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Description  |
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INTRODUCTION
This invention relates to detection of proteins, and more particularly to a
method of rapidly detecting presence of specific antibodies in a solution.
I. Giaever application Ser No. 384,113, filed July 30, 1973, and assigned
to the instant assignee, describes and claims an improved method and
apparatus for detecting and purifying proteins and antibodies. As pointed
out in the Giaever application, immunological reactions are highly
specific biochemical reactions in which a first protein, denominated an
antigen, combines with a second protein specific to the antigen,
denominated an antibody, to form an immunologically-complexed protein.
Immunological reactions within an organism, such as an animal, are vital
to the animal in combating disease. Through a process not fully
understood, entry of a foreign protein, i.e., the antigen, causes the
organism to produce antibody proteins specific to the antigen. The
antibody protein molecules have available chemical bonding sites which
complement those on the antigen molecule so that the antigen and antibody
chemically combine to form an immunologically-complexed protein.
The Giaever application points out that collection and purification of
immunologically-active proteins has, in the past, depended upon the
precipitating or agglutinating characteristic of the proteins resulting
from the immunological complexing reaction. One example of such reaction
has been the HCG protein pregnancy test. This test is performed by mixing
a quantity of HCG anti-serum into a urine specimen. A plurality of
polystyrene spheres which have been coated with HCG protein are then
introduced into the previously-prepared urine specimen. The polystyrene
spheres will agglutinate if, but only if, HCG protein is absent from the
urine specimen, since the HCG protein on the polystyrene spheres will
complex with the HCG anti-serum previously introduced in the specimen.
However, if HCG protein is present in the urine specimen, the protein will
complex with the previously-introduced HCG anti-serum so as to form a
precipitate, thereby precluding availability of the anti-serum for
complexing with the HCG protein on the spheres to cause the spheres to
agglutinate.
The aforementioned Giaever application states that the HCG protein
pregnancy test could be simplified by adhering HCG anti-serum onto the
polystyrene spheres and directly testing a urine specimen. In such case,
the spheres will agglutinate if, but only if, HCG protein is present in
the specimen. The Giaever application, however, points out that this
procedure has not been employed apparently because available HCG anti-sera
are complex mixtures containing a large proportion of constituents other
than HCG antibodies from the antisera. The Giaever application also notes,
as another shortcoming of agglutination tests, that the particles involved
may tend to agglomerate for any of a variety of reasons having nothing to
do with immunological agglutination, thereby lowering reliability of the
tests.
A discovery described in the Giaever application is that any arbitrary
protein will adsorb onto a substrate in a monomolecular layer only, and
that a specific antibody (or antigen) for such arbitrary protein will bond
to the protein to form a bimolecular protein layer on the substrate. More
particularly, a wafer of substrate material may be immersed in a solution
of a first protein so that a monomolecular layer of the first protein will
adhere to the substrate. The substrate thus coated is immersed in a second
solution which may contain a second protein that specifically reacts with
the first protein. The second protein, but only this protein, if present
in the second solution will form a monomolecular layer overlaying the
monomolecular layer of the first protein on the substrate. The coated
substrate, after immersion in the second solution, is examined
electrically or optically to determine whether a bimolecular or
monomolecular layer of protein is adhering thereto, thereby signifying
whether or not the second solution actually contains the second protein.
In accordance with the present invention, a first protein, or antigen, is
deposited on microscopic particles. Such particles, thus coated, exhibit a
certain electrophoretic mobility. If a protein which specifically reacts
to the first protein, i.e., an antibody, is then combined with the first
protein in a dilute solution of such antibodies, the electrophoretic
mobility of the particle drops to a much lower value, since antibody
molecules are of much lower mobility than most other proteins at normal pH
of the solution. By properly adjusting pH, a substantial mobility
difference is maintained between the antigen films, carried as a
monomolecular layer on some of the particles, and the antigen-antibody
films, carried as a bimolecular layer on others of the particles. This
approach to detection of complexing lends itself to high sensitivity
because mixing of the solution containing the particles and antibodies is
a more efficient way of making contact therebetween than by bringing a
protein solution into contact with a plane macroscopic surface. Also,
detection of molecular layer formations on microscopic surfaces avoids
insensitivities and other problems associated with agglutination
detection. As an additional advantage, substantial particle surface
coverage can be obtained in 10 to 20 minutes for concentration of protein
in the nanogram per cubic centimeter range while, for macroscopic plane
surfaces, the time period required to obtain comparable coverage with the
same dilute protein concentrations is about 10 hours.
Accordingly, one object of the invention is to provide a method for rapidly
detecting presence of a specific protein in a solution.
Another object is to provide a simple, highly sensitive method for
detecting discrete molecular layer formations of proteins.
Another object is to provide an improved method of sensitively detecting a
specific immune reaction.
Briefly, in accordance with a preferred embodiment of the invention, a
method of detecting an antigen-antibody reaction comprises the steps of
depositing an antigen on each of a plurality of microscopic particles, and
forming a dilute suspension of the particles in a solution to be tested
for presence of antibodies specific to the antigens on the particles. The
suspension is stirred, and electrophoretic mobility of the particles is
then measured.
BRIEF DESCRIPTION OF THE DRAWING
The features of the invention believed to be novel are set forth with
particularity in the appended claims. The invention itself, however, both
as to organization and method of operation, together with further objects
and advantages thereof, may best be understood by reference to the
following detailed description taken in conjunction with the accompanying
drawing in which:
The FIGURE is an isometric view of apparatus that may be employed in
practicing the method of this invention.
DESCRIPTION OF TYPICAL EMBODIMENTS
Preconditions for sensitivity of the detection method of the invention
include selection of microscopic particles to be coated with a
monomolecular protein layer, the particle concentration in solution, and
mixing procedures. Particles comprising polystyrene spheres of 0.81
microns diameter, dialyzed to remove surfactants and other contaminants,
have been employed successfully. However, the invention is not limited to
polystyrene particles since other particles to which proteins will adsorb,
such as silica particles, are also suitable. Most colloidal particles
could, in principle, be suitable since there is no requirement on shape or
uniformity of size, provided the specific gravity of the particles is
chosen so that the particles remain in suspension for at least a few
minutes to allow sufficient time for optical scattering measurements to be
made. For adequate sensitivity, test particle solutions must be quite
dilute since total particle surface area must be kept small (much less
than one square centimeter, for example). Concentration of 10.sup.6 to
10.sup.7 particles per cubic centimeter, where the particles are of 0.81
microns diameter, have proven satisfactory.
High sensitivity may not necessarily be useful if it is accompanied by
susceptibility to nonspecific effects, which generally occur in
concentrated serum or protein solutions. In this instance, washing of the
microscopic particles by centrifugation is sufficient to avoid nonspecific
mobility changes which would otherwise be caused by exposure of the
particles to such concentrated solutions. Presence of the second protein
layer, if any, may then be detected with maximal sensitivity when the
spheres are observed in a 0.005 Normal sodium chloride solution.
Mixing has been accomplished with use of conventional magnetic turbulent
stirring in a beaker and by employing electrophoretic stirring in an
optical cell. Electrophoretic stirring is especially useful since it
results in motion of the microscopic particles relative to the immediate
surrounding liquid. For optimal mixing, it may be best to combine
turbulent and electrophoretic motion, the latter requiring a high enough
electric field to produce electrophoretic motion.
The technique for detecting the antigen-antibody combination on a
microscopic surface is dependent upon changes in electrophoretic mobility
of particles which are protein-coated. A certain electrophoretic mobility
is associated with a surface formed by an antigen deposited on a particle.
If an antibody molecule is then made to combine with that protein,
mobility of the particle drops to a considerably lower value, since
antibody molecules are of much lower mobility than most other proteins at
normal pH. Additionally, the pH may be adjusted within a range of about pH
4.0 to pH 8.0, so that a substantial mobility difference is maintained
between the antigen and antigen-antibody films. Mobility of antigen-coated
particles changes by as much as a factor of 2 or 3 when antibody molecules
are made to combine with the antigen.
The electrophoretic mobilities are measured by detection of laser light
that is scattered from the particles. The scattered light exhibits a shift
in frequency as an electric field is applied to the particle solution, due
to the Doppler effect and electrophoretic motion of the particles. This
type of measurement, which is described by E. E. Uzgiris in
"Electrophoresis of Particles and Biological Cells Measured by the Doppler
Shift of Scattered Laser Light", Optics Communications 6 (September 1972)
55, allows even fractional coverage of the particle surface with antibody
molecules to be readily observed. A substantial particle surface coverage
can be obtained in 10 to 20 minutes for concentrations of protein in the
nanogram per cubic centimeter range.
An optical Doppler electrophoresis measurement system for detecting
mobility changes, such as described in the aforementioned Uzgiris Optics
Communications article, is illustrated in the FIGURE. The system comprises
an electrophoretic cell 10 including fluid containment means 11 fabricated
of a light-transmissive, fluid-impenetrable material, such as glass,
plastic or the like. A pair of closely-spaced electrodes 12 and 13 are
included in cell 10. These electrodes are preferably of rectangular shape
and have mutually parallel facing surfaces defining an interelectrode gap
not exceeding 1 millimeter in width.
Container 11 is filled with a dilute colloidal suspension containing the
microscopic particles having a layer of protein adsorbed thereon, and an
electric field is established between electrodes 12 and 13 by power supply
14. The gap between electrodes 12 and 13 is illuminated by coherent
optical energy from a laser 15. A portion of this energy is scattered by
the microscopic particles within the gap between electrodes 12 and 13 and,
because of the motion of the scattering particles in the electric field,
exhibits a Doppler frequency shift. Energy scattered at a predetermined
angle is received by optical detector 16 which is preferably a
photomultiplier tube but may be any appropriate square law detector.
Detector 16 receives the Doppler-shifted energy scattered by the particles
in suspension in the fluid inside container 11, and also receives
unshifted energy scattered by fixed scattering objects, such as a wall of
container 11. Since detector 16 receives both Doppler-shifted and
unshifted energy, and is a square law detector, its output signal is
indicative of the heterodyne product of the two frequencies received and
hence may be analyzed by conventional techniques to determine
electrophoretic mobility of the particles in cell 10 at two different
times in order to detect a reduction in mobility that may have occurred
between those two times.
The foregoing describes a method for rapidly detecting presence of a
specific protein in a solution. The method is both simple and highly
sensitive, functioning to detect discrete molecular layer formations of
proteins. Specific immune reactions may be sensitively detected thereby.
While only certain preferred features of the invention have been shown by
way of illustration, many modifications and changes will occur to those
skilled in the art. It is, therefore, to be understood that the appended
claims are intended to cover all such modifications and changes as fall
within the true spirit of the invention.
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