U.S. patent number 3,904,367 [Application Number 05/388,407] was granted by the patent office on 1975-09-09 for contrast enhancement for immunological film detection.
This patent grant is currently assigned to General Electric Company. Invention is credited to David C. Golibersuch.
United States Patent |
3,904,367 |
Golibersuch |
September 9, 1975 |
Contrast enhancement for immunological film detection
Abstract
A method for increasing the sensitivity of immunologic
diagnostic slides by increasing the contrast observable between
immunologically reacted and unreacted portions thereof. The method
is based on the fact that an arbitrary antibody functions as an
antigen when introduced into the system of a vertebrate to whom it
is a foreign protein. Specifically reactive antibodies to the
antibodies so introduced are therefore produced and may be
collected. Accordingly, a plurality of specific immunological
reactions may be performed in chain fashion. This technique is used
in accordance with one embodiment to build a plurality of
successive antigen-antibody films on a substrate to provide for
contrast between multilayer protein films and a monomolecular layer
which is in excess of the contrast obtainable between monomolecular
layers and bimolecular layers to thereby provide diagnostic
apparatus of increased sensitivity.
Inventors: |
Golibersuch; David C.
(Schenectady, NY) |
Assignee: |
General Electric Company
(Schenectady, NY)
|
Family
ID: |
27012299 |
Appl.
No.: |
05/388,407 |
Filed: |
August 15, 1973 |
Current U.S.
Class: |
436/518; 436/805;
422/69; 436/820; 422/426 |
Current CPC
Class: |
A61L
2/26 (20130101); G01N 33/54393 (20130101); Y10S
436/805 (20130101); Y10S 436/82 (20130101) |
Current International
Class: |
A61L
2/26 (20060101); G01N 33/543 (20060101); G01N
033/16 () |
Field of
Search: |
;23/23B,253R,253TP
;424/12 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chemical Abstracts, 52: 6435h-i (1958)..
|
Primary Examiner: Wolk; Morris O.
Assistant Examiner: Marantz; Sidney
Attorney, Agent or Firm: MaLossi; Leo I. Cohen; Joseph T.
Squillaro; Jerome C.
Claims
The invention claimed is:
1. A method for improving the detection sensitivity of immunologic
diagnostic slides, said slides comprising a substrate having a
layer of particles of a first protein adsorbed thereon for
specifically reacting immunologically with a second protein, said
second protein being the protein to be detected, said method
comprising the steps of:
a. contacting said slide with a fluid suspected of containing said
second protein to bond a plurality of particles of said second
protein to particles of said first protein; and then
b. contacting said slide with a solution of a third protein
immunologically reactive with said protein to be detected, said
first, second and third proteins all being different from each
other.
2. The method of claim 1 further comprising the steps of:
preparing a solution of antibodies to the third protein
immunologically reactive with the second protein to be detected;
and
c. contacting said slide resulting from step (b) with said solution
of antibodies.
3. The method of claim 1 further comprising the steps of:
preparing a series of solutions of antibodies, each solution of
said series comprising antibodies to the antibodies of the next
preceding solution of the series; and
c. serially contacting said slide resulting from step (b) with each
solution of said series.
Description
This invention relates to the immunological detection of protein by
detection of immunological complexing between a protein previously
absorbed on a surface to form a test slide and the specifically
immunologically reactive protein thereto. More particularly, this
invention relates to the improvement of contrast observable on such
immunologic diagnostic slides by the selective immunologic bonding
thereto of additional protein layers.
This application is related to the copending applications of
Giaever, Ser. No. 266,278, filed June 26, 1972; Ser. No. 384,113,
filed July 30, 1973; and Ser. No. 388,406, filed Aug. 15, 1973 now
U.S. Pat. No. 3,853,467. These related copending applications are
assigned to the assignee of this invention and are incorporated
herein by reference thereto.
The above cited copending applications, Ser. No. 266,278 and Ser.
No. 384,113 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 absorbed 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 substrate having a
monomolecular layer of one protein absorbed 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 immersion in the solution, has a
bimolecular protein layer on the substrate. If the specifically
reacting protein be absent from the solution, the slide after
immersion, has only the original monomolecular protein layer on the
substrate. Optical, electrical, and chemical means for
distinguishing between a bimolecular protein layer and a
monomolecular protein layer are taught in these related copending
applications of Giaever.
In two embodiments of diagnostic apparatus taught in these two
related copending applications of Giaever, optical distinction
between monomolecular and bimolecular protein layers is made by
unaided visual observation of the slide and is further taught
therein to provide detection sensitivities which are functions of
the structure of the slide and of the absolute thickness of the
protein layer or layers thereon. Specifically, the diagnostic slide
taught by Giaever to comprise a plurality of metal globules
attached to a substrate and having a monomolecular protein layer
thereon is taught to have maximum sensitivity for films thinner
than 200 A, while the embodiment taught by Giaever to comprise a
gold surface substrate is taught to have maximum sensitivity for
films exeeding 30 A in thickness. The chemical detection embodiment
taught by Giaever involves the use of a substrate having a surface
of a material which forms a visible amalgam with mercury and
depends for its operation on Giaever's discovery that a mercury
drop placed atop a bimolecular protein layer requires approximately
ten times as long a time to diffuse therethrough as a mercury drop
placed atop a monomolecular protein layer. The electrical detection
embodiment taught by Giaever involves the use of an electrically
conductive substrate material and the placing of an electrode atop
the protein layer thereon; the capacitance between the two
conductive members is then measured and its value is an indication
of the thickness of the dielectric protein film separating them and
hence, whether such film is monomolecular or biomolecular. It may,
therefore, be seen that the electrical and chemical embodiments
taught by Giaever require that the protein to be detected, if
present in the solution under test, form a complete second
monomolecular layer on the substrate of the diagnostic slide.
The techniques and apparatus taught by Giaever provide for rapid,
reliable, and inexpensive detection of immunologically reactive
particles. However, in the case of the gold surface substrate, the
immunologically reactive particles must exceed approximately 20 A
in diameter. More significantly, some particles, whose detection is
highly significant, are present in physiologic specimens only in
highly dilute concentrations. In this case, only a small plurality
of such particles become bonded to the diagnostic slide and their
detectability is limited.
In the special case of large particles found in dilute
concentrations, for example, viruses, Giaever has provided, in
accordance with the teachings of the above-referenced copending
application, Ser. No. 388,406, means for reliable detection of the
particles. In this special case, as taught in application Ser. No.
388,406 the test slide includes a layer of an etchable metal
underlying a layer of, for example, antibodies to the virus to be
detected. A thin layer of non-etchable metal is deposited over the
slide and any viruses bonded thereto. After etching, sites at which
any viruses have been bonded appear as voids in the slide of
sufficient size to be detected by, for example, optical
microscopy.
It is known in the immunological arts 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. This capability has been utilized in the prior art to
provide for attachment of fluorescent molecules to immunologically
complexed proteins to aid in the detection of particular species of
microorganisms. For example, antibodies to human immunoglobulins
may be prepared and chemically combined with flourescent organic
molecules such as the isothiocyanates. This socalled tagged
antibody is then used to render an immunological reaction visible
by ultraviolet microscopy. One of the most significant utilities
for such antibodies to antibodies known to the prior art is the
serological test for syphilis after the method of Coons, now known
in the art as FTA-STS procedure. In the FTA-STS procedure, a
quantity of Treponema pallidum (T. pallidum) is dried on a slide.
The slide is then immersed in a blood specimen. The slide is
subsequently immersed in a solution of tagged immunoglobulin. The
anti-human immunoglobulin does not bond to the T. pallidum;
accordingly, only if the specimen contained antibodies to T.
pallidum will the slide fluoresce when observed by ultraviolet
microscopy.
Thus, the prior art may be seen to have found utility for a single
immunological reaction. However, no utility is known in the prior
art for serially occurring immunological reactions.
It is the principal object of this invention to improve the
detection sensitivity of immunologic diagnostic slides.
A further object of this invention is to improve such detection
sensitivity by building multimolecular layers of specifically
reacting proteins on such slides.
It is another object of this invention to improve the detection
sensitivity of such slides by bonding additional protein layers
thereon in sufficient quantity to insure the presence of at least
two additional complete monomolecular protein layers on a surface
of such an immunologic diagnostic slide.
Briefly, and in accordance with one embodiment of this invention,
an immunologic test slide having as one surface thereof a
monomolecular layer of protein which is immunologically reactive
with the protein to be detached is immersed in a solution suspected
of containing the protein to be detected. After immersion in the
suspected solution, the substrate has a bimolecular protein layer
thereon consisting of the original monomolecular protein layer and
overlying monomolecular layer of the protein to be detected if the
protein to be detected was present in the suspected solution. On
the other hand, if the suspected solution did not contain the
protein of interest, the slide after immersion therein has only the
original monomolecular protein layer thereon. The slide is then
immersed in a solution of protein which reacts immunologically with
the protein of the second monomolecular layer. The slide then has a
trimolecular protein layer thereon if the suspected solution
contained the protein of interest or a monomolecular layer thereon
if the suspected solution did not contain the protein of interest.
Accordingly, detection sensitivity of the slide is improved because
of the increased contrast between a monomolecular and trimolecular
protein layer as opposed to that between a monomolecular and
bimolecular protein layer.
The novel features of this invention sought to be patented are set
forth with particularity in the appended claims. The invention,
together with further objects and advantages thereof, may be
understood from a reading of the following specification and
appended claims in view of the accompanying drawing in which:
FIG. 1 is a schematic illustration of the attachment of a plurality
of protein layers to a substrate.
FIG. 2 is an immunologic test cross-sectional elevation view of a
slide having three monomolecular protein layers thereon.
FIG. 3 is a sectional elevation view of a slide prepared in
accordance with one embodiment of this invention illustrating the
completion of a monomolecular layer on a slide.
FIG. 1 shows a substrate 10 having adsorbed thereon a plurality of
arbitrary protein molecules or other particles, such as, for
example, viruses or hepatitis associated antigen particles 11 in
accordance with the teachings of the aforementioned copending
applications of Giaever. A plurality of molecules or other
particles 11 are immunologically complexed with their specifically
reacting antibody molecules indicated generally at 21 and 22.
Antibody molecules 21 and 22 are illustrated schematically to
facilitate an explanation of the structure thereof. For
convenience, unless otherwise required by specific context, the
initial particle layer is referred to hereinafter as "molecules",
it being understood that other particles are also included in the
scope of this invention.
The five major classes of antibodies comprise the immunoglobulins
designated as IgG, IgM, IgA, IgD, and IgE. The preponderant
immunoglobulin is IgG, which constitutes approximately 85 percent
of the immunoglobulins in normal human sera and is found in roughly
similar preponderance in the normal sera of other vertebrates. An
IgG particle comprises two heavy peptide chains and two light
peptide chains joined by disulphide bonds to form a generally "Y"
shaped antibody molecule. IgG antibody molecules are
immunologically divalent; that is to say, each molecule has two
antibody active sites. The active sites 21a1 and 21a 2 of molecule
21 and 22a1 and 22a2 of molecule 22 are located at the extreme ends
of the arms of the Y-shaped molecule. The active sites provide for
immunologic bonding of the antibody to a molecule of its associated
antigen, as shown, for example, in FIG. 1 where active sites 21a1,
22a1, and 22a2 are bonded to a plurality of protein molecules 11.
As previously pointed out, antibody molecules also function as
antigens when introduced into a vertebrate system to which they are
a foreign protein. They accordingly stimulate the production of
antibodies specific to the anitbody qua antigen. An antibody to IgG
antibody is another IgG antibody, and accordingly also has two
active sites. These active sites bond to particular antigenically
active sites on the first antibody. The antigenically active sites
are known alternatively as antigenic determinants or epitopes. As
previously stated, the number of antibody active sites of an IgG
antibody is known to be 2. The number of epitopes associated with
an IgG molecule is not precisely known but the number of epitopes
is known to be at least 2. Epitopes are known to be present on the
tail of the Y-shaped molecule and are probably also present on the
arms thereof. For the purposes of illustrating the principle of the
operation of this invention, three epitopes are shown disposed
along the heavy peptide chains of each antibody molecule
illustrated in FIG. 1. The epitopes of antibody molecule 21 are
designated as 21e1, 21e2, and 21e3. The epitopes of antibody
molecule 22 are designated as 22e1, 22e2, and 22e3.
Thus, the adsorption of molecules 11 onto substrate 10 and the
immersion of the resulting immunologic test slide into a solution
of antibodies to protein 11 to immunologically bond molecules 21
and 22 thereto constitutes the practice of the aforementioned
inventions of Giaever. In accordance with the practice of this
invention, a quantity of solution of antibodies of type represented
by 21 and 22 is injected into a vertebrate to stimulate the
production of antibodies thereto. The antibodies produced are
collected by means known in the art, and a solution thereof is
provided for use in the improved diagnostic procedure in accordance
with this invention. The slide is immersed in a solution of the
anti-antibodies and if molecules 21 and 22 are bonded to molecules
11, a plurality of molecules 31, 32, 33, 34, and 35 become
immunologically bonded to molecules 21 and 22. If, on the other
hand, antibodies of the type of molecules 21 and 22 are absent from
the solution into which the slide was first dipped, molecules 31-35
will be absent from the slide after immersion into the solution
containing them because molecules 31-35 react specifically with
molecules 21 and 22 and will not attach to molecules 11.
Accordingly, at this stage, the procedure in accordance with this
invention, the detectability of molecules 21 and 22 is improved in
that their presence is determined by distinguishing between a layer
of molecules 11 and a layer of molecules 11, 21, 22, 31, 32, 33,
34, and 35 as opposed to distinguishing between a layer of
molecules 11 and a layer of molecules 11, 21, and 22. Further, in
accordance with this invention, the process may be repeated to add
additional protein layers to further increase detection
sensitivity, as for example, the selective bonding of molecules 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, and 53 to molecules 31,
32, 33, 34, and 35.
Antibody molecules 31, 32, 33, 34, and 35 have respectively active
sites 31a1, 31a2, 32a1, 32a2, 33a1, 33a 2, 34a1, 34a2, 35a 1, and
35a 2. Antibody 31 is immunologically bonded to antibody 21 by the
attachment of active site 31a 2 to epitope 21e2. Antibody 32 is
immunologically bonded to antibody 21 by the attachment of active
site 32a1 to epitope 21e1. Antibody 33 is immunologically bonded to
anitbody 22 by the attachment of active site 33a2 to epitope 22e2;
antibody 34 is bonded to antibody 22 by the attachment of active
site 34a 1 to epitope 22e1; antibody 35 is bonded to antibody 32 by
the attachment of active site 35a1 to epitope 22e3. As shown on the
lelf-hand side of FIG. 1, with regard to the protein chain
beginning with antibody 21, each antibody is shown as having two
active epitopes. That is, each antibody qua antigen binds two
antibodies thereto. Thus, antibody 21 has antibodies 31 and 32
bonded thereto which in turn have antibodies 41, 42, 43, and 44
bonded thereto. This corresponds to the known minimum number of
active epitopes per IgG antibody molecule. On the right-hand side
of FIG. 1, the protein chain beginning with antibody 22 is
illustrated as having three active epitopes per antibody. Thus, it
may be seen that the sensitivity improvement obtained in accordance
with this invention is proportional to the number of active
epitopes per antibody molecule and the number of immersions of the
slide into successive antibody solutions.
In one example of sera immunological reactions performed in
accordance with this invention, a layer of bovine serum albumin was
adsorbed on a substrate. The resulting slide was then immersed in a
solution of rabbit anti-serum to bovine serum albumin, after which
the slide contained the original bovine serum albumin layer having
immunologically bonded thereto a layer of rabbit IgG immunoglobulin
antibodes to bovine serum albumin. The slide was next immersed in a
solution of goat anti-serum to rabbit IgG, after which the slide
contained a layer of bovine serum albumin, a layer of rabbit
anti-BSA IgG, and a layer of goat anti-rabbit IgG antibodies. The
next step was the immersion of the slide in a solution of rabbit
anti-serum to goat IgG to add a fourth layer of rabbit anti-goat
IgG molecules to the slide. Clearly, the last two steps of goat
anti-serum to rabbit IgG, followed by rabbit anti-serum to goat IgG
may be repeated serially for as many operations as desired to
thereby build an n-molecular layer protein film.
In another example of the application of this technique, a quantity
of hepatitis associated antigen particles was adsorbed on a
substrate. The resulting slide was then immersed in a human serum
specimen. The slide was next immersed in a solution of rabbit
antibodies to human hepatitis antibody. Accordingly, the slide, if
the human test serum contained hepatitis antibody, has thereon
hepatitis associated antigen having hepatitis antibody
immunologically bonded thereto and rabbit anti-human hepatitis
antibody immunologically bonded to the hepatitis antibody. On the
other hand, if the serum sample were negative, the slide would have
only the original hepatitis associated antigen particles thereon.
This technique in accordance with this invention was found to
significantly improve the detectability of human hepatitis over
that obtainable by the bonding of hepatitis antibody to hepatitis
associated antigen alone. This example points out that the
technique of this invention has particular utility in cases in
which the initial protein including particle deposited on the slide
is substantially larger than its specifically reacting protein.
FIG. 2 is a sectional elevation view illustrating a multimolecular
protein film on a slide. Substrate 60 may, for example, in
accordance with the first example cited above, initially have a
monomolecular layer 61 of bovine serum albumin adsorbed thereon to
constitute an immunologic diagnostic slide. Immersion in rabbit
anti-serum to bovine serum albumin will adhere a second
monomolecular layer 62 over layer 61 by specific immunologic
reaction. Subsequent immersion in goat anti-serum to rabbit IgG
will immunologically bond monomolecular layer 63 to layer 62 if
layer 62 is present. Similarly, layer 64 comprising rabbit
anti-goat IgG is bonded to the slide by immersion in rabbit
anti-serum to goat IgG. As indicated above, the process may be
continued but since the bonding of layers is a specific immunologic
reaction, the process will be irrevocably interrupted if any one
layer does not form. Specifically, the structure of FIG. 2 has
utility for high sensitivity detection of the protein comprising
layer 62. Layer 61 is attached to slide 60 by adsorption which is a
non-specific process. All subsequent processes, however, are
specific. Accordingly, if a test specimen contains the specifically
reacting protein to the protein of layer 61, layer 62 forms and an
n-molecular protein layer can be formed thereover in accordance
with this invention. If, on the other hand, the test specimen does
not contain protein immunologically reactive with the protein of
layer 61, layer 62 does not form and no subsequent layers can be
formed. Therefore, a protein of interest in a specimen may be
detected with any desired degree of sensitivity by distinguishing
between a monomolecular protein layer on a substrate and a layer of
any desired thickness.
FIG. 3 is a cross-sectional elevation view of a slide illustrating
another utility for the technique of this invention. FIG. 3
illustrates the utility of this invention in the detection of
proteins which are present in physiologic specimens in
concentrations too dilute to provide for the formation of a
complete layer on a slide. As an example utilizing specific
proteins and not by way of limitation to those proteins, a
substrate 70 has absorbed adsorbed a monomolecular layer of human
hepatitis antibodies 71. The resulting slide is immersed in a blood
specimen and a plurality of hepatitis associated antigen particles
77 become immunologically bonded thereto if they are present in the
specimen. The slide is then immersed in a solution of monkey
hepatitis antibody to bond a layer of molecules 72 thereof on such
hepatitis associated antigen particles as may be bonded to the
slide. The slide is next immersed into a solution of rabbit
anti-serum to monkey IgG to bond a layer of molecules 73 thereof
over the monkey hepatitis anitbody molecules. It is clear that the
layer of molecules 72 could be formed of human hepatitis antibody
71. However, if this were done, the next layer would have to be
anti-human IgG molecules which would bond not only to those
antibodies surrounding the hepatitis associated antigen particles
77 but also to the original layer of molecules 71 thereby defeating
the purpose of the technique of this invention. The next immersion
of the slide may be, for example, into goat anti-serum to rabbit
IgG to form a layer of molecules 74 thereof on the slide. This
process is repeated a number of times as indicated generally by the
volume shown as 75 until the point is reached at which a last
monomolecular protein layer 76 completely fills the spaces between
particles 77 on the slide. This embodiment of this invention
provides for the formation of a protein layer of at least
bimolecular thickness over the entirety of the slide and has
particular utility to the chemical and electrical detection methods
disclosed in the aforementioned copending applications of Giaever
as discussed above.
While this invention has been described with reference to
particular embodiments and examples, other modifications and
variations will occur to those skilled in the art, in view of the
above teachings. Accordingly, it should be understood that within
the scope of the appended claims, the invention may be practiced
otherwise than is specifically described.
* * * * *