U.S. patent number 3,853,467 [Application Number 05/388,406] was granted by the patent office on 1974-12-10 for method and apparatus for immunological detection of biological particles.
This patent grant is currently assigned to General Electric Company. Invention is credited to Ivar Giaever.
United States Patent |
3,853,467 |
Giaever |
December 10, 1974 |
METHOD AND APPARATUS FOR IMMUNOLOGICAL DETECTION OF BIOLOGICAL
PARTICLES
Abstract
Method and apparatus for the detection of biological particles
such as viruses, bacteria, and other cells by detection of the
occurrence of an immunological reaction between the particle to be
detected and its antibody. In one embodiment a substrate having an
etchable metal surface is coated with a layer of antibodies to the
biological particle to be detected. The antibody coated substrate
is exposed to a fluid suspected of containing the biological
particles to be detected. After such exposure, the structure is
coated with a layer of a non-etachable metal of sufficient
thickness to cover the antibody layer but of insufficient thickness
to completely coat the biological particles and form a continuous
film. The structure is then exposed to an etchant. After etching,
positions at which biological particles had been immunologically
bonded to their antibodies are observable as voids in the
structure.
Inventors: |
Giaever; Ivar (Schenectady,
NY) |
Assignee: |
General Electric Company
(Schenectady, NY)
|
Family
ID: |
23533993 |
Appl.
No.: |
05/388,406 |
Filed: |
August 15, 1973 |
Current U.S.
Class: |
435/5; 422/504;
435/7.21; 436/525; 436/806; 216/108; 216/84; 435/287.2; 435/287.9;
428/433; 435/7.32; 436/805 |
Current CPC
Class: |
C12Q
1/70 (20130101); G01N 33/553 (20130101); Y10S
436/805 (20130101); Y10S 436/806 (20130101) |
Current International
Class: |
C12Q
1/70 (20060101); G01N 33/551 (20060101); G01N
33/553 (20060101); G01n 021/04 (); G01n 031/06 ();
G01n 033/16 () |
Field of
Search: |
;23/23B,253R,253TP
;195/13.5R ;424/12 ;117/71R |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
chemical Abstracts, 63:3532d (1965). .
Chemical Abstracts, 63:10483b (1965). .
Chemical Abstracts, 65:2821g (1966). .
Chemical Abstracts, 73:48541m (1970). .
G. M. Edelman et al., Proc. Nat. Acad. Sci., 68, (9), 2153-2157
(Sept. 1971)..
|
Primary Examiner: Wolk; Morris O.
Assistant Examiner: Marantz; Sidney
Attorney, Agent or Firm: Edelson; Paul I. Cohen; Joseph T.
Squillaro; Jerome C.
Claims
The invention claimed is:
1. Apparatus for detecting biological particles of a particular
species in a fluid comprising:
a substrate;
a metal film on a surface of said substrate;
a layer of antibodies to said species of biological particle, said
antibodies being adsorbed on said metal film;
means for applying a second metal layer over said layer of
antibodies and any of said biological particles bonded thereto;
and
means for selectively etching said biological particles, said metal
film, and said antibodies.
2. The apparatus of claim 1 wherein:
said means for selectively etching comprises an acid;
said metal film comprises a metal which is etchable by said acid;
and
said second metal layer comprises a metal which is resistant to
etching by said acid.
3. The apparatus of claim 2 wherein said second metal layer has
voids therein, said voids being disposed about the peripheries of
said biological particles.
4. The apparatus of claim 3 wherein said substrate comprises a
light-transmissive material which is resistant to etching by said
acid.
5. The apparatus of claim 4 including additionally means for
directing light onto a surface of said substrate.
6. The apparatus of claim 5 wherein said means for directing light
onto a surface of said substrate comprises:
an enclosure having first and second end members, said second end
member having an aperture therein;
a lamp within said enclosure adjacent said first end member;
and
means attached to a surface of said second end member external to
said enclosure for receiving said substrate and for positioning
said substrate adjacent said aperture.
7. The apparatus of claim 6 further including means external to
said enclosure for receiving light transmitted through said
substrate and for indicating the quantity of light energy
received.
8. A method for detecting biological particles of a particular
species in a fluid comprising the steps of:
immersing a substrate having a metal surface into a solution of
antibodies to said species of biological particle to adhere a layer
of said antibodies thereon;
immersing said substrate having said antibodies thereon into said
fluid whereby a plurality of said biological particles, if present
in said fluid, immunologically bond to said antibodies;
applying a second metal layer over said layer of antibodies and any
of said biological particles;
immersing said substrate in an etchant solution; and
examining said substrate to determine whether any of said
biological particles had bonded to said substrate.
9. The method of claim 8 further including the step of:
bonding a metal film to a wafer of light-transmissive material to
form said substrate.
10. The method of claim 8 wherein said applying step more
particularly comprises the steps of:
immersing said substrate in a solution of a salt of said second
metal;
immersing a block of said second metal in said solution of salt;
and
applying an electrical potential difference between said block and
said metal surface to electroplate said second metal onto said
substrate.
11. The method of claim 8 wherein said applying step comprises
evaporating said second metal onto said substrate.
12. The method of claim 8 wherein said applying step further
includes the step of monitoring the deposition of said second metal
to insure that said second metal layer is discontinuous about the
peripheries of said biological particles.
13. The method of claim 8 wherein said examining step comprises
microscopic examination of said substrate.
14. The method of claim 8 wherein said examining step more
particularly comprises:
backlighting said substrate; and
visually observing as points of transmitted light locations on said
substrate at which said biological particles had been bonded.
15. The method of claim 8 wherein said examining step more
particularly comprises:
backlighting said substrate;
receiving and integrating light transmitted through said substrate;
and
indicating the integrated quantity of said light to provide a
measure of the concentration of said biological particles in said
fluid.
16. Apparatus for detecting biological particles of a particular
species in a fluid comprising:
a substrate:
an etchable film on a surface of said substrate;
a layer of antibodies to said species of biological particle, said
antibodies being adsorbed on said etchable film;
means for applying a layer of non-etchable material over said layer
of antibodies and any of said biological particles bonded thereto;
and
means for selectively etching said biological particles, said
etchable film, and said antibodies.
17. The apparatus of claim 16 wherein:
said means for selectively etching comprises an acid;
said etchable film comprises a metal which is etchable by said
acid; and
said layer of non-etchable material comprises a metal which is
resistant to etching by said acid.
18. The apparatus of claim 16 wherein said etchable film is opaque
and said non-etchable material is light transmissive.
Description
This invention relates to immunological detection of biological
particles. More particularly, this invention relates to
immunological detection of biological particles such as viruses,
bacteria, and other cells which are very much larger than molecules
of their specifically reacting antibody proteins. This invention
further has particular utility in the immunologic detection of
particles whose presence in physiologic fluids in very low
concentrations is of diagnostic interest, as for example,
viruses.
This application is related to the copending application of
Giaever, Ser. No. 266,278, filed June 26, 1972, and the copending
application of Giaever, Ser. No. 384,113, filed July 30, 1973.
These copending applications are commonly assigned with this
application.
The related copending applications cited above 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. This discovery is exploited for
diagnostic purposes in accordance with the teachings of the
aforementioned related copending applications by means disclosed
therein for efficiently and economically distinguishing between a
monomolecular protein layer and a bimolecular protein layer on a
substrate.
The diagnostic methods disclosed in the aforementioned copending
applications require the formation of a substantially complete
biomolecular protein layer for detection. While in theory, any
immunologically reactive particle will form a detectably complete
bimolecular layer when exposed to a slide coated with a
monomolecular layer of its specifically reacting protein, in cases
in which the particle to be detected is present in very dilute
concentration in a fluid, detection in accordance with the
teachings of the referenced copending applications may be
unacceptably time-consuming.
A number of biologic particles are of significant diagnostic
interest in substantially dilute concentrations. A prime example of
these are the viruses. It is important to be able to detect the
presence of viruses in body fluids in concentrations on the order
of several thousand virus particles per cubic centimeter. Viruses
are uniformly immunologically reactive. Viruses are not detectable
by means of optical microscopic examination.
It is accordingly an object of this invention to provide method and
apparatus for the immunologic detection of large biological
particles.
It is another object of this invention to provide such method and
apparatus which is highly sensitive so that particles present in
highly dilute concentrations may be detected within short periods
of time.
A further object of this invention is to provide such method and
apparatus which provides for both the detection of such biological
particles and for a determination of the concentration thereof in a
fluid specimen.
Yet another object of the invention is to provide such method and
apparatus which is sufficiently sensitive to provide for the
detection of a single biological particle.
Briefly, and in accordance with one embodiment of this invention, a
wafer of substrate material has a film of etchable metal on one
surface thereof. The metal film is coated with a monomolecular
layer of antibodies to the biological particle to be detected. The
substrate is then immersed in a fluid specimen to be tested for the
presence of the particular species of biological particle of
interest. Subsequently, a layer of etch-resistant metal is applied
to the substrate over the layer of antibodies and any biological
particles bonded thereto. The substrate is then immersed in an
etchant bath which dissolves the biological particles, those
antibodies immediately underlying the biological particles, and a
portion of the etchable metal film. After etching, the substrate is
examined and points at which the biological particles to be
detected had been bonded thereto are observed as voids in the
non-etchable metal layer on the substrate.
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 sectional elevation view of apparatus in accordance
with this invention showing a substrate having an etchable film
thereon, a monomolecular antibody layer over the metal film, and a
biological particle immunologically bonded to the antibody
layer.
FIG. 2 is a sectional elevation view of the apparatus in FIG. 1
having additionally a non-etchable metal layer thereover and
further illustrating means for applying the non-etchable layer in
accordance with one embodiment of this invention.
FIG. 3 illustrates the etching of the apparatus of FIG. 2.
FIG. 4 is an isometric view, partially cut away, of apparatus in
accordance with one embodiment of this invention for examining the
etched apparatus of FIG. 3.
FIG. 1 is a sectional elevation view of a diagnostic slide in
accordance with this invention indicated generally at 30 and
comprising a light-transmissive substrate member 10 which may be
glass, mica, plastic, fused silica, quartz, or similar material,
with glass being preferred, having a layer 11 of an etchable
material which is preferably a film of an opaque etchable metal
such as, for example, indium, and hereinafter, for convenience,
referred to as "metal," attached to one surface thereof. Metal
layer 11 may be conveniently appled to substrate member 10 by
evaporation as one example. The metal coated slide is then immersed
in a solution of antibodies to the biological particle to be
detected to adsorb a monomolecular layer of antibodies 12 on the
surface of etchable metal film 11; this completes the fabrication
of a diagnostic slide. The diagnostic procedure utilizing this
slide comprises immersing the slide into a physiologic fluid
specimen to be tested for the presence of biological particles of
the species of interest. If the specimen contains particles of the
species of interest, some of the particles, as 13 in FIG. 1, become
immunologically bonded to antibody molecules 12 on the slide. Only
particles specifically reactive with the antibodies will
immunologically complex therewith.
The next step in accordance with this invention is illustrated in
FIG. 2 and comprises the deposition of a non-etchable layer over
the structure of FIG. 1. The non-etchable layer is preferably a
light-transmissive, non-etchable material and may be glass or other
suitable oxide or a metal such as gold. In accordance with one
embodiment of this invention, diagnostic slide 30 is immersed in an
electrolyte bath (not shown) and an electrical potential is applied
by a source 17 thereof between metal film 11 and a quantity 16 of
non-etchable metal which is also immersed in the electrolyte
solution to plate a quantity 14 of non-etchable metal onto the
structure shown in FIG. 1. For reasons more fully set forth
hereinafter, metal 16 is preferably gold. It is important in
practicing this invention to carefully control the deposition of
non-etchable metal coating 14 to insure that the deposited metal is
discontinuous about the periphery of particle 13 as shown in FIG. 2
by gap 15. In an alternative embodiment of this invention,
non-etchable metal may be applied by evaporation, again preferably
gold. In this case, also the deposition process is monitored to
insure discontinuties of the non-etchable metal coating about the
peripheries of each biological particle bonded to the slide.
FIG. 3 illustrates the next step in the practice of this invention
and comprises the immersion of diagnostic slide 30 after the
deposition of the non-etchable metal layer thereon into a container
19 of a quantity 20 of an etchant solution, such as an acid.
Etchant 20 dissolves the biological particles bonded to the slide,
the immediately underlying antibody which had been complexed
therewith and the underlying portion of etchable metal film leaving
a void 18 in the slide structure and exposing a portion of
light-transmissive substrate 10. Etchant 20 further undercuts a
portion of the structure surrounding void 18 by dissolving
additional quantities of etchable metal 11 to produce an expansion
of void 18 into area 21. This undercutting serves to provide
amplification of the detectability of void 18 when the non-etchable
metal is gold. This is the reason for preferring gold as the
non-etchable metal and results from the fact that a thin gold layer
such as is applied in accordance with this invention is quite light
transmissive in the absence of an underlying layer of another metal
but is substantially opaque when the underlying layer is present.
Accordingly, after removal from the etchant bath, slide structure
30 exhibits light-transmissive regions corresponding to the points
at which biological particles have been bonded thereto, which
light-transmissive regions have dimensions determined by the
diameter of regions 21 which are substantially greater than the
dimensions of voids 18. At other regions diagnostic slide 30
remains opaque.
Following the etch procedure, slide 30 is examined to determine
whether or not biological particles had been immunologically bonded
thereto and consequently whether or not the test specimen contained
the specific biological particles of interest. A virus particle,
for example, is typically 1,000 A in diameter. By the use of gold
metal backed by etchable metal which is undercut in the etch
process as discussed above, light-transmissive regions in excess of
10,000 A in diameter are produced at sites at which biological
particles had been immunologically bonded to their specific
antibodies on the slide. This provides for reading of the slide to
detect particles in the specimen by optical microscopy.
Furthermore, if desired, a counting grid may be superimposed over
the microscopic image to thereby provide for determination of
concentration of the particles of interest in the specimen.
FIG. 4 illustrates alternative examination procedures to optical
microscopy in accordance with this invention. In FIG. 4 an
enclosure 31 has a bracket 32 on one side thereof for supporting
slide 30 adjacent an opening (not shown) in enclosure 31. An
illumination source such as incandescent lamp 33 is positioned
within enclosure 31 to direct light against slide 30. If desired, a
reflector 34 may be employed to increase the light intensity
impinging on slide 30. By use of such a light box, a diagnostic
slide 30 processed as hereinbefore described may be examined by
unaided visual observation in a slightly darkened room. The sites
at which biological particles have been immunologically bonded to
the slide appear as clearly distinct speckles of illumination when
so viewed. Obviously, quantitative information regarding the
concentration of particles in the specimen may be obtained by
counting the speckles of illumination and this may be aided by
placing a grid structure over the outer surfaces of slide 30 in
retaining means 32. Alternatively, an integrating, indicating
photoresponsive device 35 may be employed to provide quantitative
information on the concentration of biological particles in the
specimen. Device 35 may, for example, conveniently comprise a lens
system for focusing light transmitted through slide 30 onto the
photocathode of a photomultiplier tube operating in the current
mode having a millimeter connected in series with the
photomultiplier tube.
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.
* * * * *