Note: Descriptions are shown in the official language in which they were submitted.
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IMPROVED SUBSTRATE FOR IMMUNOLOGICAL TESTS
AND METHOD OF FABRICATION THEREOF
My invention relates to an improved substrate utilized for
detecting an immunological reaction between a first biological
particle and a second biological particle specific to the first,
and the method of fabrication thereof, and in particular, to
a substrate which provides improved contrast, visible to
the unaided eye, between mono and biomolecular layers of the
immunologically reactive biological particles on the surface
of the substrate.
This application is related to my Canadian applications
S.N. 172,639 entitled "Method and Apparatus for Detection and
Purification of Proteins and Antibodies", filed May 29, 1973
and S.N. 204,262 entitled "Improved Method and Apparatus for
Detection and Purification of Proteins and Antibodies" filed
July 8, 1974 and assigned as herein. Other
publications related to the present invention are "Optical
Measurement of the Thickness of a Film Adsorbed From a Solution".
authors I. Langmur et al, Journal of the American Chemical ~-
Society, volume 59 (July-December-l937) page 1406, "Immunologic and
Enzymatic Reactions Carried Out at a Solid-Liquid Interface" by
A. Rothen, Physiological Chemistry and Physics, Volume 5, (1973)
Pages 243-258 and "Interactions Among Human Blood Proteins at
Interfaces", L. Vroman et al, Federation Proceedings, volume 30,
No. 5 (September-October, 1971) pages 1494-1502.
Immunological reactions are highly specific biochemical
reactions in which a first immunologically reactive biological
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particle (generally a protein) known as the antigen combines
(links) with a second protein specific to the antigen, and
known as the antibody, to form an immunologically complexed
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 foreign 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 time. The antibody
protein molecules have ~vailable chemical combining or binding
sites which complement those of the antigen molecule so that the
antigen and antibody chemically link or bond to form an --
immunologically complexed protein.
Most antigens are proteins or contain proteins as am ~ ;~
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 co-pending 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
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 having 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
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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
protein thereon. Optical, electrical and chemical means for
distinguishing between bimolecular and monomolecular
biological particle layers are taught in the related co-pending
applications and have different degrees of sensitivity and
economy.
Because antibodies are produced by biological systems ~-~
in response to invasions thereof by foreign proteins, the
detection of antibod~es in a biological system is of medical
diagnostic value in determining the antigens ~o 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 antigens 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 blooddonors.
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 a foreign
protein. For example, many antibodies are found in the
blood serum of animals and human beings which have been `~
exposed to the corresponding antigens. Many antigens, ~
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however, may be controllably produced in laboratory
cultures. How~ever, some antigenQ, for example, hepatitis-
associated-antigens, are at present, like antibodies~ only
obtainable from the higher living biological systems.
S It 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.
Accordingly, specifically reacting antibodies to a given --
antibody may be readily produced in such vertebrate system.
Although the substrates (slides) described in the
hereinabove referen~ed patent applications are satisfactory
in their performance with many of the immunologically reactive
biological particles, for certain diseases such as hepatitis,
my previous slides have not provided the desired high degree
of contrast between single and double layers of the hepatitis
(antigen and antibody) molecules.
Therefore, a principal object of my invention is to
provide a simple and improved device for detecting immunological
reactions occurring at a solid surface by direct visual
observation.
Another object of my invention is to provide an
improved substrate which obtains improved contrast between
single and double layers of immunologically reactive ~i
biological particles.
A further object of my invention is to provide
a relatively si~ple method for fabricating the improved `
substrate.
Briefly, and in accordance with the objects of my ;
invention, I fabricate my improved slide by evaporating
small globules of indium on the surface of`a suitable substrate
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which may conveniently be a glass slide. Alternatively, the
indium may be evaporated onto the ~urface of the slide as a
thick film of constant thickness. Subsequently, a thin film
of gold is evaporated over the indium and some alloying of the
indium and gold occurs in this step. Finally, the coated
slide is heated in air sufficiently to complete the alloying
and to obtain some oxidation of the indium and thereby form
an indium oxide film on the outer surface of the substrate
coating. The degree of oxidation of the indium determines the
color of such oxide film which is significantly differently
sensitive to single and double layers of particular
immunologically reactive biological particles being detected
such that the contrast is readily visible to the unaided eye.
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 drawing
wherein:
FIGURE lA is an elevation view of an intermediate
structure of the preferred embodiment of my improved substrate;
FIGURE lB i8 an elevation view of the preferred
substrate after final fabrication; and
FIGURE 2 is an elevation view of a second embodiment
of my improved substrate.
Referring now to FIGURE la, there is shown an elevation
view of a substrate 10 having a substantially flat top surface
and being fabricated of a suitable material which may be a metal,
glass, plastic or similar material. Substrate 10 is preferably
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in the form of a glass slide such as a conventional microscope
cover glass 25 millimeters square and 10 mils thick, the glass
slide being preferred primarily due to its low cost and
ready commercial availability. After selection of the substrate,
the top surface thereof is coated with a plurality of metal
globules 11 by evaporating a metal, for example, indium, onto the
substrate. Typically, the indium is evaporated slowly from
a tantalum boat in the evaporator onto the glass substrate in
an ordinary vacuum of about 5 x 10 5 mm of mercury. Because
the indium atoms have high mobility on the surface of the
substrate and do not wet the glass substrate significantly, the
indium evaporated onto the glass slide agglomerates into small -
unequal size particles. Some other metals, such as tin, having
similar characteristics so that they will also form globules
on the substrate when evaporated thereon, and are oxidizible,
can be used, and the particular metal used is dependent
on the particular immunologically reactive biological
solution (including its pH) being investigated. The slow
evaporation of the indium is necessary in order to obtain the
globules, the evaporating process taking approximately 3 to 5
minutes. The indium globules have average diameters on the
order of 3000 A and are closély spaced together, having an
average maximum spacing of approximately lt2"diameter. A
significantly faster evaporation of the indium (i.e., in the
order of 30 seconds) results in a deposit on the substrate of
a film of relatively constant thickness, as illustrated in
FIGURE 2.
After the indium globules 11 have been evaporated
on substrate 10, a thin substantially constant thickness
film 12 of gold is evaporated over the indium globules, and
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during this evaporation step some alloying of the indium
and gold occurs. Other metals than gold may be utilized, such
as cvpper or silver when testing other type body fluids.
However, if the immunological test to be conducted requires
the use of human serum, as is often the case, it has been
found that such other metals are attacked through some
chemical reaction and therefore are unsatisfactory. The gold
deposition can be accomplished at a faster rate than the indium
deposition step, and again may be accomplished by evaporating
the gold from a tantalum boat in an ordinary vacuum of about
5 x 10 5 mm of mercury. The gold film 12 is of thickness in
the order of 1000 A for the best contrast between a monomolecular
layer of hepatitis B antigen (HBAg) and a bimolecular layer of
such antigen and its specific antibody (HBAb) for an indium
layer of average thickness of 2000 A. Thus, the average thickness
of the indium layer (i.e., a constant thickness layer that would
be obtained from globules 11) is approximately twice the
thickness of the gold film 12. The intermediate structure
of the coated substrate after evaporation of the gold film
thereon is similar to that illustrated in FIGURE la with the
gold film 12 forming an irregular or uneven undulating
pattern (due to the indium globules~ that diffracts incident
light, and with the understanding that some alloying of the
indium and gold has occurred as described in my referenced
application S.N. 204,262. The evaporation of the indium
and gold onto the substrate can be accomplished in any
suitable evaporator, a typical evaporator being model type
CV-18 manufactured by Consolidated Vacuum Corp., Rochester,
N.Y. A Deposit Thickness Monitor, model DTM-3 and Deposit
Rate Control, model DRC, both manufactured by Sloan
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Instrument Corp., Santa Barbara, California were utilized with
the aforementioned evaporator for obtaining the desired thickness
and rate of deposition of the indium and gold metals being
evaporated. Due to the maximum current limitation in the
aforementioned evaporator, the gold could not be evaporated
as rapidly as it could be in a higher power evaporator, and
such evaporation prOcesstherefore took about 3 minutes.
After the gold has been evaporated on the indium
globule-coated substrate, the coated substrate is removed
from the evaporator and placed in a suitable electric furnace
for heating in an air atmosphere sufficiently to obtain some
oxidation of the indium and thereby form an indium oxide film
on the outer surface of the substrate coating having the
irregular pattern of t~e gold film in FIG. la. This
oxidation step also completes the alloying of the indium and
gold. The degree of oxidation of the indium determines the
color of such oxide film. The various degrees of oxidation produce
different colored slides having different sensitivities for
different thicknesses of the layers of the biological particles,
and each differently colored slide is differently sensitive to
single and double layers of particular biological particles. The
oxidation accomplished by heating for approximately 150 minutes -
at 325& yields an indium oxide film having greenish-gold or
bronze color. A lesser degree of oxidation produced by heating
325C for 100 minutes produces a reddish-gold color. A
greater oxidation of the indium produced by heating at 325C for
30-45 minutes produces a blue color. The greenish-gold (bronze)
color is presently the preferred color for HAA as will be
described hereinafter, and such- oxide film has a thickness
30 in the order of several hundred Angstrom. ~ -
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In the case of only indium globules evaporated on the
substrate, as taught in my hereinabove referenced patent
application S.N. 204,262, the detection of a single or double
layer of imm~nologically reactive biological particles thereon
is obtained by light transmission, that is, the test is
accomplished by direct visual observation of the light
transmitted through the slide. In the case of my present
gold-indium alloy and indium oxide coated substrate, the
biological particle layers are detected by reflected light
since the irregular surface of the indium oxide diffracts the
incident light. The coated substrate, after the indium
oxidation process, appears as in FIGURE lB. The 25 millimeter
square coated slides may then be cut into approximately 4 equal
squares for use in the immunological tests to be described
hereinafter. Obviously, a commercial production of my slides
would probably begin with a much larger size glass, and could
be cut to any desired size slides. The cutting process may
utilize conventional glass cutting techniques such as scoring
the glass with a diamond scribe, and then breaking the glass
along the scored lines.
My finished substrate, as illustrated in FIGURE lB
(or 2) is placed on a suitable support and a monomolecular layer
of a first immunologically reactive biological particle is
adhered onto the coated surrace of the substrate. The
adherence of the first biological particles may be accomplished
by depositing a single drop of a first solution of the first
biological particle on the substrate coated surface. The
first biological particle is selected on the basis of its
being specific to particular second biological particles which
will form the second layer on the substrate surface if
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they are present in a solution to be tested. The first
particles may be produced in laboratory cultures or obtained
from the higher living biological systems as described hereinabove,
and are generally commercially available in highly purified
5 form, and if not available commercially, may be purified
chemically. The 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.
The substrate is preferably stored in a moist chamber for a
time interval sufficient so that the first biological
particles in the drop of the first solution are adsorbed
onto the coated surface of substrate 10 and form a substantially
complete monomole~ular layer in the pattern of the drop in
accordance with the teachings of the aforementioned Canadian
patent applications of Giaever. The time interval (generally
up to 1 hour) for the formation of the monomolecular layer
on substrate 10 is an inverse function of the concentration
of the first particle in the solution. The area size of the
monomolecular layer on the substrate coated surface is
20 preferably as small as practicable, and is generally in the -
range of 1 square millimeter to 1 square centimeter in order
to conserve the amount of biological material used in the
process. A rinsing of the coated surface of substrate 10
is often recommended after the fbrmation of the monomolecular
layer thereon in order to minimize nonspecific adsorption. The
monomolecular layer coated substrate is then dried, if the
slide is to be shipped commercially or s~ored, preferably by
blowing air at room temperature across the substrate in order -~
to speed the drying process. If the slide is to be used
immediately, there is no need to dry it after the rinsing. For
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commercial use by others, the metallized slide could be sold by
the slide manufacturer with or without the first monomolecular
layer thereon. The spot on the substrate coated surface caused
by the monomolecular layer pattern is generally barely, if at
all, visible to the unaided eye.
The monomolecular layer coated substrate is then
exposed to a second solution suspected of containing second
immunologically reactive biological particles specific to
the first in a direct test for such second particles. This
exposure is generally aceomplished by ~mmersing the
monomolecular l~yer coflted substrate in the second solution
for a time interval which is again an inverse function
of the concentration of the second biological particles
in the second solution. Since the concentration of the
second particles is generally much less than the concentration
of the first particle in the first solution, the immersen~
step generally takes much longer than the time interval
for for~ing the first monomolecular layer, and may take
up to 24 hours. Presence of the second biological particles
in the second solution results in the formation of a second
substantially complete monomolecular layer on the pattern
(generally a round spot) established on the coated substrate by
the first monomolecular layer as a result of the immunologica
reaction wherein the second particles become bound to the
first particles.
After the coated substrate has been sufficiently exposed
to the second solution, the substrate is removed therefrom
and may be immediately visually examined. Alternatively, and
more generally, the substrate after removal from the second
solution is ~gain rinsed with a suitable solution which, in many
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cases, may be water or a salt solutlon thereof, and the slide is
then dried. The direct visual observation of the coated
substrate is made by detecting the reflection off the coated
surface of the substrflte due to the light diffraction occurring
5 at the oxidized surfsce, rather than by detecting the light
transmitted therethrough. Absence of the second biological
particles in the second solution results in only the presence
of the monomolecular layer on the coated substrate surface and,
as noted hereinabove, such single layer of biological particles is
10 barely, if at all, visible on my improved slides. However,
presence of the second particles in the second solution develops
the second layer described hereinabove and produces ~
surprisingly different colored spot on the substrate so that the ~ -
contrast between single and double layers of immunologically
15 reactive biologic~l particles is very pronounced.
As noted above, the different degrees of oxidation
of the indium produce different colored slides, and it has
been found that different colored slides have different -
sensitivities for different thicknesses of particular
20 biological particle layers. Thus, a particular colored slide
is selected for the particular biologic~l particle being
investigated since such p~rticular colored slide has the
best sensitivity for such biological particle system.
The greenish-gold (bronze) colored slide has been found
25 to have the highest sensitivity for detecting the difference
between single and double layers of many types of immunologically
reactive biolo~ical pflrticle systems, that is, provides the
highest degree of contrast for detecting the double layer which
appears as a purplish spot on the slide. Although this particular
30 color of the indium oxide film h~s been described as being
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obtained by heating the slide in air for 150 minutes at 325C,
it should be obvious that a comparable color ~xide film
may be obtained by heating the slide at a higher temperature for
a shorter time, or at a lower temperature for a longer time.
FIGURE 2 illustrates a second embodiment of my improved
substrate. This embodiment is fabricated in the same manner
as the first embodiment except for the first step. In the
first step, a thick continuous (as opposed to the noncontinuous
~ film of globules in FIGURE 1~) film of indium of substantially
constant thickness is evaporated on the top surface of substrate
10. This thick film, in the order of 3000 A thickness, is
obtained by evaporating the indium at a much faster rate than
in the case of the first embodiment~ the evaporation interval
being approximately 30 seconds. Alternatively, the gold can be
evaporated ~n the substrate before the indium. The finished
structure of the FIGURE 2 embodiment thus includes substrate
10, a layer 3~ of indium-gold alloy of substantially constant
thickness and an indium oxide film 13 which is flat. The
light diffraction produced from t~e indium oxide film 13 in the
FIGURE 2 embodiment results from gold particles dispersed therein
during the oxidation process. Such gold particles contribute
to a high dielectric constant and therefore promote very visible
interference colors. Such gold particles are probably present in
the indium oxide film in FIGURE lb.
A specific application of my improved substrate for the
detection of hepatitis B antigen (B Ag) and antibody (HsAb)
will now be described. This particular biological system
requires a very sensitive test since the HBAg is a large size
protein (mo~ecular weight of 5 x 106) whereas the HBAb is small
(M.W. of 1.6 x 105) so that the added thickness of an HBAb
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monomolecular layer to one of HBag does not produce a l~rge
I chflnge in thickness. Experiments involving single and double
; layers of the HBAg and sAb molecules on the anodized tantalum
slides described in the hereinabove referenced Rothen article
S were not very satisfactory since a Qufficient contrast between
the single and double layers for a sensitive test was not
obtained. However, the use of my improved substrate described
herein provided a high degree of contrast between the single
~nd double layers so thst the layers were detected with at least
the same sensitivity as in standard radioimmunoassay tests.
IQ the tests for detecting the hepatitis B antigen and
antibody, the first step requires the adsorption of a
monomolecular layer of B Ag onto the coated surface of
my substrate. Since the HBAg is readily available in purified
~orm, a drop of a common solution thereof (such as a salt solution)
is applied onto the coated surface of the slide. The drop of
this first solution is maintained on the substrate surface for
a time interval sufficient to obtain a substantially complete
monomolecular layer over the area of the drop, and may be
in the order of 15 to 30 minutes with the slide being stored in
a moist chamber to prevent evaporation of the drop. The slide
is then rinsed with distilled water and may subsequently be gently -
blown dry with compressed air, although this drying step is
aot essential. The HBAg has now been adsorbed from the drop
of solution in a small monomolecular layer spot on the coated
slide surface and is barely, if at all, visible to the unaided
eye.
The s Ag monomolecular layered slide is next exposed to a
second solution suspected of containing the HsAb. This second
solution is generally a human serum sample. The exposure
is generally accomplished by immersing the slide in the second
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solution for an interval up to about 24 hours due to the con-
centration of the specific antibody in the serum sample being low,
if at all present. The slide is then again rinsed and dried
and visually examined with the naked eye. A purplish spot on the
slide indicates presence of a second monomolecuJ.ar layer (i.e.,
the HBAb layer) whsreas absence of the spot indicates absence
of B Ab in the human serum sample in a direct test therefor.
The direct test for HBAg can be accomplished in a similar
manner as described above, with HsAb being adsorbed on the
coated slide surface as a small spot forming the ~irst monomolecular
layer, and H~Ag in the second solution (human serum sample), i~
present, forming the second monomolecular layer.
An indirect or inhibition test for the detection of
HBAg may also be demonstrated using my improved slide.
The principle of the inhibition test is that HBAg particles,
if present in sufficient quantity~ will neutralize free
B Ab in solution. This reaction will prevent the antibodies
from forming observable complexes (i.e., a bimolecular layer)
when the slide with the antigen spot (first monomolecular layer)
is exposed to the solution.
The inhibition test is accomplished as ~ollows:
A monomolecular layer spot on HBAg is adsorbed on the coated
slide surface as in the direct test described hereinabove.
he second solution is prepared by adding the 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 ti~ interval sufficient
for the HBAb to complex with HBAg in the human sample, if the
antigen is present therein. The vial is preferably agitated to
increase the rate of complexing. Finally, the HBAg monomolecular
spot covered slide is immersed in the second solution, and after
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a suitable period of time (again up to 24 hours), the slide
is removed, rinsed, dried and visually examined. The results
of this inhibition test are the opposite of the direct test,
that is, presence of the HBAg in the human serum sample ~
5 produces no purplish spot on the slide, i.e., produces no - ;
second monomoleclular layer on the slide, whereas presence of
` such purplish spot indicates absence of the HBAg in the
` human serum sample.
The inhibition test for the detection of ~BAb 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 all of the above tests, the HBAb may be obtained from
human serum of a patient k~own to have hepatitis B, or it may
be developed in a goat, rabbit or other suitable animal by
;~ injection thereof with the HBAg, waiting a suitable incubation
`; period such as two weeks, and then drawing bloJd containing
the specific antibody from the animal and separating the antibody
from the remaining blood particles.
The same significant visual contrast of a purplish spot
against a bronze color background was found for a CEA (cancer
embryonic antigen) and anti-CEA double layer and for a BSA
(bovine serum albumin) and anti-BSA double layer as compared
to a barely, if at all, visible spot for a single layer of the
CEA or BS~ particles. In the case of a blue colored slide,
the double layer spot was white.
From the foregoing description, it can be appre~ated
that my invention makes available an improved substrate for
detecting immunological reactions occurring at the surface
thereof by direct visual observation with the unaided eye,
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as well as a method for fabricating the improved substrate.
The improved substrate has a metallized coating including
an alloy of two metals and an oxide of one of such metals.
Thus, the two metals can be identified as a base metal and noble
metal since only the base metal can be oxidized, and is overcoated
by the second (noble) metal. Also, there is no reason to limit
the coating to two metals, one of the materials can be of the
semiconductor type,and the materials are not necessarily limited
to only two, there may be more. In the case of the metallized
coating being an indium-gold alloy and indium oxide, such
coated substrate provides significantly improved contrast
between single and double monomolecular layers of immunologically
reactive biological particles including a single layer of
hepatitis B antigen and a second single layer of its specific
hepatitis B antibody. Different degrees of oxidation of the
indium may be found more useful with other types of pairs of
immunologically reactive biological particles for increasing
.; .
the contrast thereof between single and double layers. The
coated substrate of my invention is a simple, relatively inexpen-
sive device which is easily fabricated in accordance withanother aspect of my invention. The major advantage of my
invention is the significantly improved contrast between single
and double layers of immunologically reactive biological particles
which thereby permits detection of such immunological reaction
by direct observation visible to the unaided eye.
Having described my invention with reference to two
particular embodiments, it is believed obvious that modification
and variation of my invention is possible in the light of the
above teachings. Thus, the coating on the substrate may be
formed of an alloy of two mor mo-e metals (or metal and
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semiconductor other than the indium and gold, with the base
metal forming the outer oxidized surface. Such other alloy-
oxide coated substrate may be found to obtain better contrast
between single and double layers of some immunologically
reactive biological particles other than the hepatitis type
than if the indium-gold slide was used. That is, each pair of
`~ immunologically reactive biological particles are detected with the
greatest contrast between single an~d double layers thereof with
a specific substrate fabricated in accordance with my invention.
Finally, the irregular surfaced slide constituting my first
embodiment could obviously also be fabricated by starting with an
irregular surfaced substrate and evaporating constant thickness
layers of indium and gold thereon. 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 as defined by the following claims.
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