to the invention must be capable of providing
radiation at relatively short wavelengths, i.e., providing
-~6- ~
~S.1;2~7~
`:
electromagnetic radiation of at least 100 nm width and having
an upper bounds slightly more than the mean diameter of the
particles used in the method of the invention. In a preferred
embodiment the electromagnetic radiation source is a quartz-
halogen lamp.
The radiation from electromagnetic radiation source 3
is concentrated and collimated to form a beam 7 by means of
mirror 1 and lenses 5. The representation of the mirror 1 and
the lens 5 in Figure 1 is schematic only, it being und6rstood
that those skilled in the art could devise a variety of different
mirror and lens configurations to thereby concentrate and colli-
mate the radiation to form a beam.
Filter 9 modifies the beam by eliminating the radiation
having wavelengths greater than the mean diameter of the
particles (generally about 0.45~ m for latex particles) and, as
recited, by transmitting radiation below the mean diameter over
a wavelength range of at least lOO;nm, e.g., 300 to 450 nm for
latex particles. As is known to those skilled in the art, glass
(other than quartz~ absorbs most electromagnetic radiation
having a wavelength below about 300 nm, although if this ~adia-
tion energy were available, one would desire not to eliminate
this radiation. In a preferred embodiment the filter is a so-
called low-pass optical filter, i.e., a filter which is so
selected and manufactured to transmit essentially all radiation
below a characteristic wavelength (here the mean diameter of
~,
the particles) down to very short wavelengths at which the
matrix material comprising the filter exhibits absorption.
Of course, as one in the art can appreciate, one can ad~ust to
a given mean diameter for a set of particles in suspension
by changing the filter, and if necessary, the light radiation
` source.
-27-
j~ ~
The cuvette fixture 11 shown schematically in Figure
1 is more particularly described by reference to Figures 2,
3, 4 and 5. In Figure 2, cuvette fixture 11 provides a means
for positioning a cuvette 17 (not shown - see Figure 3) contain-
incJ a liquid sample 1~ (not shown - see Figure 3) prepared
according to the method of the invention to allow the filtered
beam to be transmitted through the cuvette and sample.
The cuvette fixture comprises a tubular incident
radiation beam aperture and at least two tubular radiation-
receiving apertures. As noted above, and as shown in Figures1-5, cuvette fixture 11 comprises tubular incident radiation
beam aperture 13 and tubular radiation-receiving apertures 19,
21 and 23. The incident radiation beam aperture and the
radiation-receiving beam apertures are coplanar, and inter-
sect along the axis of cuvette opening 15, to which they are
perpendicular.
' Furthermore, in addition to being perpendicular to the
axis of cuvette opening 15, the axes of the radiation-receiving
apertures are also fixed at predetermined angles with respect to
the axis of the incident radiation beam aperture 13. In the
preferred embodiment shown in Figure 2, the predetermined
angles of radiation-receiving apertures 19, 21 and 23 have
arbitrarily been chosen to be 0 (parallel), 30 and 90,
respectively. One s~illed in the art could locate apertures
at different angles if desired. For the latex agglutination
tests, it is recommended that the O (or colorimetric) position
be used, for the detection of globulins, the 30 light
scattering position is recommended, finally, for the detection
of precipitates and turbidity, the 90 nephelometric position
is recommended.
~eturning to Figure 3, in the metllod of the invention
-28-
the cuve~te 17, containing the sample 18, is positioned within
the tubular cuvette opening 15 so as to occupy the space between
the incident radiation beam aperture 13 and the tubular
radiation-receiving apertures.
Cuvettes for various samples should ke reproducibly
positioned within the tubular cuvette opening to insure uniform-
ity in the distance of the radiation transmission through the
sample and cuvette. Reproducible positioning of the cuvette can
be accomplished by a variety of binding techniques, such as,
: 10 for example, that shown in Figures 3, 4 and 5.
Cuvette-holder assembly 11 is comprised of two
principal parts: a cylindrical base portion 50 and a cuvette
holder-positioner assembly 51. Base portion 50 is attached to
the floor plate 64 of the instrument case or housing (not shown).
Above the radiation apertures 13, 19, 21 and 23, the base 50
is provided with a vertically extending cylindrical hole 53
to receive a tightly fitted cylindrical base formed on the
bottom end of cuvette holder-positioner 55.
A vertically extending rectangular hole 59 in-
positioner 51 is in axial alignment`with cylindrical hole 15in base fixture 50 as is best seen in Figures 3, 4 and 5.
One wall of hole 59 has a rectangular axially extend-
ing recess 60, with flanges 56 extending partialiy into the
opening between hole 59 and recess 60. The interior surface
63 of flanges 56 are inclined toward one another to form V-
notch-positioning surfaces for a cuvette (best seen in Figure 5).
On the rear wall 66 of hole 59, opposite flanges 56,
is a leaf spring 67 secured in a groove 69. Spring 67 is bowed
inwardly near its mid-point, as seen in Figure 4, and at that
location, there are two angularly extending fingers 71. Fingers
71 engage the cuvette and force it against surfaces 63, thus
-2~-
po~sitiollir~l t~ cu~t:te concentrically in hole 15 of base 50.
Cuvette holder-positioner 51 extends upwardly through
an opening 73 in top plate 61 of the instrument housing, and a
hin~ed li(i 57 closes the hole 5g and covers cuvette 17.
The bottom end of cuvette 17 rests on floor plate 64
in the center of hole 15.
It will be apparent to those in the art that the
cuvette fixture 11 as described will position all sample-holding
cuvettes of the same standard size and shape accurately and
identically in the path of the electromagnetic radiation.
In an apparatus according to the invention as shown
in Figures 3-4, cuvette 17 rests on the floor plate 64 in the
center of hole 15.
Sample-containing cuvettes are inserted into and
removed from the apparatus by means of lid 57 (here Bausch &
Lomb 33-31-27~ being pivoted about hinge 58.
Attached to the cuvette fixture through one or more
: of the tubular radiation-receiving apertures are one or more
receptor-conveyor means (receptor assemblies) for conducting
the radiation transmitted through thè cuvette and sample to one
or more signal processing circuits according to the invention.
Referring to Figures 1, 3 and particularly 6, the
receptor-conveyor means 25 (receptor assembly) comprises
preferably a suitable flexible light transmission means, here
a fiber-optic bundle 27. The fiber-optic bundle is terminated
at one end (cuvette fixture end) by a substantially coaxial
tubular detector cone 24, having an orifice 29 at the end of
the detector cone opposite to the end of the fiber-optic bundle
27. The dimensions of the detector cone are such that the
admittance angle 0 for radiation transmitted through orifice
2~ to the fiber-optic bundle is from about 5 to about 7.
-30-
',`3
The prefel~ed admittance anc~le ~ is about 6, which is con-
veniently acllieved by positioning the fibcr-optic bundle at a
distance of about four times (4d) the orifice diameter (d) from
orifice 29. ~le detector cone 24 has the orifice 29 at the
viewing or cuv~tte fixture end. Tlle diameter of -the orifice
29 is suitably machined so as to permit an optimum amount of
light into the cor-e 24 to the bundle 27 without permitting
diffracted li~ht from the cuvette to reach the bundle 27. The
diameter of the orifice 29 is constructed to allow the maximum
light transmission through the contents of the cuvette without
allowing the scattering of light to reach the bundle caused
by any imperfections in the cuvet-te, generally, the diameter
is preferably from about .35 to about .6 the diameter of the
cone 24 (here about .037 inches is preferable), at the existing
space relationships of the cone 24 from the cuvette 17 and the
diameter of the cuvette 17. The fiber-optic bundle (diameter:
here .Og3 inches not including the width of the ferrule) is
inserted at the opposite end as noted (distance of about 4d).
This cone eliminates the necessity of an optical lens assembly
and azimuth mount used in the literature. The angle 0 must
not be so large as to entertain reflected light from the imper-
fect cuvettes often obtained in the field, but must not be so
small as to block off light so as to require a specialized
and unduly expensive light source. Detector cone 24, which is
made of metal, such as brass, or other suitable material, is
inserted by the user in a sealing engageMent and locked with
one of the radiation-receiving apertures. Fiber-optic bundle
27 is covered by a protective layer 28.
The other end of fiber-optic bundle 27 is terminated
at signal processing circuit 33. Referring to Figure 7, showing
a novel advanced signal processing circuit accorc~ing to the
invention, radiation that is transmitted from detector cone 24
through fiber-optic bundle 27 (not shown) impinges on a
suitably-housed photodiode 35. Preferably, a silicon photo-
diode is employed (here UDT Pin SD/SB). The radiation signal
received by photodiode 35 is transferred, once the apparatus
has been calibrated, using a sample containing a known quantity
of an immunochemical substance as a standard, by means of the
circuit shown, and particularly by means of a zero control sub-
circuit 37 (zero control by Helipot, 8136R50K) and a novel gain
control subcircuit 39 (gain control by Helipot, 8136R50K), to an
electrical signal transmitted to meter 43 (not shown~. The
circuit is especially useful in that it enables the photometric
values measured by the invention to be amplified for detection
readout. In general, the means (33 and 43 of Figure 1) for
detecting and measuring the transmitted beams comprises a
photodiode-meter signal processing circuit including a means for
amplifying a transmitted beam for a standard to a full-scale
meter reading.
For measurement of HCG, infra, by a P~EGNOSTICON
Slide Test, any suitable circuit is ~unctional that enables
the operator to zero the instrument with a 0 IU HCG/l and then
amplify the difference in transmitted or scattered light between
the O IU HCG/l and 4500 IU HCG/l and display this value as a
full scale reading.
- Figure 8 (see Example III) is a graph illustrating a
; typical straight-line relationship between samples of known
concentrations of an immunochemical suhstance and the correspond-
ing meter readings. Therefore, using the method of the
invention, the concentration of an immunochemical substance
: ,.
in a sample of unknown concentration can be determined directly
from the meter reading.
-32-
The ~etector core portion of the receptor-conveyor
means (receptol assembly) is engaged with a particular radiation-
receiving aperture based on the particular electromagnetic
radiation property -that it is desired to measure. The preferred
radiation-receivin~ aperture positions are at 0 for colori-
metric determinatiolls, 30 for scattered radiation determina-
tions, and 90 for nephelometric determinations, such as,
for example, wherein a fluorescent marker is employed (FIA),
or for measuring total protein in urine, or for turbidimetry
measurements, respectively.
The novel apparatus and method can also be used, or
example, on fluorescent immunoassays, providing that the 90
tor about 90 radiation receiving aperture is used, and
the fluorescent pigment has emission/excitation over 400 nm
(example-fluorescein isocyanate, emission 492 nm, excitation
520 nm) which is larger than the mean diameter of the particles.
A method for detecting and measuring a predetermined
specifically-bindable immunochemical substance according to
the invention first comprises providing, in an immunoassay tech-
nique for a liquid sample, a component compLising a suspensionof particles which may be agglutinated. Following the prescribeo
immunochemical reaction where agglutination may or may not have
occurred, the electromagnetic radiation transmission properties
of the sample are determined.
If the instrument is used for non-immunochemical
purposes, such as for determining the clarity of liquids (for
instance, wine), a filter after the light source need not ~
ernployed if the correct ~suitable~ electromagnetic radiation
source is chosen corresporlding to the designated purpose. It
is well within the knowledge~ of those skilled in the photometric
arts what radiation source should be selected for a particular
73
purpose the instrument is to be employed for, how wide the
wavelength range that must be provided by the radiation ~ource, -
as well as the intensity that must be provided by the radiation
source. Generally, one skilled in the art chooses for a
particular purpose a radiation source having a sufficient wave-
length range for the purpose intended, and if necessary, will
employ a filter as, for example, that described above.
Although the invention has been described with res- ~ `
pect to the specific embodiments above, numerous variations
and modifications will become evident to those skilled in the
art, without departing from the scope and spirit of the inven- `
,
tion as described above, defined in the appended claims, and -
as shown in the following Examples:
~- EXAMPLE I
In one preferred embodiment of the invention, the
immunochemical substance is detected and measured by measuring
the electromagnetic radiation properties of a ample pre-pared
using a "competitive" latex-agglutination method, here the
PREGNOSTICON Slide Test kit by Or~anon Inc., W~st Orange,
~ 20 ~-J-
x~ Basically, according to the NOSTICON* method, a liquid
suspension of particles coated with an immunochemical substance
having the same immunochemical properties as the immunochemical
)t~ ~
substance being detected and measured is prepared. The immuno-
chemical substance used to coat the particles may be the
identical immunochemical substance being detected and measured.
, ~s
In a preferred embodiment, the particles in suspension are
; latex particles.
In the PREG~OSTICON ~ Slide Test, a latex agglutina-
, . .
~ 30 tion inhibition test, a solution is prepared by mixing a
, .. .
* Trade Mark
-34-
suitable reagent (antl-E-~CG serum) sucl~ as shown below capable
of specifically binding the immunochemical substance with a
suitable liquid (urine) as shown below for which it is desired
to detect and measure the immunochemical substance.
Then, the test solution is combined with the liquid
suspension (latex). After allowing sufficient time for
a~glutination to occur, the electro~agnetic radiation trans-
mission properties are determined. The combined reagents, i.e.,
agglutination reaction, are suitably diluted with an appropriate
buffer solution and mixed to facilitate the determination.
The PREGNOSTICON ~ Slide Test is a special applica-
tion of the above NOSTICON* method and of an antigen-antibody
reaction based on the principle of the Wide and Gemzell
Pregnancy Test (Acta Endocrenologica 35, 1960), ~hich is
designed to demonstrate the presence of human chorionic
gonadotropin (HCG) in urine. HCG is the antigen, and serum
from rabbits immunized against HCG is the antibody.
According to the method here, polystyrene latex
particles having a mean diameter of about 0.45 ~m are washed
in a 0.1 M borate buffer and then exposed to a pre-coating
solution of bovine serum albumin. After further borate buffer
washing, the latex particles are resuspended in a solution of
human chorionic gonadotropin (HCG) and a period of sensitization
follows.
The particles here in Example I were s~bsequently
washed in borate buffer and placed in a final suspension fluid
having a pH of 8.2. A dilution of ra~hit anti-human chorionic
gonadotropin serum was pre~ared so that in the presence of 1-2
IU HCG/ml (1000-2000 IU HCG/l) contained in a urine specimen,
agglutination would be inhibited.
* Trade Mar~
-35-
To ~)el~form these latex inhibition tests using the
novel instrument, 0.05 ml of antiserum dilution was pipetted and
mi~ed in a cuvette with 0.05 ml of urine specimen for a period
of 30 se~onds after which 0.05 ml of the latex suspension was
added by pipette and the reaction mixture agitated for two
minutes. ~or pipetting operations, a micropipettor is used,
set for 50 ~1 delivery, with disposable tips. Ten milliliters
(10.0 ml) of a 0.1 M boLate buffer were added and the cuvette
contents were mixed by inversion of the covered cuvettes two
times. The reaction mixture was then placed in the cuvette
fixture of the novel apparatus for readout of the amount of
agglutination. Instead of a borate buffer, one may use a
phosphate buffer, or a citrate buffer whose ionic strength does
not exceed 0.3 M.
In the case of inhibition of agglutination (a positive
test for HC~) light transmission will be impeded by the homo-
geneous suspension, in a negative test, the latex and antiserum
will form agglutinates leading to more light transmission
; through the contents of the cuvette. Bausch and Lomb
Spectronic 20 ~ cuvettes (or disposable 13 mm x 100 mm culture
tubes) can be used for the novel apparatus of the invention.
Tests were conducted using the PREGNOSTICON ~ Slide Test kit
reagents above with different levels of HCG (IU/l) in borate
buffer, the tests were read on the novel apparatus using a
receptor-conveyor means (see 25 in Figure 1) at an angle of
30 (i.e., means 25 placed in aperture 21) with respect to the
extension of the axis of incident radiation beam aperture 13
of Figure 1. As shown in Figure 9, the data show an almost
linear response by the instrument to varying levels of HCG
(IU/l). Buffer dilution volumes less than ten milliliters
(10.0 ml) may be used with similar success.
-36-
~ L~ ~' 3
E~AMPLE Il
A test using PREGNOSTICON ~ Slide Test reagents was
performed in the same manner as Example l, however, actual
urine samples, positive and negative for pregnancy were tested
on the novel apparatus at 0 , 30, and 90 to the extension of
the axis of the incident radiation beam aperture 13 of Figure
1. The data below clearly shows that at 0 and 30, an average
detectable difference of 20% - 25% can be measured between the
positive and negative clinical urine samples.
Table II
PREGNOSTICON ~ SLIDE TEST CLINICAL TEST DATA
POSITIVE CLINICAL URINE
ANGLE
SAMPLE 0 30 90 MANUAL
49981.80 4.86 +
66 48285.31 4.92 +
87 49684.59 4.85 +
88 48784.67 4.75 +
NEGATIVE CLINICAL URINE
JK 61963.51 4~35
MM 62161.96 4.24
DS-2 63760.81 4.29
DS 64361~29 4.21
MD 70267.47 4.12
Test Reacted in Tube
Test Diluted to 10 ml c Borate Buffer
Readings at 450 nm on Light-Scattering Test Stand
. Readings are W/cm2 x 10 8,
-37-
EXAMPLE III
The purpose of this Example was to demonstrate the
linear response of the novel instrument to varying levels of
~CG (IU/l) in borate buffer. For this test of HCG calibration,
the samples were prepared as exactly with Examples I and II.
The 0 HCG test was p~aced in the instrument and the zero con-
trol was adjusted for zero on the readout meter; the 4500 IU/l
test placed in the instrument and the gain control was adjusted
for a full-scale reading on the readout meter. Five known
10 replicates of HCG dilutions from 0 to 4500 IU/l were tested
on the novel instrument, as shown in Figure 8. Linearity of
the response of the novel apparatus to varying levels of HCG
~ (IU/l) was demonstrated.
- In the protocol, each sample was diluted with 10 ml
of a 0.1 M borate solution as in Example 1. All of the
readings were at the 0 angle with a 450 nm filter, with five
replicates taken of each HCG value. The equation y =
-1.38 + 0.02x was determined by a linear regression.
EXAMPLE IV
This example describes a modification of the
GO~OSTICON ~ DRI-DOT ~ latex agglutination test. Polystyrene
latex particles having a mean diameter of 0.60 ~m are washed
in a 0.1 M borate buffer and then exposed to a pre-coating
solution of bovine serum albumin. After further borate buffer
washing, the latex particles are resuspended in a solution of
gonococcal antigen (Gc9) and a period of sensitization follows.
The particles are subsequently washed in a 0.1 M borate buffer
and placed in a final suspension fluid.
To block or neutralize non-specific antibodies found
in some human sera, an absorbing antigen prepared by combining
the antigens of guinea pig extrac~ and beef erythrocyte stroma
-3~-
is employed.
To perform the modified GONOSTICON ~ DRI-DOT ~ test
using the novel apparatus of the invention, a 0.05 ml sample of
human serum to be tested for the presence of gonococcal anti-
~ody is mixed in a cuvette with 0.05 ml of absorbing antigen.
To this is added 0.05 ml of the GONOSTICON ~ sensitized latex.
The reaction mixture is agitated for two minutes, and 5.0 ml
of 0.1 M borate buffer are added. The covered tube is then
inverted twice for final mixing. The reaction mixture was then
placed in the cuvette fixture of the novel apparatus for read-
out. The 0~ position of the radiation receiving aperture was
used.
If agglutination occurs due to the prese-nce of anti-
body in the sample and its combination with the latex antigen
more light will be transmitted, in the absence of gonococcal
antibody there will be no agglutination and the homogeneous
suspension will impede light transmission through the contents
of the cuvette.
EXAMPLE V
This example describes a modification of the
RHEUMANOSTOCON ~ Slide latex agglutination test. Latex parti-
cles are prepared to receive a sensitizing coating of, in this
instance, gamma globulin after being washed in a buffer solu-
tion, under the same conditions as above. After exposure of
the latex to the gamma glo~ulin for a period of time, the latex
is washed to remove excess gamma globulin and taken up in a
final suspension fluid having an alkaline pH (8.0 - 8.5~.
To perform the modified RHEUMANOSTICON ~ Slide test
using the novel instrument of the invention, a 0.05 ml of serum
sample suspected of having RF activity is mixed in a cuvette
with 0.05 ml of RHEUMANOST~CON ~ latex suspension. The reaction
-3"- 1
mixture is mixed by agitation after which 5.0 ml of glycine
buffer of an effective concentration is added. The covered tube
is then inverted twice for final mixing and the reaction mix-
ture placed in the cuvette fixture of the novel apparatus.
Again, the 0 position of the radiation receiving aperture
was used.
If agglutination occurs due to the presence of the
~F in the sample and its combination with the latex antigen,
more light will be transmitted; in the absence of RF there will
be no agglutination, and the homogeneous suspension will impede
light transmission through the contents of the tube.
EXAMPLE VI
-
In one embodiment the circuitry of Figure 7 may be
modified in order to present an easy to recognize display on
the outside of the novel device a positive or negative result
of the test. ~or example, in the PREGNOSTICON ~ Slide Test
protocol aforementioned in Examples I-III, the circuit can
easily be modified to give a "amber light-green light" detec-
tion system. The use of this new circuit is as follows: a
negative control sample test, i.e., 0 IU/liter HCG, is placed
in the cuvette fixture; a zero-set button is pressed, automatic-
ally setting the instrument detection circuit to zero, and
turning on the green light. Next, a borderline sample test,
i.e., 1000 IU/liter HCG, is placed in the cuvette fixture and
the green light ~ill automatically turn on the gain control
is adjusted until the panel meter reads 1000. The threshold
control is adjusted until the green light turns off and the
amber light turns on' the same control is backed off until
the green light turns back on. In this mode any sample test
of 1000 IU/liter HCG or less will turn on the green light;
any sample test greater than 1000 IU~liter HCG will turn on
-~0- 1
1S.~2~7'3
., ~ .
the amber light: only one light will be on at any one time.
Difficulties with the novel device operation of the "amber
light-green light" system because of random temporary
instabilities of the light transmission source (3 in Figure 1 -
for example a quartz-halogen lamp) or line voltage perturba-
7'~'tions have been corrected by modif^cation of the circuitry of
Figure 7 to incorporate a standard 110 VAC voltage regulator.
In Figure 10 a signal processing and zero circuit isohown which not only encompasses much of Figure 7 but is
modified to give a "amber light-green light" indication for
technical personnel. More specifically, when a light restric-
ting object (such as a latex suspension of particles, turbidity
sample, colored solution, etc.) is placed in the path of light
¦~ striking the photodiode 35, the output current of the photo-
diode 35 is proportional to the amount of light striking it.
This output current is amplifled by a current to voltage
amplifier 79. The ~ltpUt of amplifier 79 will be a negative
i`: :. .
voltage that is proportionaL to the~current times the gain.
This negative voltage is applied to a summing amplifier circuit
; 20 81, which is summed with an opposite polarity voltage from a
digital to an analog circuit 83. ~This voltage from the digital
to analog circuit 83 is the automatic zeroing voltage and is
generated by pressing an auto zero button and releasing it.
When depressed and held in, an automatic zero button
84 grounds the input to nand gate 85. The output of qate 85
goes to a +15 volts, setting "flip-flop" amplifier 87 output
to a ~1~ volts. This enables nand gate 89 to allow the clock
pulse to pass through. The clock pulse is generated by
,:~ .
amplifier 91 and amplifier 93 connected as a multivibrator,
running at a fre~uency of approximately 3000HZ. The output of
- amplifier 85 also drives digital counter 95. When gate ~S output
i -41-
.
73
is +15 volts, it sets digital counter 95, a 10 bit output, to
zero. The 10 bit binary output drives digital to analog con-
verter 83. The analog output from converter 83 will be zero
volts when the 10 bit binary output digital counter 95 is at all
zeros. When the automatic zero button 84 is now released, the
output of gate 85 goes to zero volts, allowing digital counter
95 to start counting the clock pulses from gate 89 output. As
binary number from counter 95 increases, the analog output from
converter 83 increases accordingly. The analog output from
converter 83 drives the summing amplifier 81. When the output
from converter 83 is equal to the output of amplifier 79, and
- is the opposite polarity, the output of amplifier 81 will be
zero volts, also the output of amplifier 103 will be zero volts.
The comparator 99 detects the zero volts and its output switches
to a +15 volts driving amplifier 87 (flip-flop) output to zero
volts. This disables gate 89, stopping the clock pulses from
driving digital counter 95. The binary output from digital
counter 95 is now the stored digital value to drive the digital
to analog converter 83. The circuit has now been automatically
set at zero. The digital value for `the zero sample that has
been placed in the instrument is now stored in digital counter
95, the calibration meter will read zero and the green indicator
lamp 128 will be on.
Successive readings for samples in the instrument
will be with respect to the zeroing voltage driving the summing
amplifier 81. The summing amplifier 81 has a gain control
potentiometer 39 in its feedback circuit. If the light
measuring ohject allows more light to pass with respect to the
one used to zero the circuit, the output of amplifier 81 will
be positive. The output of this summing amplifier 81 drives
amp3ifier 101, an absolute value circuit. When the output of
-42- ,
:`
amplifier 81 is positive, the output of amplifier 101 is -
negative, reverse biasing a diode 105. This allows the positive
signal to pass through resistor 107 into the non-inverting
amplifier 103, resulting in a positive output. When the output
of amplifier 81 is negative, the output of amplifier 101 i9
positive, forward biasing the diode 105. This allows the
positive signal to pass through the diode into the non-inverting
,......... .
amplifier 103. The resulting output of amplifier 103 is always
positive and drives a comparator circuit 109. This comparator
``~ 10 circuit 109 compares the oueput from the amplifier 103 to the
~ voltage that is set on the threshold control potentiometer 111. ~ -~
.
Whenever the output voltage of amplifier 103 is less positive
~: .
`,G", than the positive voltage set on the potentiometer 111, the
output of circuit 109 will be a positive 15 volts and represents
a negative sample. When the output of amplifier 103 is more
~; positive than the positive voltage set on the threshold
potentiometer 111, the output of amplifier 109 will be zero volts
and represents a positive sample.
When the output of amplifier 109 is at zero volts,
r~,~
20 the transistor 113 is turned on, causing transistor 115 to
conduct and to light the amber light emitting diode indicator
127, indicating a positive sample. When the output of amplifier
109 is at a +15 volts, transistors 113 and 115 are in a non-
conducting condition and the positive indicator is off. The ~15
,~,s,~,
volts from amplifier 109 drives a comparator circuit ~mplifier
117, through resistor 119. Whenever the input to resistor 119
.; ~
is a +15 volts, the output of amplifier 117 is a ~15 volts
causing transistor 121 to conduct, turning on the green light
,.. .
: emitting diode indicator 128 for a negative sample. The switch
, .
123 is a normally-open switch located at the base of the cuvette
~ detection fixture.
::;
-43-
When ~1 cuvette is not in the dc~tection fixture,
s~itch 123 is open and the output of comparator 125 is +15 volts,
causing tLallSiStor 127 to conduct. When transistor 127 is in a
conducting mode, both LED circuits a~e disabled. When a
cuvette is fully inserted into the detection fixture, switch 123
is depressed and held closed, driving comparator 125 to zero
volts. This drives transistor 127 into a nonconducting mode,
thus permitting the LED circuits to function according to the
amount of light passed through the cuvette samples of the
threshold control potentiometer 111 voltage setting.
The following parts represent the preferred devices
for key components in Figure 10:
79, 81 AD-741 by Analog Devices, Inc.
103, 101 MC-1458 by Motorola
99, 109, 117, 125 LM-339 by Analog Devices, Inc.
87 MC-14013 hy Motorola
85, 93, 91, 89 MC-14011 by Motorola
MC-14040 by Motorola
83 DAC-03-DOXl by Precision Monollthic Inc
A sample test tube placed in the instrument activates
switch 123 to a closed condition. The output of amplifier 125
goes to zero volts which drives transistor 127 into a non-
conducting condition enabling the light emitting diode indicators
to function.
As one skilled in the art can now appreciate, the
novel features of the invention may have many applications in
photometry (for studying molecular and micellor weights of
compounds, particle size and size distribution shapes of macro-
molecules, interactions in solutionsr etc., as mentioned above),
hence, in its broadest aspect the novel photometer may be used
for any purpose related to detecting and measuring electro-
magnetic radiation at predetermined angles through a liquid
sample, and comprises:
(a~ a suitable electromagnetic radiation source
-44-
capable of providirlg radi.ation over a predetermined waveiength
range
(b) means for concentrating and collimating radiation
from said electromagnetic radiation source to form a beam:
(c) means for (i) positioning a sample-containing
cuvette and for ~ii) allowing the filtered beam incident on the
cuvette to be transmitted through the cuvette and sample, and
for (iii) receiving a portion of the filtered beam transmitted
through the sample at two or more predetermined angles with
respect to the beam, comprising:
a cuvette fixture comprising a tubular incident
radiation beam aperture, a tubular cuvette opening, and at
least two tubular radiation-receiving apertures, wherein the
axes of all the apertures are coplanar and intersect along the
axis of the cuvette opening and are perpendicular to the cuvette
opening, and wherein the axes of the radiation-receiving
apertures are fixed at predetermined angles with respect to
the axes of (1) the incident radiation beam aperture and (2)
the tubular cuvette opening, and attached thereto, and
a receptor-conveyor means comprising a tubular fiber-
o~tic bundle terminated at one end by a substantially coaxial
tubular detector cone, wherein the detector cone is (1) inserted
in sealing engagement with one of said radiation beam receiving
apertures, and (2) has a substantially coaxial orifice at the
end of the detector cone remote from the bundle, with dimensions
such that electromagnetic radiation is transmitted at about a 6
admittance angle from the orifice to said fiber-optic bundle,
and being at the intersection of the receiving aperture and
the cuvette opening, and
(e~ means with the non-terminated end of the fiber-
optic bundle and with the cuvette-positioning mealls for
-~5-
cletect inc3 an-~ mc~asuL^incl sul)stantially only a portion of the
transmitt~d beam.
-- 4
