Note: Descriptions are shown in the official language in which they were submitted.
RD-6640
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~y invention relates to a method and apparatus
for detecting an immunological reaction on a solid surface
wi-th the unaided eye and obtaining a durahle record thereof,
and in particular, for detecting the reaction as the
result of a double diffusion in a layer of gel on a
metallized solid surface and without requiring a staining
process.
This application is related to my Canadian
applications S.N. 221,149 filed March 3, 1975 entitled
"Method and Apparatus for De-tecting Immunologically
Reactive Biological Particles", S.N. 221,150 filed
March 3, 1975 entitled "Method and Apparatus for
Determination oE Concentration of Immunologically
Reactive Biological Particles", S.N. 221,153 filed
March 3, 1975 entitled "Method and Apparatus for
Quantitative Test for Immunologically Reactive Biological
Particles", S.N. 172,639 filed May 29, 1973 entitled
"Method and Apparatus for Detection and Purification of
. Proteins and Antibodies", S.N. 204,262 filed July 8, 1974
entitled "Improved Method and Apparatus for Detec-tion
and Purification of Proteins and Antibodies", and
S.N. 218,333 filed January 21, 1975 entitled "Improved
Substrate for Immunological Tests and Method of
Fabrication Thereof", all assigned as herein.
Immunological reactions are highly specific
biochemical reactions in whcih a first immunologically
reactive biological particle (generally a pro-tein)
known as the antigen, combines (links) with a second
protein specific to the antigen, and known
as the antibody, to form an immunologi.cally complexed
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protein. Immunological reactions taking place within a
biological system, such as an animal or human being, are
vital in combatting disease. In a biological system, the entry
of a Eoreign protein, i.e., the antigen, causes the biological
system to produce the specific antibody proteins to the antigen
in a process not fully understood at this ~ime. The antibody
protein molecules have available chemical combining or binding i
sites which complement those of the antigen molecule so that
the antigen and antibody link or bond to form an
immunologically complexed protein.
Most antigens are proteins or contain proteins as an
essential part, whereas all antibodies are proteins. Proteins
are large molecules of high molecular weight, i.e., are
polymers consisting of chains of variable numbers of amino
acids. The above-cited Canadian applications disclose that
an arbitrary protein will adhere to a substrate in a
monomolecular layer only, and that no other arbitrary protein
will adhere to the protein layer. On the other hand, the
specifically reacting protein to the first protein adsorbed
; 20 onto the substrate will immunologically bond thereto. In
accordance with the teachings of those applications, this
discovery is exploited to provide medical diagnostic apparatus
in which a slide havin~ a monomolecular layer of one protein
adsorbed thereon is used to test suspected solutions for the
presence of the specifically reacting protein thereto. If
the specifically reacting protein is present in the solution,
, the slide after exposure to the solution has a bimolecular
protein layer thereon. If the specifically reacting
protein be absent from the solution, the slide after
exposure to the solution has only the original monomolecular
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layer thereon. Optical, electrical and chemical means for
distinguishing between bimolecular and monomolecular
biological particle layers are taught in the related canadian
applications and have different degrees o sensitivity and
economy.
Because antibodies are produced by biological systems
in response to invasions thereof by foreign proteins, the
detection of antibodies in a biological system is o medical
diagnostic valuP in determining the antigens to which the system
has been exposed. A typical example of diagnostic detection
of antibodies is the detection of antibodies to syphilis or
gonorrhea in human serum. Conversely, the detection of
certain antigen~ in a biological system also has medical
diagnostic value; examples of diagnostic detection of antigens
include detection of HCG-protein molecules in urine as a
test for pregnancy, and detection of hepatitis-associated~
antigen (HAA) molecules in the blood of prospective blood
donors.
In order to perform such diagnostic tests, the
appropriate protein of the immunologically reacting pair
must be obtained. The only known source of an antibody
protein is a living biological system. More particularly,
only vertebrates are known at this time to exhibit
immunological reactions to the introduction of ~ foreign
protein. For example, many antibodi~s are found in ~he
.; . ,
blood serum of animals and human beings which have been
exposed to the corresponding antigens. Many antigens, however,
may be controllably produced in laboratory cultures. However,
~ome antigens, for example, hepatitis-associated-antigens, are
at present, like antibodies, only obtalnable from the
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higher living biological systems.
I~ is known in the immunological art that antibody
molecules function as antigens when introduced into the
system of a vertebrate to whom they are foreign proteins.
Accordlngly, specifically reacting antibodies to a given
antibody may be readily produced in such vertebrate system.
Double diffusion immunological experimen~s have been
carried out in the prior art in gel in which specimens
contalning antigens and their antibodies are applied to
different wells in the gel and diffuse-toward each other to form
a complexed protein precipitate line in the gel. This -
prior art technique is generally known as the Ouchterlony
technique. However, the precipitate formed in the gel is
only a temporary record of the immunologic reaction since
the gel soon deteriorates through normal drying out (desiccates)
due to high water content. A further disadvantage of the gel
being used as the immunologic reaction medium is that undesired
bacteria growth readily develops in the gel during the
time it is stored in a suitable environment which would
otherwise prevent the deteriorartion of the gel. Finally, the
sensitivity of the immunological experiments in the gel
is relatively low and the precipitate line is often not
visible to the unaided eye until the gel is suitably stained
with a protein material such as Amido Black as described
2~5 in the book "Methods in Immunology and Immunochemistry",
Vol. III, edited by C.A. Williams and M.V. Chase,
Academic Press, pages 153 and 169. This staining ~rocess
adds another step in the method for detecting such immunologic
reaction. Also, as noted on page 151 in the above-identified
book. "It is important to have the bottoms of the wells
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completely sealed with agar to prevent leakage of antigen
or antibody between the gel and surface of the plate".
Finally, although the substrates (slides) described
in my hereinabove-referenced patent applications are
~atisfactory in their performance for detecting a bimolecular
layer of immunologically reactive biological particles,
such substrates are not, by themselves, well adapted for the
double diffusion technique described hereinabove. This result
also occurs with another type of metallized slide known in the
prior art, the anodized tantalum slide described in the
articles "Interactions Among Human Blood Proteins at Interfaces",
authors L. Vroman et al, Federation Proceedings, Vol. 30, No. 5
(September-October, 1971~ pages 1494-1502 and "Three Simple
Ways to Detect Antibody-Antigen Complex on Flat Sur:Eaces",
authors A.L. Adams et al, Journal of Immunological Methods 3
(1973) pages 227-232, which is, however, less sensitive than my
indium-gold alloy indium oxide slide disclosed and claimed
in my above-referenced Canadian application S.N. ~/8~33J
~, especially in the detection of hepatitis. Anokher article related
to prior art metallized slides is "Immunologic and Enzymatic
Reactions Carried Out at a Solid-Liquid Interface", by
Alexandre Rothen, Ph~siological Chemistry and Physics 5,
(1973~ pages 243-258.
Therefore, a principal object of my invention is to
j 25 provide an improved method and apparatus for the double
d~ffu~ion detection of immunological reactions utilizing a
gel as the diffusing medium.
Another object of my invention is to provide a
simple method and apparatus for det~cting immunologically
reactive biological particles by a double diffusion
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process without the need for staining the gel in which
specimens containing the particles diffuse.
A further object of my invention is to provide a
simple method and apparatus for producing a durable record
of the precipitate line formed by immunological reaction between
the particles which is visible to the unaicled eye with good
contrast.
Briefly, and in accordance with ~he objects of ~y
invention, I provide a method and apparatus for detecting second
immunologically reactive biological particles in a ~est -
solution by direct visual observation of a complexed protein
precipltate line formed on a metallized solid surface as the
result of an immunological reaction. The metallized solid
surface is initially covered with a very thin layer of gel
and two or more wells are formed completely through the gel.
Then, a specimen of a first solution containing first
immunologically reactive biological particles is deposited in
a first of the wells, and a specimen of a test solution
suspected of containing second biological particles which are
specific to the first particles is deposited in a second well
spaced from the first, and the two specimens are allowed to
diffuse. During the diffusion process, the biological
particles permeate the gel and a complexed protein precipitate
line orms at the intersection of the diffusing first and
~econd biological particles. The precipitate line is
visible with good contrast to the unaided eye without requiring
'( ~ the use of a staining material and forms a durable record of the
`, detected reaction. The apparatus of the metallized solid
i surface and gel layer is maintained in a moi~st chamber during
the diffusion p~oces~ and obtains the detection of the second
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~049401 RD-6640
biological particles with a sensitivity substantially
better than that which can be obtained with the conventional
double diffusion in gel technique. The metallized solid
surface can be that of a metallized slide or the surface
of a metalliæed glass or plastic dish as typical examples.
The features of my invention which I desire to protect
herein are pointed out with particularity in the appended
claims. The invention itself, however, both as to its
organization and method of operation together with
further objects and advantages thereof, may best be
understood by reference to the following description taken
in connection with the accompanying drawings wherein:
FIGURE la is a plan view of a metallized dish-gel
layer apparatus in accordance with my invention prior to
depositing specimens into the wells formed through the gel
layer;
FIGURE lb is an elevation view, in section, of the
apparatus illustrated in FIGURE la taken along line lb-lb;
FIGURE 2a is a plan view of the apparatus of FIGURE la
after diffusion of the specimens and ormation of
precipitate lines;
FIGURE 2b is an elevation view, in section, of the
apparatus illustrated in FIGURE 2a taken along line 2b-2b;
FIGURE 3a is a plan view of a metallized substrate-gel
layer apparatus in accordance with my invention prior to
- depositing the specimen6 into the wells formed through the gel
layer;
FIGU~E 3b is an elevation view, in section, of thè
apparatus illustrated in FIGURE 3a taken along line 3b-3b;
FIGURE 4a is a plan view of the apparatus of FIGURE 3a
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after diffusion of the specimens and formation of the
precipitate line; and
FIGURE 4b is an elevation view, in section, of the
apparatus illustrated in FIGURE 4a taken aLong line 4b-4b.
Referring now to FIGURES la and lb, there are
shown the apparatus, in accordance with my invention,
for detecting immunologically reactive biological particles
as a result of an immunological reaction thereof occurring on
a metallized solid surface. In particular, this first
embodiment of my apparatus consists of a suitable container
10 such as a small glass, metal or plastic dish or tray or
other type container fabricated of a suitable solid material
and having a generally vertically extending lip portion for
containing a liquid medium within the container. Metal
particles are evaporated on the inner bottom surface of
container 10 to form a non-continuous layer 11 of such metal
particles to thereby provide a metallized surface on the
; inside of container member 10. A typical example of the
metallization of container 10 is a non-continuous layer of
indium globules of average thickness in the order of 2000 to
4000 Angstrom. Alternatively, a continuous film of a single
metal, alloy of two metals, or a single metal or alloy of
two metals with an oxide film of one of such metals may
aiso be utilized for the metallization of the surface of
container 10. The criteria is that a solid surface of some type
member on which a very thin layer of gel can be formed is
metallized for purposes of improving the sensitivity of
` my apparatus in the detection of an immunological reaction
~'' precipitate line which is subsequently formed on the
metallized surrace so that the precipitate line is capable of
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being observed with the unaided eye with good contrast and
without the need for staining the gel as is often done in
the prior art double diffusion in gel imm~mological test
apparatus. As an example of the alloy metallization, a
convenient alloy is that of indium and gold. A typical
example of single metal and oxide metallization is indium with
an indium oxide film of a few hundred Angstrom thickness, or
nickel-nickel oxide. Finally, a typical rnetallizatîon
of an alloy of two met:als and oxide film of one of -the metals
is a gold-indium alloy and indium oxide film wherein such
metallization is developed from a continuous or non-continuous
layer of indium particles as described ardclaimed in my
. ~, .
f'~`, above-identified Canadian patent application S.N. ~/~J333
The metallized container 10 is then placed on a
sultable support and a smalL quantity of gel is poured into
container 10 sufficient for covering the metallized
surface thereof to a depth less than one millimeter. The
most common gels suitable for the immunologic reaction
tests are agar and agarose. The gel solution that is poured
into container 10 may be a salt solution, distilled water solution
or buffered solution thereof depending upon the biological
particle solutions to be utilized in the test as described on
pages 147 and 148 of the above-referenced book "Methods in
Immunology and Immunochemistry". After the gel has solidified,
a~plurality of closely spaced wells 13a-e are formed through
the thin layer 12 of gel in any convenient manner such as
` described on page 149 of the above-referenced book. The major
distinctions between my invention and the prior art diffusion
in gel apparatus as descrihed in the above-referenced book are:
(1) My apparatus requires a metallized solid surface since
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the visible precipitate line resulting from an immunological
reaction of biological particles is formed on the metallized
surface (although the precipitate line i.s also formed in
the gel). In the prior art apparatus no metallized solid
surface is required although a means for merely supporting the
gel layer is used.
(2) My gel layer is substantially thinner than the prior
art gel layer which is of at least 1 mm thickness and is
described in the above-referenced book as being in the range
of 1-3 mm. This significant change in thickness results
from the fact that in my apparatus the gel is utilized merely
as a diffusion medium, i.e., for purposes of holding moisture
in an immobile state so that specimens of immunologically
reactive biological particles can diffuse along the metallized-
solld-surface to form reproducible precipitatelines thereon.
In the prior art apparatus the precipitate line is formed
::
in the gel and therefore requires a thick~r layer of gel
in order to form a sufficiently thick precipitate line to make
it visible.
(3) As a result of (2) the precipitate line formed with my
invention becomes a durable, and can be a permanent, record of
the immunologic reaction, and requires no staining to be
visible. In the prior art apparatus the gel often requires
I staining in order for the precipitate line to be visible (with
much less contrast than in my invention) to the unaided eye.
(4) In my invention the wells are formed comple~ely through
the gel layer. In the prlor art apparatus as noted on page
151 in the abovP-referenced book, the bottoms of the wells
must be sealed from the surface of a plate on which the gel
is supported. Although my apparatus operates satisfactorily
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with the bottom of the wells also being sealed from the
metallized surface of container 10, such sealing of the
bottom of the wells is not necessary, and it is preferred to
form the wells completely through the layer of gel as indicated
in all of my figures. This significant distinction between the
wells results from the fact that ~he visib]Le precipitate line
in my apparatus is formed on a metallized solid surface whereas
in the prior art it is formed within the gel itself.
The wells 13a-e formed through gel layer 12 are
generally circular in cross section and are generally of equal
diameter as small as one millimeter and as large as several
millimeters. My apparatus just prior to the specimens of
immunologically reactive biological particles being deposited
into the wells is as shown in FIGURES la and lb.
The gel covered metallized solid surface assembly is
then placed in a moist chamber and a specimen of a first
solution ~ontaining first immunologically reactive biological
particles is deposited in a first well, for example, centrally
located well 13a. Each of the specimens described here:in
may consist of one or more drops of the corresponding solution.
Immediately after the first specimen is deposited in well 13a,
or at the same tlme, a specimen of a first test solution suspected
of containing second immunologically reactive biological particles
which are specific to the first particles is deposited in well
13c and the two specimens are allowed to diffuse in the gel.
The ~irst and test solutions generally also contain other
(nonspecific) biological particles, a typical example being a first
solution of rabbit anti-serum and a test solution of human serum~
~ ~During the diffusion of the two specimens in the gel, the first
,,, :
l 30 and other ~nonspecific) biological particles in the first specimen
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permeate the gel and are adsorbed onto the metallized solid
surface to form a monomolecular layer 13a' thereof as
illustrated in FIGURES 2a and 2b. In like manner, presence of
the second particles in the first test specimen results in the
second and other (nonspecific) biological particles permeating
the gel and being adsorbed onto the metallized solid surface
to form a monomolecular layer 13c' thereof. Along the
region of intersection of the two diE~usiny, specimens
there is formed a complexed protein precipitate line
14 which is several layers thick and results from an
i~munologic reaction of the first and second particles. The
specimens difuse in the gel radially outward from the wells
to form circular patterns such that precipitate line 14 is a
straight or curved line depending on the types of particles
and concentrations thereof. The time for completion of the
diffusion and formation of the precipitate line is a Eunction
of the types of first and second particles involved, the
concentrations of each particle in its respective solution, the
temperature and the spacing of the wells in the gel. Thus,
a close spacing of the wells results in the difEusing particles
intersecting more rapidly and thereby forming the
precipitate line 14 more rapidly than if the wells were
spaced further apart. The wells may be spaced apart as little
as several millimeters. The time for diffusion of the
specimens in the gel and formation of the precipitate line is
,
generally several hours, although the process can be
' speeded up to several mintures if electrophoresis is employed.
Since the moisture is held immobile in the gel, a controlled
~' diffusion of the specimen occurs in the gel to thereby obtain
reproducible results.
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After formation of precipitate line 14 on the metallized
surface of solid member 10, the layer 12 of gel is peeled
or otherwise removed from the metallized solid surface.
The metallized surface with the precipitatle line 14 adhered
thereon is then rinse~, typically with distilled water and dried
preferably by blowing air at room temperature across the
metallized solid surface. The metallized solid surface is
then visually examined by direct visual observation in that
the unaided eye is employed -to ~serve the light reflected
off or transmitted through the metallized surface. The
indium particle slide is viewed by transmitted light whereas
the indium-g~ld alloy, indium oxide slide is viewed by reflected
light. The color of the precipitate depends primarily on the
color of the metallized surface.
15The complexed protein precipitate line 14 is visible with
good contrast to the unaided eye. A smaller amount of the
biological particles is needed ~o obtain a visually detected
precipitate line on the metallized solid surface as compared
to the amount of particles needed to form such precipitate line
in the gel in the prior art. Thus, my invention results in the
detection of immunologic reactions and the biological particles
involved therein to a sensitivity which is considerably better
than that obtained with conventional double diffusion
: in gel techniques. Finally, no staining of the precipitate is
required, as distinguished from the prior art double diffusion
techniques in order to visually detect the precipitate line,
: .
and the contrast is also si~nificantly better than that
obtained with the prior art techniques.
In the detection method described hereinabove, it was
assum~d that the first solution was a known solution
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containing the first biological particles. Alternatively,
both the first and second solutions may be test solutions
suspected of containing the first and second particles in
which case formation of the precipitate line would indicate that
such particles were, indeed, contained within the respective
solutions whereas absence of the precipitatle line would
merely indicate that one or both of the solutions did not
contain their respective particles. In the case of the known
solution containing the first particles, such first particles -
may be produced in laboratory cultures or obtained from the higher
living biological systems as described hereinabove, and are
commercially available in highly purified form, and if not
available commercially, may be purified chemically. A typical
solution of the first biological particles may be a salt
solution of water or other liquid appropriate to, and not reactive
with, the first biological particles, or a human serum sample.
The biological particles referred to hereinabove as
first and second biological particles may be antigens,
antibodies, viruses, bacteria, hormones, enzymes or other
biological particles which can be readily grown or otherwise
isolated and collected or are present in human serum or
other solution being tested. A typical example of particular
biological particles which are detected by the method and in
the apparatus described hereinabove is hepatitis B antigen
~HB~)as the first biological particles and antibodies to
hepatitis (HBAb) as the second biological particles.
In many cases, the specimen of first particles will be
I
a specimen containing the particular antigens such as HBAg. ~-
In such case, the test soLution would bea drop of human serum
taken from a patient suspected of having had hepatitis
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10494Ql
B, and in a direct test therefore, the presence of antibodies
(HBAb) would be detected by direct visual observance
of precipitate line 14. Alternatively, the particles in
the first specimen can be antibodies to a particular disease,
and in a direct test, the presence of antigens to such
antibodies in the serum sample would be determined by my
detection test.
An indirect or inhibition test for the detection of
particular immunologically reactive biological particles
; 10 may also be conducted with my apparatus. The principle
of the inhibition test is that the first particles, if presen~
in sufficient quantity, will neutralize free second particles
in solution. Thus, in the inhibition test, HBAg particles,
if present in sufficient quantity, will neutralize free
antibodies to hepatitis B in solution. This reaction will
prevent the antibodies from forming observable complexes
with HBAg when the test specimen is deposited in well 13b in
gel layer 12.
The inhibition test for an antigen, and specifically
HBAg is accomplished as follows: A specimen of known solution
of HBAg is deposited in well 13a of gel layer 12 and the
HBAg and other particles present in the solution are
; adsorbed as a monomolecular layer 13a on the metallized surface
of solid member 10 as in the direct test described hereinabove.
The test solution is prepared by adding a human serum sample
to be tested to a solution of HBAb in a vial or other suitable
container. The vial is then stored for a time interval
.
sufficient for the HBAb to complex with HBAg in the human '
serum sample, if the antLgen is present therein. The vial
Ls preferably agitated to increase the rate of complexing.
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Finally, a specimen of the test solution is deposited in
well 13c of gel layer 12, and after a suitable period of
time or the diffusion of the specimens, gel layer 12 is
peeled from solid member 10 and the metallized surface of
5 member 10 is visually examined. The results of the inhibition ~-
test are the opposite of the direct test, that is, presence
of HBAg in the human serum sample produces no precipitate
line 14 whereas presence oi such precipitate line indicates
absence of HBAg in the human serum sample.
The inhibition test for the detection of HBAb is performed
similarly to the inhibition test for HBAg with the obvious
substitution of the antigen for antibody and antibody for
antigen in each of the steps.
In the above heptatitis tests, the HBAb may be obtained
from human serum of a patient known to have had hepatitis
B, or it may be developed in a goat, rabbit or other suitable
animal by injection therof with the HBAg, waiting a suitable
incubation period such as two weeks, and then drawing blood
contalning the ~pecific antibody from the animal and separating
the antibody from the remaining blood particles.
In the case where the first solution is known to contain
the first biological particles, the specimen of such first
301ution is deposited in centrally located well 13a, and specimens
of various test solutions suspected of containing the second
biological particles are deposited into the surrounding wells
, 13b, c, d, and e. In each case of sufficient concentration of
the second particles in the corresponding test solution, a
straight or curved precipitate line is formed at the intersection
of the outwardly diffusin~ irst and second immunologically
reactive - biological particles, and is a detection
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test for the presence of the second biological particles
in the test solutions. Thus, as depicted in FIGURES 2a and
2b, specimens of three diferent test solutions deposited into
wells 13b, c and e contain the second particles due to the
formation o the illustrated precipitate lines whereas the
specimen of a fourth test solution deposlted into well 13d either
did not contain the second particles, or contained it in
too dilute a quantity to be detected. In ~he case where the
first solution i3 known to containt~e first biological
particles, and one of the other solutions contains a known
concentration of the second particles, a specimen of the first
~ solution i8 deposited into centrally located well 13a and a
; 9pecimen of the "standard solution" (known concentration of
second par~icle~) is deposited into one of the surrounding wells,
say well 13c. The relative position of the precipitate line
14 ormed on the metallized solid surface between wells 13a
and 13c is then the standard against which the relative
positions of any other precipitate lines, formed as the result
of specimens of test solutions suspected of containing the
second biological particles being deposited in the other
surrounding wells 13b, 13d, 13e, are compared in order to
determine the concentration of the second particles in ~ch
test solutions. Thus, since the position of precipitate line
lS between 13a and 13e wells is closer to well 13a than is
the "~tandard" precipitate line 14, this indicates that the
! .
concentration of the second particles in the specimen depo~ited
lnto well 13e is greater than the "standard" concentration.
, In ~his latter (concentration~ test, the surrounding w~lIs
ar~ equi-distant from central welI 13a.
Referrin~ now to FI~U~ES 3a and 3b, there is shown a second
embodiment of my apparatus wherein the metallized so:Lid
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surface member is now a metallized substrate or slide of
the type described in my above-referenced Canadian application
S.N. ~/8;3~3 In particular, substrate 30 has a substantially
flat top surface and is fabricated of a suitable material
which may be a metal, glass, plastic, or similar material.
Substrate 30 is preferably in the form of a glass slide
such as a conventional microscope cover glass that is readily
commercially available. The top flat surface of substrate 30
is metallized in accordance with the teachings disclosed in
my above-identified patent applications. As examples of
such teachings, the metallization may consist of (l~ a non-
continuous layer, i.e., metal particles or globules with indium
being a typical metal, or (2) a first layer of the indium
globules overlayed with a thin gold film, or (3) a layer of
; 15 the indium globules (or a constant thickness continuous layer
of indium) overlayed with a thin film of gold which is alloyed
with the indium and a thin oxide film of the indium,
or (4) a metal such as nickel and o~;ide film t~lereof~
The indium particle metallization is often the preferred
embodiment for generally equal size particles whereas the
indium-gold alloy and indium oxide coated substrate is often
the preferred embodiment for very differently sized particles
such as when testing for hepatitis. Following the teaching
of the above-referenced patent applications, the non~continuous
layer of indium particle metallization requires use of a light- -
transmissive substrate material such as glass or plastic, and
the indlum particles evaporated on the substrate surface have
diameters on the order of 1000 Angstrom althOugh the precise
size of the particles is not critical as long as they
have diameters equal to a large fraction of a wavelength of
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visible light. The color of the indium particle metallization
is a light brown, In the case of the indi~lm-gold alloy, indium
oxide metallization, the thickness of the indium is approximately
twice the thickness of the gold when initially deposited (indium
thickness is approximately 2000 A, gold is approximately
1000 A) and the indium oxide film is several hundred Angstrom
to obtain a bronze color of such film. As noted in my patent
application S.N. ~/~)333 the degree of oxidation of the
indi~n metal determines the color of the oxidized film so that
various degrees of oxidation produce different colored slides
having different sensitivities for different thicknesses of the
layers of the biological particles.
In the case of the metallized coating 31 on the top surface
of substr~Le 30 being formed of glo~ules alone or globules of a
first metal such as indium, a film of a second metal such as gold
and the oxide film of indium, the top surface of such
metallized coating is slightly irregular. Alternatively, such
metallized coating when formed with a continuous, constant
thickness layer of the indium, film of gold and the indium
oxide, has a top surface that is substantially flat. Either
type of metallized substrate 30 may be utilized in this
second embodiment of my invention. Substrate 3~ may be as
small as a half inch square. Further details of the substrate
metallization and fabrication thereof are disclosed in my
above-referenced patent applications~
My apparatus employing metallized substrate 30 is
` fabricated in the same manner as my first embodiment. Thus,
a thin layer (less than 1 mm) of gel 12 is formed on the
metallized surface of the substrate and two or more wells 13a,
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13b are formed, preferably completely through the gel layer.
The apparatus is then utilized in the same manner as my
first embodiment in that a specimen con~aining first
immunologically reactive biological particLes (and other non-
specific particles) is deposited into well 13a and a testspecimen suspected of containing second (and other nonspecific)
immunologically reactive biological particles specific to the
first particles is deposited into well 13b. The apparatus is
then maintained in a mdst chamber f~atime interval sufficient
for the two specimens to diffuse through the gel so that
a monolayer 13a' of the first and other nonspecific particles
is adsorbed onto the metallized surface 31 of the
substrate and, in a like manner it is evident tha~
a monolayer 13b' of any second and other nonspecific particles
is also adsorbed onto the metallized surface and at the
intersection of the two diffusing specimens a complexed protein
precipitate line 14 is formed which, after removal of the
gel layer, is clearly visible to the unaided eye by observing
the light reflected off or transmitted through the metallized
surface 31. After removal o the gel layer, the precipitate
line 14 remains adhered on the metallized surface and again
forms a durable record of the immunological reaction between
the first and second immunologically reactive biological particles.
The complexed protein precipitate line 14 is again several
layers thick and is a straight or curved line. After the gel
.
is peeled from the metallized surface of the substrate, such
surface is again rinsed with distilled water and dried as in
the case of my first embodiment. In the case of the indium
particle metallization the precipitate is a much darker shade
of brown as compared to the light brown background. In the
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case of a bronze color indium oxide film as the outermost
surface of the metallization layer 31, the precipitate is
a purplish line which is clearly dis~inguished from the bronze
color background.
In each of the two embodiments of my inven~ion described
hereinabove, it is noted that no staining of the gel is required
in order to make visible the precipitate line 14. Also,
the greater sensitivity of my apparatus, in that the plurality
of layers of biological particles which form the precipitate
line 14 are more easily detectable, makes my testing method
more sensitive. That is, the precipitate line is more readily
visible (or the same amount of biological particles) on the
metallized substrate than in the gel, and therefore a smaller
amount of such particles can be detected with my apparatus.
Since my apparatus i9 more sensitive than the apparatus
used in the Ouchterlony ~echnique, a lesser amount of the
first biological particles and smaller specimens of the
test solution need be deposited into the wells in the gel
layer in my apparatus and therefore an economy in the case of
such particles is realized which may be particularly significant
; in the case where the first particles are obtained from a costly
laboratory process, and, or, where the physical condltion of
the patient is so poor that the taking of a larger specimen
I from him may be detrimental to his condition. Finally, the~ 25 precipitate line formed on my metallized solid surface forms
a durable and even permanent record of the immunological
reaction which is not true in the Ouchterlony ~echnique unless
the non~p`ecific particles are first removed in a water bath
,, requiring approximately 24 hours, then staining the gel,
another washing process to remove the stain in the gel material,!
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but not from the precipitate line, and finally drying the
gel in a slow delicate process.
From the foregoing description, it can be appreciated
that my invention makes available an improved double diffusion
method and apparatus for detecting immunologically reactive
biological particles in a test solution by direct visual
observation of the metallized surface of a solid member on which
a complexed protein precipitate is formed as a result of an
immunological reaction between first biological particles and
the particular biological particles being investigated and
which are specific to the first particles. My method and
apparatus are very simple in that only a thin layer of gel
with suitable wells formed therethrough is required on the
metallized solid member for diffusion of the specimens and the
unique and highly sensitive properties of the metallized solid
member, and in particular the metallized substrate, thereby
avoids the need for staining the gel or substrate in order
to detect the precipitate line by direct visual observation.
- As a result, I have provided a simple method wherein the
previously described metallized slide described in the
hereinabove-referenced patent applications can now be adapted
for use with a double diffusion of specimens in a thin gel
layer for detecting the biological particles. Since the
metallized slides, in particular, can be fabricated repetitively
25~ with identical characteristics, the results of the detection
of the biological particles in accordance with my present
, invention are very consistent and can serve many useful
I purposes, especially in the medical diagnostic field in the
analysis of human serum, for example, for the detection of
various antibodies and antigens therein. Since the visual
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contrast between the precipitate line and monomolecular layer
of biological particles is very distinct when utilizing my
metallized solid surface, tha detection is accomplished by
direct observation with the unaided eye and therefore does not
require elaborate test equipment and obtains the precipitate
llne in durable form.
Having described my invention with reference to two
specific embodiments, it is believed obvious that modification
and variation of my in~rention is possible in the light of the
above teachings.- Thus, the shape and size of the substrate or
solid member and thin layer of gel may be varied and virtually
any pair of immunologically reactive biological particles which
will immunologically react with each other can be detected
with my apparatus. Further, my metallized substrate, if
suficiently large, can be employed to detect the presence
of the second biological particles in more ~han one test
solution by depositing the specimens thereof in other
wells formed through the thin gel layer surrounding a central
well in which the specimen known to contain the first
particles is deposited as in the first embodiment illustrated
in FIGURES la and 2a. The presence of the second particles
in each test solution is then detected by observing the
formation on the metallized substrate of precipitate lines
formed by the first particles in the central diffusion
immunologically reacting with the second particles in the
respective intersecting surrounding diffusions. A measure of
the concentration of the second particles in the test solutions
can also be obtained in this manner if one of the second
solutions (i.e., a standard solution) contains a known
concentration of the second particles. A good approximation
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of the concentration can be estimated by comparing the
relative position of each precipitate line (rel.ative to the
distance between the wells in which the first particle
specimen and each test solution specimen is deposited) to that
5 of the relative position of the precipitate line formed by the
standard solution specimen. Further, metallizations other than
the indium and indium-gold alloy, indium oxide may be found
to obtain better contrast of the precipitate line on the
metallized surface for some specific biological particles.
10 Also the irregular surface embodiment of my metallized slide
could obviously also be fabricated by starting with an
irregular surfaced substrate and evaporating constant thickness
layers of a metal such as indium thereon. Finally, it should
be evident that my apparatus may also be utilized for
15 determining the concentration of the second biological particles
by first adsorbing a monomolecular layer of the first
biological particles along substantially the entire metallized
surface of the solid member 10 or substrate 30, and then
forming the thin gel layer 12 on top of the first particle
20 layer in complete contact therewith. The specimen of the
test solution is then deposited into a well formed in the gel
layer, and diffusion of such specimen results in an immunologic
reaction whereby a monomolecular layer of only the second
biological particles is formed on top o-f the first particle
25 layer in the shape of a small circular spot if the test solution
contains such second particles. The diameter of the second
layer spot, which is visible with good contrast to the
unaided eye as a purplish spot in the case of a bronze color
; metallized slide, is related to the concentration of the ~
; 30 second particles in the test solution. Thus, in the figures, J
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and especially in the case of the metallized substrate 30,
coating 31 on substrate 30 includes both the metallization and
monomolecular layer of first biological particles. A specimen
of a first test solution is then deposited into well 13a
and a specimen of a second test solution, or a standard solution
(i.e., solution of known concentration of the second particl~s)
is deposited into well 13b, and 13a', 13b' are the circular
spot monomolecular layers of the second particles. Thus, my
apparatus as fabricated may also consist of a metallizecl substrate
10 with a monomolecular layer of first immunologically reactive -
biological particles that adhere therein and a thin layer
of gel. After formation of the second layer spot(s) the gel layer
is removed, the metallized slide is rinsed, dried, and then
visually examined with the unaided eye and the diameter(s) (or
areas) of the second layer spot(s) is measured (and compared to
a standard) for determining the second particle concentration.
It is, therefore, to be understood that changes may be made
in the particular embodiment of my invention as described
which are within the full intended scope of the invention
88 defined by the following claim8.
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