U.S. patent number 3,926,564 [Application Number 05/445,204] was granted by the patent office on 1975-12-16 for substrate for immunological tests and method of fabrication thereof.
This patent grant is currently assigned to General Electric Company. Invention is credited to Ivar Giaever.
United States Patent |
3,926,564 |
Giaever |
December 16, 1975 |
Substrate for immunological tests and method of fabrication
thereof
Abstract
Substrates for providing contrast, visible to the unaided eye,
between single and double layers of immunologically reactive
biological particles are fabricated by depositing an alloy of
indium and gold on glass slides, and then heating the slides in air
at different temperatures or time intervals to cause various
degrees of oxidation of the indium. 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.
Inventors: |
Giaever; Ivar (Schenectady,
NY) |
Assignee: |
General Electric Company
(Schenectady, NY)
|
Family
ID: |
23767988 |
Appl.
No.: |
05/445,204 |
Filed: |
February 25, 1974 |
Current U.S.
Class: |
422/426;
204/192.15; 427/250; 428/336; 428/434; 428/469; 428/478.2; 436/525;
436/805; 436/809; 436/811; 436/820; 436/807 |
Current CPC
Class: |
G01N
33/553 (20130101); Y10S 436/805 (20130101); Y10T
428/31768 (20150401); Y10S 436/809 (20130101); Y10S
436/807 (20130101); Y10S 436/82 (20130101); Y10T
428/265 (20150115); Y10S 436/811 (20130101) |
Current International
Class: |
G01N
33/551 (20060101); G01N 33/553 (20060101); G01N
021/06 (); G01N 033/16 () |
Field of
Search: |
;23/23B,253R,259,253TP
;204/192 ;117/107 ;128/2 ;424/12 ;427/250 ;428/336,434,469,474 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chemical Abstracts, 67:48743e (1967). .
Chemical Abstracts, 72:16733p (1970)..
|
Primary Examiner: Wolk; Morris O.
Assistant Examiner: Marantz; Sidney
Attorney, Agent or Firm: MaLossi; Leo I. Cohen; Joseph T.
Squillaro; Jerome C.
Claims
What I claim as new and desire to secure by Letters Patent of the
United States is:
1. A medical diagnostic device comprising:
a substrate member and
a composite metallized coating attached to the surface of said
substrate member, said coating containing an alloy having a noble
metal constituent and an oxidizable metallic constituent and having
present in the outer surface thereof oxide content derived from
said oxidizable metallic constituent, said coating being free of
oxide of said noble metal constitutent.
2. The device set forth in claim 1 wherein the noble metal
constituent is gold.
3. The device set forth in claim 1 wherein the oxidizable metallic
constituent is indium, the noble metal constituent is gold, and the
oxide content is of indium oxide.
4. The device set forth in claim 1 wherein the surface of the
substrate receiving the coating is flat.
5. The device set forth in claim 1 wherein the substrate is a glass
material.
6. The device set forth in claim 1 wherein the oxidizable metallic
component is tin.
7. The device set forth in claim 1 wherein the noble metal
constitutent is silver.
8. The device set forth in claim 1 wherein the oxidizable metallic
constituent is indium.
9. The device set forth in claim 8 wherein the color of the coating
is bronze.
10. The device set forth in claim 1 wherein the outer surface of
the coating is slightly irregular.
11. The device set forth in claim 10 wherein the oxide portion of
the coating is about several hundred Angstrom thick.
12. The device set forth in claim 1 wherein the outer surface of
the coating is flat.
13. The device set forth in claim 12 wherein unoxidized particles
of the noble metal constituent are dispersed within the oxide
portion of the coating.
14. A medical diagnostic device comprising:
a substrate member,
a composite metallized coating attached to the surface of said
substrate member, said coating containing an alloy having a noble
metal constituent and an oxidizable metallic constituent and having
present in the outer surface thereof oxide content derived from
said oxidizable metallic constituent, said coating being free of
oxide of said noble metal constituent and
a monomolecular layer of immunologically reactive protein overlying
at least a portion of said coating.
15. The device set forth in claim 14 wherein
the protein is an antigen.
16. The device set forth in claim 14 wherein
the protein is an antibody.
17. The device set forth in claim 14 and further comprising
a layer of second immunologically reactive protein bound to the
first monomolecular layer wherein the second protein is specific to
the first protein, the double layer of first and second proteins
being visible to the unaided eye as a spot of color distinct from
the color of the outer surface of the coating.
18. The method of making a diagnostic device for determining the
presence or absence of a specific protein in a biological sample
comprising the steps of:
coating surface area of a substrate with noble metal and oxidizable
metal,
heating said coated substrate in an oxidizing atmosphere to convert
to the oxidized state a portion of said oxidizable metal at the
surface of the coating and to simultaneously cause substantial
alloying between oxidizable metal and nobel metal and
contacting said surface with a solution of a specifically reactive
protein to said specific protein to apply a monomolecular layer of
said specifically reacting protein to at least a portion of said
surface.
19. The method set forth in claim 18 wherein the noble metal and
oxidizable metal are applied in layers with the thickness of the
oxidizable metal being approximately twice the thickness of the
noble metal when initially deposited.
20. The method set forth in claim 18 wherein the oxidizable metal
is applied as small globules on at least a portion of the surface
of the substrate.
21. The method set forth in claim 20 wherein
the noble metal is applied as a thin substantially constant
thickness film over the oxidizable metal globules.
22. The method set forth in claim 18 wherein indium is applied by
evaporation as the oxidizable metal.
23. The method set forth in claim 22 wherein
the step of evaporating indium consists of evaporating the indium
in a vacuum of approximately 5 .times. 10.sup..sup.-5 mm of mercury
for a time interval of 3 to 5 minutes.
24. The method set forth in claim 22 wherein gold is applied by
evaporation over the indium as the noble metal.
25. The method set forth in claim 24 wherein
the thickness of the indium is approximately 2,000 A, and
the thickness of the gold is approximately 1,000 A.
26. The method set forth in claim 24 wherein
the step of heating the metal coated substrate consists of heating
in air for approximately 150 minutes at 325.degree..
Description
My invention relates to an 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
monomolecular and biomolecular layers of immunologically reactive
biological particles on the surface of the substrate.
This application is related to my copending applications Ser. No.
266,278, entitled "Method and Apparatus for Detection and
Purification of Proteins and Antibodies," filed June 26, 1972 and
Ser. No. 384,113 entitled "Improved Method and Apparatus for
Detection and Purification of Proteins and Antibodies" filed July
30, 1973 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 Americal Chemical Society, volume 59 (July-December-1937)
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, "Three Simple Ways to
Detect Antibody-Antigen Complex on Flat Surfaces," authors A. L.
Adams et al., Journal of Immunological Methods 3 (1973) pages
227-232 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 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 available
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 an essential
part, whereas all antibodies are proteins. Proteins are large
molecules of high molecular weight, i.e., 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 devices 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 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
protein 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
antibodies in a biological system is of medical diagnostic value 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 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 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 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, however, may be controllably
produced in laboratory cultures. However, some antigens, for
example, hepatitis-associated-antigens, are at present, like
antibodies, only obtainable from the higher living biological
systems.
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
referenced patent applications are satisfactory in their
performance with many of the immunologically reactive proteins, 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.
Also, the anodized tantalum slide described in the
hereinabove-referenced articles "Interactions Among Human Blood
Proteins at Interfaces" and "Three Simple Ways to Detect
Antibody-Antigen Complex on Flat Surfaces" has been found to be
less sensitive than my slide to be hereinafter described,
especially in the detection of hepatitis.
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 biological particles.
A further object of my invention is to provide a relative simple
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 which may conveniently be a
glass slide. Alternatively, the indium may be evaporated onto the
surface 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, more specifically proteins, 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:
FIG. 1a is an elevation view of an intermediate structure of the
preferred embodiment of my improved substrate;
FIG. 1b is an elevation view of the preferred substrate after final
fabrication; and
FIG. 2 is an elevation view of a second embodiment of my improved
substrate .
Referring now to FIG. 1a, 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 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 .times. 10.sup..sup.-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
3,000 A and are closely spaced together, having an average maximum
spacing of approximately 1/2 inch 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 FIG. 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 during this evaporation
step some alloying of the indium and gold occurs. Other metals than
gold may be utilized, such as copper 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 .times.
10.sup..sup.-5 mm of mercury. The gold film 12 is of thickness in
the order of 1,000 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 2,000 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 FIG. 1a 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 Ser. No. 384,113. 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 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 aformentioned evaporator, the
gold could not be evaporated as rapidly as it could be in a higher
power evaporator, and such evaporation process therefore 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 13 on the outer surface of the
substrate coating having the irregular pattern of the gold film in
FIG. 1a. 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.degree.C yields an indium oxide film having greenish-gold or
bronze color. A lesser degree of oxidation produced by heating
325.degree.C for 100 minutes produces a reddish-gold color. A
greater oxidation of the indium produced by heating at 325.degree.C
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 in the order of
several hundred Angstrom.
In the case of only indium globules evaporated on the substrate, as
taught in my hereinabove referenced patent application Ser. No.
384,113, the detection of a single or double layer of
immunologically reactive proteins 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,
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 FIG. 1b. The 25 millimeter square coated slides may
then be cut into approximately four 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 FIG. 1b (or 2) is placed
on a suitable support and a monomolecular layer of a first
immunologically reactive biological particle is adhered onto the
coated surface 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 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 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 monomolecular layer in the pattern of the drop in
accordance with the teachings of the aforementioned co-pending U.S.
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 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 formation 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 stored, 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 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
accomplished by immersing the monomolecular layer coated 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 immersent step
generally takes much longer than the time interval for forming 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 immunological 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 again
rinsed with a suitable solution which, in many cases, may be water
or salt solution 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 substrate due to the light
diffraction occurring at the oxidized surface, rather than be
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 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 a
surprisingly different colored spot on the substrate so that the
contrast between single and double layers of immunologically
reactive biological 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 biological particle layers. Thus, a
particular colored slide is selected for the particular biological
particle being investigated since such particular colored slide has
the best sensitivity for such biological particle system. The
greenish-gold (bronze) color slide has been found to have the
highest sensitivity for detecting the difference between single and
double layers of many types of immunologically reactive biological
particle 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 color of the indium oxide film
has been described as being obtained by heating the slide in air
for 150 minutes at 325.degree.C, it should be obvious that an oxide
film of comparable color may be obtained by heating the slide at a
higher temperature for a shorter time, or at a lower temperature
for a longer time.
FIG. 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
FIG. 1a) film of indium of substantially constant thickness is
evaporated on the top surface of substrate 10. This thick film, in
the order of 3,000 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 on the substrate
before the indium. The finished structure of the FIG. 2 embodiment
thus includes substrate 10, a layer 30 of indium-gold alloy of
substantially constant thickness and an indium oxide film 13 which
is flat. The light diffraction produced from the indium oxide film
13 in the FIG. 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 FIG. 1b.
A specific application of my improved substrate for the detection
of hepatitis B antigen (HBAg) and antibody (HBAb) will now be
described. This particular biological system requires a very
sensitive test since the HBAg is a large size protein (molecular
weight of 5 .times. 10.sup.6) whereas the HBAb is small (M.W. of
1.6 .times. 10.sup.5) so that the added thickness of an HBAb
monomolecular layer to one of HBAg does not produce a large change
in thickness. Experiments involving single and double layers of the
HBAb and HBab molecules on the anodized tantalum slides described
in the hereinabove referenced Rothen article were not very
satisfactory since a sufficient 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 and double layers so that the
layers were detected with at least the same sensitivity as in
standard radioimmunoassay tests.
In the tests for detecting the hepatitis B antigen and antibody,
the first step requires the adsorption of a monomolecular layer of
HBAg onto the coated surface of my substrate. Since the HBAg is
readily available in purified form, 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 raised with distilled water and may subsequently
be gently blown dry with compressed air, although this drying step
is not 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 HBAg monomolecular layered slide is next exposed to a second
solution suspected of containing the HBAb. This second solution is
generally a human serum sample. The exposure is generally
accomplished by immersing the slide in the second solution for an
interval up to about 24 hours due to the concentration of the
specific antibody in the serum sample being low, it 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 monomolecular layer (i.e., the HBAb layer) whereas absence
of the spot indicates absence of HBAb 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 HBAb being adsorbed on the coated slide
surface as a small spot forming the first monomolecular layer, and
HBAg in the second solution (human serum sample), if 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 HBAb 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 follows: A monomolecular
layer spot on HBAg is adsorbed on the coated slide surface as in
the direct test described hereinabove. The 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 time 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 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 produces no purplish spot on the slide, i.e., produces no
second monomolecular 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 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 all of the above tests, the HBAb may be obtained from human
serum of a patient known 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 blood 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 BSA particles.
In the case of a blue colored slide, the double layer spot was
white.
From the foregoing description, it can be appreciated 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, 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 inexpensive
device which is easily fabricated in accordance with another 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
or more metals (or metal and semiconductor other than the indium
and gold) including a base metal, 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 and 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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