Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
10484Q9 RD-6735
My invention relates to a method for detecting large size
immunologically reactive proteins and small size reactive
proteins specific to the first protein, and in particular,
for improving the contrast between single and double layers
of the protein molecules.
This application is related to my Canadian application
S.N.~o7~o ~ , filed ~ 5~ /q~ , entitled "Method for
Binding Antibodies To a Surface Such That They Remain Active"
Immunological reactions are highly specific biochemical
reactions in which a first protein known as the antigen com-
bines 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 sys-
tem such as an animal or a human being are vital in combating
disease. In a biological system, the entry of a foreign pro-
tein, 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 pro-
tein molecules have available chemical combining or binding
sites which complement those on the antigen molecule so that
the antigen and antibody chemically combine or bond to form
the immunologically complexed protein.
Because antibodies are produced by biological systems
in response to invasion thereof by foreign proteins, the
detection of antibodies present in a biological system is
of medical diagnostic value in determining the antigons
to which the system has been exposed. Conversely, the
detection of certain antigens in a biological system also
has medical diagnostic values; examples of diagnostic
detection of antigens include detection of HCG
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proteln molecules in urine as a test for pregnancy, and
detection of hepatitis-sssociated antigen (HAA) molecules
in blood of prospective blo~d donors.
Tn order to perform such dlagnostic tests, the
apprapriate protein of at least an immunologically reacting
pair must be obtained. The only known source of sn 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
blqod serum of animals and human beings which have been
exposed to this corresponding antigen. Many antigens,
however, ~y be co~trollably prod~ced in laboratory
cultures. However, some antigens, fo~ example, hepatitis-
associated ~ntigen~, are at present, like antibodies,only obtainable from the higher living biological systems.
Most antigens are proteins or contain proteins as
an essential part, whereas all antibodies are proteins.
Since proteins are large molecules of high molecular
welght, i.e., are polymers consisting of chains of variable
number~ of amino ncids, the antigen and antibody protein
may each have several combining sites~ The ~ive ma~Dr
classes of antibodies (immunoglobulins Ig-G, Ig M, Ig A,
Ig E and Tg D) are each apparently characteriz~d by at
lea6t two heavy (lcng) peptide chains of amino acids
and at least two light ~short) peptide chains of the
acids wherein the bond between the amino acid units is
known as a peptide bond. These heavy and light peptide
chains are oriented in the general shape of the letter '~"
and the active or combining sites are the extreme ends of
10484~9 RD-6735
the two arms of the Y-shaped antibody for the Ig G
antibody.
Immunological reactions can be detected by various
techniques lncluding the use of a suitable substrate
S ~uch as a metallized glass or metal slide. Expos~re of the
substrate to a solution of antigen will result in the
antigens being physically adsorbed in a dense monomolecular
layer onto the surface of the sub~trate. Subsequent
expo8ure of the antigen-coated substrate to a serum
containing antibodies towards the antigen re~ults in the
i~munological reaction wherein the antibodies selectively
attach themselves to the antigens by means of the binding
sites on the antibody molecule which complement tho~e on
the antigen molecule to thereby form at least a partial
bimolecular layer of immunologically complexed protein
on the substrate surface. The problem which often arises
i~ the inability to distinguish between the monomolecular
and bimolecular layers of protein on the substrate, especially
if the ad~orbed (antigen) layer is relatively thick as in
the case of HAA layer since the hepatitis-associated
antigen molecule is at least ten times as large as the
antibody to the HAA.
Therefore, one of the principal objects of my
invention is to provide a new method in surface immunological
te~ts for improving the contrast between single and double
protein layers when one of the layers consists of large
~ize proteins.
Another ob~ect of my invention i8 to provide the
$mproved contrast when the layer adsorbed on the surface
con8igt~ of large antigen proteins.
RD-6735
~1~)484Q9
A further object of my invention is to provide
improved contrast between a monomolecular and bimolecular
layer by means of a third molecular layer of protein
coated on a substrate when the adsorbed-first layer consists
of large size antigens and the third layer is an antibody
to the antibody in the second layer.
Briefly, and in accordance with the objects of
my invention, I add an immunologically inert protein to
a solution containing a relatively large size immunologically
reactive antigen protein. A substrate is then immersed
in the solution and the surface of the substrate is coated,
by adsorption, with a monomolecular layer of the antigen
protein molecules and inert protein molecules wherein
each antigen molecule is generally surrounded by inert
protein molecul~s. Subsequent immersion of the coated
substrate in a second solution containing a relatively
small size immunologically reactive antibody protein
specific to the antigen protein results in the antibody
molecules chemically bonding with the antigen molecules
to form at least a partial bimolecular layer on the
substrate. The quantity of inert protein molecules
in the first layer is sufficient so that the spacing
between antigen molecules permits more antibody molecules
to be bound to the antigen molecules than if the antigen
molecules were densely packed together, and thereby
produces a much greater change in contrast between single
and double layers of the two proteins coated on a substrate.
Thus, my invention provides a simple procedure for
distinguishing between a single layer of a large size
antigen, such as hepatitis-associated antigen, and a
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double layer which include~ a second layer of a smal1 size
antibody such as the hepatitis antibody, and thus can be
utilized in the analysis of the second solution to determine
the presence of the antibody therein. ~~ .
The features of my invention which I desire to protect
~erein are pointed out with particularity in the appended
claim~. 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
ln connection with the accompanying drawing wherein like
; parts in each of the severai figures are identified by the
same reference character, and wherein:
FIGURE 1 is an elevation view of a substrate after
- 15 it has been immersed first in a~solution of a large size
antigen by a known method and then in a solution of a
smaller size antibody specific to the antigen;
FIGURE 2 is an elevation view of the substrate after
it has undergone two immersion steps as in FIGURE l,-but
with the first immersion being in accordance with my
- invention;
FIGURE 3 is an elevation view of the substrate of
FIGURE 2 after a subsequent immunological reaction wherein
the coated substrate is immersed in a solution containing
an antibody to the antibody in the second solution; and
FIGURE 4 is a plan view of a substrate having a small
region thereon of active-inert proteins surrounded by '
inert protein in accordance with my invention.
Referring now to.FIGURE 1, there is shown a highly
magnified elevation view of a portion o~ diagnostic
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~04~409
apparatus in the form of a thin wafer 10 of a suitable
substrate material which may be metal, glass, mica, plastic,
fused silica, quartz or similar material, with metal being
preferred as having the greatest difference in refractive
index to protein and preferably is in the form of a metal
or metallized glass slide. Substrate 10, when immersed
in a first solution generally of salt water containing a
first protein of interest, which may be biologically an
antigen or antibody, is adsorbed onto the substrate in a
monomolecular layer 11. For the purpose of my invention, the
layer 11 adsorbed on the surface of substrate 10 will herein-
after be described as an antigen layer. Any protein will
adsorb in such monomolecular layer, but no further adsorption
will take place, that is, the protein will attach to the
substrate, but will not attach to itself. Thus, the antigen
protein layer 11 can only be monomolecular and not of
greater thickness. The time required to completely coat
the substrate with the antigen protein is a function of
the concentration of the protein in the solution, the
degree of agitation of the solution and the solution
temperature. As an example, a concentration of 1 milligram
per cubic centimeter of hepatitis-associated antigen
solution completely coats a slide in approximately lO
minutes with a monomolecular antigen protein layer.
After the monomolecular layer of antigen protein 11
has formed over substantially the entire surface of
the substrate 10, the coated substrate is removed
from the solution of the antigen protein, and is next
immersed in a second solution containing, or suspected
of containing, the specifically reacting antibody
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~04~4~9
protein to the antigen protein. For purposes of my
invention, it will be assumed that the protein 11 adsorbed
on the surface of substrate 10 is always of greater size
than the protein which is immunologically reactive there-
with and forms a second layer on the substrate. The
second solution may contain many constituents in addition
to the specifically reacting smaller antibody protein whose
presence it is desired to detect. However, no protein
other than the specifically reacting antibody protein
will adhere to the first antigen protein layer~on the
substrate. Thus, only if the specifically reacting antibody
protein is present in the second solution will immunological
complexing between the antigen and its specifically
reacting antibody take place and the substrate will,
after a time, have a bimolecular protein layer thereon.
The time required for the adhesion of the second (antibody)
molecular layer 12 onto the coated substrate is again
a function of the concentration of the specifically
reacting protein in the solution, the degree of solution
agitation, and solution temperature. For antibodies in
blood serum, this timing may be as long as one day. The
second layer may be only a partial one, or substantially
complete, depending upon the above three enumerated factors.
As illustrated in FIGURE 1, in the case wherein the
antigen layer 11 substantially completely coats substrate
10, i.e., the spacing between adjacent antigen molecules
is minimal, the number of antibody molecules 12 that can
bind to any particular antigen molecule is limited due to
the limited surface of the antigen molecule which is
available for such immunological complexing. This
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1 048409
substrate coating process described in my Canadian application,
S.N. 172,639, entitled "Method and Apparatus for Detection
and Purification of Proteins and Antibodies", filed May 29,
1973, and assigned to the assignee of the present invention.
Due to the monomolecular antigen layer 11 being
adsorbed over substantially the entire surface of
substrate 10 in FIGUR~ 1, the second layer 12 of the
smaller size specific antibody for such antigen bonds
thereto in a thin layer to form the bimolecular layer.
However, due to the proteins in the second layer 12 being
of smaller size than the proteins in the first layer 11
and also due to the second layer being much less dense
than the first tantigen) layer, it is clearly evident
that there can be considerable difficulty in distinguishing
between this single and double layer of protein on the
substrate 10. A good example of this situation is the
case of first layer 11 being hepatitis-associated antigen
and the second layer 12 being an antibody specific to HAA.
Examination of the coated substrate with an optical instrument
such as the ellipsometer does not readily allow one to
distinguish between the single (antigen) and a partial
or dilute double (antigen-antibody) complex) layer on
the substrate and thus it cannot readily be determined
whether the second solution did indeed contain any of the
antibodies to HAA and thus such diagnostic test is of
little value. In the present invention, I have discovered
a method by which a much ~reater number of antibody
molecules in the second layer 12 can be bonded to the
antigen molecule in the first layer 11 to thereby produce
a much greater change in contrast between the single and
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RD-6735
double layers. The basis of my present discovery is
illustrated in FIGURE 2 and is involved in the method of
forming the initial monomolecular layer of antigen molecules
11 which are adsorbed onto the surface of substrate 10.
For purposes of illustration, the antibodies depicted
herein are of the Ig G class, but there are no reasons
why antibodies of the other four major classes enumerated
above cannot be utilized in my invention.
After selection of the substrate 10 on which the
- 10 bimolecular immunologically complex film is to bfifo ~ edgenera y o s~1t water
such substrate is immersed-in a-first solutio~ containing
the antigen as in the case of the method described
hereinabove with reference to FIGURE 1~ But in
contradistinction with such previous method, an immuno-
logically inert protein has been added to the first
solution prior to imme~sion of the substrate therein
such that the adsorption layer formed on the surface
of substrate 10 consists of the relatively large
reactive antigen protein molecules 11 separated from
each other, and surrounded along the substrate surface,
by inert protein molecules 20 as seen in FIG~RE 2. The
inert protein molecules 20 are of a size at least
80mewhat smaller, and preferably substantially smaller
than the antigen molecules 11 in order to allow the
greatest surface area (and therefore more combining
8ites) to remain available on the antigen molecules 11 for
subsequent bonding with antibody molecules. The quantity
of the immunologically inert protein 20 added to the
~olution is sufficient so that, in general, the inert
protein 20 covers 1/2 to 9/10 of the area of the complete
adsorption layer, that is, the antigen corresponding covers
_g_ .
16~484Q9
KD-6735
l/2 to 1/lO of the area, although this is not a limitation
on my invention since the average spacing between adjacent
antigen molecules is also determined by the number of active
sites on the particular antigen molecule and the manner in
which such molecules adhere to the substrate, i.e., the
number of antigenic active sites which remain exposed. Thus,
in the case wherein the antibody molecule 12 is very much
smaller than the antigen, a larger spacing of the antigen
molecules is desirable, whereas if the antibody molecule is
only slightly smaller than the antigen, a smaller spacing
between antigen molecules may be tolerated in order to obtain
the maximum benefit from my invention. As a result of the
large size of the antigen protein relative to the inert
protein molecule, a major portion of the surface of the anti-
gen molecule will remain exposed and has its corresponding
bonding sites available for combining with antibody molecules
in a subsequent immunological reaction.
The (antigen-inert protein) monomolecular coated sub-
strate 10 is then removed from the first solution and is then
immersed in a second solution containing the antibodies
specific to the antigen in the first solution. As a result
of the greater surface area (and more antigenic sites)
remaining exposed on the antigens due to the antigens being
surrounded by the smaller inert protein in FIGURE 2, as
compared to FIGURE 1, the antigen molecules in FIGURE 2 can
more readily immunologically combine with a greater number
of antibody molecules than in FIGURE 1.
Thus, as seen in FIGURE 2, a greater number of antibody
molecules 12 will, in general, combine with each antigen
3~ molecule than in the FIGURE 1 case, and it is obvious from a
comparison of FIGURES 1 and 2 that a much greater change in
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~0484~9
RD-6735
contrsst between the single and double layers is obtaine~ in
the FIGURE 2 embodiment of my invention (due tO the greater
number of antibody molecules that are in the second layer).
For sn exsmple of my invention, the first solution has a con-
centration of hepatitis-associated antigen (HAA) and bovine
serwm albumin (BSA) inert protein sufficient to have the HAA
cover 1/4 of the surface area in the monomolecular layer and
the BSA inert protein cover the remaining 3/4 area and the
8econd solution is a dilute solution of the HAA antibody. The
method described hereinabove can thus be utilized in the
analy$is of a solution for readily detecting the presence of
an antibody specific to a particular relatively large size
antigen.
The double layer coated substrate of FIGURE 2 can sub-
sequently be immersed-in a third solution or a serum contain-
ing antibodies 30 to the antibodies 12 in the second layer
in order to build up a third layer as illustrated in FIGUKE 3.
Since each antibody molecule 12 has several antigenic deter-
minants, it can, in general, combine with several molecules
30 that are antibodies to the antibody 12 to thereby form a
relatively dense third layer of such second antibody molecules
30. The presence of the dense third layer 30 is indicative of
the presence of the second layer 12 and thus provides an even
monomolecular and
greater contrast between the/bimolecular antigen-first anti-
body layers. In this latter case, the inert protein mole-
cules 20 between the large antigen molecules 11 in the first
layer also aid in preventing sticking to the substrate of the
non-specific protein matter in the serum which contains the
antibodies to the antibody of such antigen. Thus, there is
a two-fold gain in that I obtain more specifically bonded
antibodies for each antigen molecule and there are less
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~0484Q9 RD-6735
non-specifically bonded protein on the substrate, all result-
ing from the use of the inert protein for spacing the large
antigsn molecules from each other and for covering the sur-
face of the substrate surrounding each antigen molecule with
such inert protein. The procedure used in forming the third
layer in FIGURE 3 is, therefore, useful in the analysis of the
second solution suspected of containing antibodies 12 since
the subsequent immersion of the coated substrate in the third
solution will provide considerably enhanced contrast between
the monomolecular layer and bimolecular (actually now a
trimolecular layer if a bimolecular layer exists due to the
second solution in fact containing the antibody 12).
The presence of the second (antibody 12) protein layer
in the FIGURE 2 embodiment, which is also indicated by means
of the third (antibody 30) protein layer in the FIGURE 3
embodiment can be readily verified by viewing the coated
substrate with an optical instrument such as an ellipsometer.
Alternatively, and as described in greatex detail in my afore-
mentioned Canadian application S.N. 172,639, as well as
my Canadian application S.N. 204,262, filed July 8, 1974,
the protein layers can also be examined electrically by
measuring the electric capacitance of a capacitor having
conducting plates formed by the metal or metal coated sub-
strate and a mercury drop or other suitable electrode, and
the capacitor dielectric being the protein layers. Also, as
described in my canadian applications, the protein layers can
be examined optically by unaided visual observation by deter-
mining the length of time before a visible amalgam is formed
between a drop of mercury and metal film coated on the sub-
strate with the protein layers therebetween. Finally, and
most importantly, the protein layers can be examined optically
1048409
RD-~735
by reflected light or transmitted light as explained in my
Canadian applications. In this latter optical examination
by reflected or transmitted light, the following is a first
(transmitted light) technique which has successfully been
used: The substrate 10 which must be a light transmissive
substrate such as glass, plastic, fused silica, mica, quartz,
or the like, and is preferably glass, with microscope slides
being a conveniently available source, is first coated with
a plurality of metal globules by evaporating a metal, for
example, indium, onto the substrate. For example, the indium
is evaporated slowly from a tantalum boat onto the glass sub-
strate 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 sig-
nificantly, the indium evaporated onto the substrate agglom-
erates into small particles. Any metal having similar
characteristics so that it will form globules on the sub-
strate when evaporated thereon may be used. In addition to
indium, gold, silver, tin, and lead have been successfully
used. The evaporation of metal is continued until the sub-
strate appears light brown in color. At this point, the
metal globules have diameters on the order of 1000 ~. The
precise size of the globules is not cirtical but they must
have diameters equal to a large fraction of a wavelength of
visible light. The next step is to immerse the globule-
covered substrate 10 in a first solution of a first immuno-
logically reactive protein such as the antigen 11 and the
inert protein 20. The first reactive protein and inert
protein again adhere in a monomolecular layer over the sub-
strate and the metal globules thereon. When a monomolecular
layer has formed, the coated substrate is then removed from
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RD-6735
the first solution and immersed in a second solution con-
taining (or suspected of containing) the specifically reacting
protein 12 to the first protein and results(in the presence of
such protein 12 in the second solution) in the substrate and
metal globules having a bimolecular protein layer adhering
thereto similar to that shown in FIGURE 2 (i.e., without the
metal globules). The coated substrate is then removed from
the second solut-ion and immersed in a third solution which
contains a reactive protein to the reactive protein in the
second solution (such as an antibody to the antibody) to form
a third layer (or partial layer) on the substrate if the
second layer is present. The coated substrate is then viewed
by transmitted light, and a determination is made from the
appearance of the coated substrate as to the thickness of the
protein layer adhering thereto and accordingly as to the
presence or absence of the second protein 12. The detection
of protein layers corresponds to variations in the shade of
brown which is observed in the coated substrate. These vari-
ations are quite pronounced and the detection of protein
layers is therefore a simple straightforward procedure. The
particles alone on the substrate appear as a first shade of
brown, the particles coated with a monomolecular protein
layer appear as a darker shade of brown, the particles covered
with a bimolecular protein layer appear as a still darker
shade of brown, and the particles covered with a trimolecular
protein layer appear darkçr still. This detection method is
based on the fact that electromagnetic radiation is scattered
to a large degree by conducting spheres having diameters equal
to a large fraction of a wavelength of the incident energy
and that in the case of scattering from such spheres, the
scattering is strongly influenced by a thin dielectric coating
-14-
~048409
RD-6735
applied to the spheres. A second technique for optical
examination by reflected light which has successfully been
used is as follows: A gold substrate, which, for reasons of
economy, is preferably a thin gold layer plated onto another
metal, has adsorbed thereon a monomolecular layer of the
first reactive protein 11 and inert protein 20 after immer-
sion in the hereinabove identified first solution. Gold has
an absorption band within the visible spectrum, and this
fact accounts for the characteristic color of gold and pro-
~ides for the operation of this particular optical examin-
ation technique. The gold substrate may conveniently be a
: glass slide coated with a thin indium layer and overcoated
the
- with the gold layer wherein/indium layer improves both the
adhesion between the glass and~ the gold as well as the
optical characteristics of the slide. The relative reflect-
ivLty of the gold substrate as a function of wavelength
results in the substrate having the characteristic bright
yellow color of gold metal in the absence of any protein
layer a & ering thereon. In the presence of a monomoiecular
layer on the substrate, the appearance of the test slide
(substrate) has a dull yellow appearance. After the test
slide has been exposed to a solution cobtaining the immuno-
logical reacting protein 12 to the protein 11 which is in the
first layer along with the inert protein 20, the test slide,
has a bimolecular protein layer thereon and a reflectivity
characteristic which provides a greenish appearance. A
third protein layer provides an even more greenish appearance.
In tests which have been performed to date, it appears that
the optical examinations of the coated substrate by reflected
or transmitted light and which employ a substrate including
metal globules or a gold substrate are the most generally
-15-
1048409 RD-6735
useful. Furthermore, it has been determined that the~e two
techniques have different sensitivities as functions o~ the
thicknesses of protein film~ of interest. Specifically, the
greatest sensitivity of the technique having a substrate
including metal globules occurs with films having thicknesses
below approximately 200 A . The gold ~ubstrate technique
has the greatest sensitivity for films exceeding 30 A in
thickne3s. The partlcular detection method employed thus
determines the type of substrate 10 utilized in each analysis,
that is, whether the substrate iB a metal or metallized glass
~lide, with a flst metal (gold, as one example) coating or
metal (indlum, as one example) globules on the surface, as
again explained in my copending applications. For purposes of
slmplification, I have illustrated the sub~trate 10 herein
as having a flat surface, although it i~ to be understood that
the surface to which the antigen layer adsorbs could also
contain the aforementioned metal globules.
The cor.tra~t between single and double protein layers
can be enhanced to an even greater extent by depositing the
antigen and inert protein on substrate 10 as a single drop of
the solution, and subeequently immersing the drop-coated
sub~trate into a solution containing only the inert protein 20
so as to form a monomolecular layer of the small antigen-inert
protein area completely surrounded by the inert protein as
depicted in FIGURE 4. This feature results in overcoming
the problem of nonspecific adsorption in the case where the
antibodies are in a serum since the inert protein along the
remaining surface of the substrate prevents the nonspec~fic
proteins in the serum from adhering to the substrate and
thereby improve the contrast.
The simple procedures described hereinabove have now
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lO9U3 ~ 9 RD-6735
resulted in the ability to detect hepatitis by a test whic~.
is as sensitive as the standard radioimmuneassy tes~.
Finally, the procedure described hereinabove is important
in identifying viruses and enzymes immunologically. All anti-
~ens of the virus type are larger than their specific anti-
bodies, whereas the antigens of the enzyme type may be larger
or smaller, depending upon the particular type of enzyme.
A procedure for identifying a particular virus or large
size enzyme ~ known as the inhibition test, is accom-
plished in the following manner: The particular virus or
large size enzyme is put into a first solution (generally
salt water) with the inert protein and the substrate is then
immersed therein, or alternatively, a drop of the solution
is placed on the substrate and then the substrate is
immersed in a solution of only the inert protein. After
a monomolecular layer of the virus or enzyme (i.e., antigen)
and inert protein has been adsorbed on the substrate, the
coated substrate is removed from the first solution.
Samples of human serum to be tested for the presence of the
~0 particular virus or enzyme are prepared by mixing therein
a quantity of known antibodies to the particular virus or
enzyme sufficient to be immunologically removed from the
mixture if the virus or enzyme is present in the serum
sample. The monomolecular coated substrate is then immersed
in,or exposed to, the previously prepared serum sample and
upon removal therefrom is examined in accordance with any
of the procedu~es described hereinabove. If inspection of
the substrate indicates the presence of only a monomolecular
layer thereon, then it is known that the human serum under
test originally cont~ined the particular virus or enzyme.
However, if a bimolecular layer is detected, then this
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10484~9 RD-6735
indicates that Lhe serum did not originally contain such
virus or large size enzyme antigen since the antibodLes
did not immunologically combine with their specific antigen
tvirus or enzyme) in the serum sample, and therefore were
free to combine with the antigen of the first layer
adsorbed on the substrate.
From the foregoing description, it can be appreciated
that my invention makes available a new method for improv-
ing the contrast in surface immunological tests between
10 - single and double layers of protein by the addition of an
immunologically inert protein of sufficient quantity to a
first solution containing an immunologically reactive
; antigen protein. ~-~ ~ ~
Having described my invention with reference to the
par~icular embodiments and examples, it is believed obvious
that modification and variation of my invention is possible
- in the light of the above teachings. Thus, the inert
protein could be in a solution separate from the antigen,
and the substrate would be immersed therein as a preliminar~
step, or as an intermediate step between immersions in a
dilute solution of the antigen and the antibody solution.
As another approach, in the case where the antibodies are
in a serum, a dilute solution of the antigen would be
utilized in forming an incomplete first layer on the
substrate, and the inert protein would be omitted in such
solution since the nonspecific protein in the serum would
function as the inert protein in adhering to the substrate
8nd thereby act as the spacing agents between the ant~gen
molecules. Finally, although ~he reactive protein
adsorbed on the substrate surface has been described
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1~)48409
RD-6735
hereinabove as being an antigen, it should be evident that
such first lay~r reactive protein could be an antibody,
and the second layer protein would then be the specific
antigen, the latter arrangement being useful in cases
S wherein an antibody molecule is larger than its specific
antigen molecule, an example being the antibody to insulin.
It is, therefore, to be understood that changes may be
made in a particular embodiment of my invention as
described which is within the full intended scope of the
invention as defined by the following claims.
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