Note: Descriptions are shown in the official language in which they were submitted.
DESCRIPTION
Solid phase biological diagnostic assay
Technical Fleld
This invention relates to a method ~or
assaying agueous samples for specific biological or
immunological substances.
Background Art
Soluble biological su~stances attached to
carriexs have many uses in diagnostic ~ests, enzyme
processes, and affinity purifications. For example,
attachment of antibodies or antigens to a carriex
allows their immunological partners to be easily
removed from a mixture of many s~bstances.
Similarly, attaching enzymes ~o a carxier allows them
to be easily removea ~rom a rea~tion mixture or to be
used in a continuous flow process. ~eterogeneous
radioimmunoassays and enzyme immunoassays rely on
a~tachment of one or ~ore of ~he reactants to a solid
phase to enable separation from the free reactants.
Agglutination assays ~to determine the presence of an
anti~en or an antibody in a fluid) utilize indicator
or carrier particles ~on which are carried the
~ ~3~
app~opriate immunological material) in order to make
the.immunological complex more easily visible.
Separation and identification of cells, cellular
constituents, and bacteria are aided by antibodies ox
antigens coupled to solids. Biological particles
will, for example, specifically adhere to solids
coated with appropriate antibodies and antigens so
that separation fxom other particles can be affected.
Identificatio~ of biological particles can be made
1~ through khe specific adherence of small particles
coated with appropriate antibody or antigen. These
small particles can incorporate a substance such as a
fluorescent dye, radioactive tracer, or electron
dense substance which make their presence more
readily detectable.
The currently available simple procedures for
bioactive material testing, for field test methods
(e.g., over the counter pregnancy test kits, rely
primarily on agglutination reactions or on enzyme
catalyzed color reactions. These proceaures use one
of several core materials in their reaction, i.e.,
they use sheep's red blood cells, latex particles, or
killed Staphylococcus cells as carriers. Such
procedures are quite fast, usually taking less than
2~ an hour to complete. However, such procedures have
several significant drawbacks. First, the biological
core materials, when utilized, have production
difficulties that can lead to non-reproducible test
results for specific bioactive materials. Second,
the ma~imum sensitivity level for reproducible,
reliable results, is in the microgram per milliliter
concentration range. This is far above the 1-10
nanogram per milliliter sensitivity which is required
~LS~
for early hormonal, viral or bacteriological analyte
detection.
The agglutination procedures often require
the careful manipulation of two solutions so that a
successful reaction will occur to cause
agglutination. Manipulations usually take the form
of stirring small volumes of the solution on a
specially designed flat surface and then waiting for
the agglutinates to appear, or mixing two solutions
by rocking a specially designed flat surace back and
forth steadily until agglutinates appear. In another
form of common agglutination test two regents are
mixed in a test tube. As the agglutinate forms, it
becomes insoluble and precipitates out to form a
pattern on the tube bottom. This is observed and is
accorded a negative or positive classification by its
shape. The appearance of the agglutination reaction
is in no way standardized and is therefore easily
misinterpreted by persons with inadequate training or
2~ instruction. The agglutination procedures are very
technique dependent. Generally it requires about two
hours of training to qualify an operator already
familiar with laboratory techniques to carry out the
slide-type test. When the agglutination procedure is
carried out in a test tube as described above, it is
extremely sensitive to vibration, temperature changes
and mi~ing techniques.
The enzyme catalyzed reaction of the prior
art require preparation of biological subs-trates for
the enzymatic reaction and careful manipulation of
; several reagent substances to first start the
reaction and then, after a rather precise time
interval, to stop the reaction before an observation
can be completed. The enzyme test p~ocedures are
sensitive to tempera~ure ehanges. During the
reaction ~ncubation the ambient temperature must be
assumed to be ab~ut 22C. The reactions are very
difficult to stop, sometimes requiring ~he use of
strong bases, e.g., 10 N sodium hydroxide for this
purpose. And, they are quite ~ime dependent. That
is, they are ki~etic reactions that must be closely
timed for accurate and precise test results
~he prior art field tests, or home use ~estsg
for bioactive materials thus have a number of very
serious problems.
Disclosure Of Invention
The present invention is directed ~o
overcom;ng one or more of the problems as set ~crth
above.
In accorda~ce with one embodiment of the
present invention, there is provided a method for assay-
ing an aqueous sample containing a specifically binding
biomaterial having a binding site which is a specific
binding partner to a biological substance by observation
in light including a se~ected wavelength in the visual
range, said specifically binding bioma~erial being in
association with other biomaterials, with increased speed,
ease of assaying, specificity and sensitivity, comprising:
(1) contacting a solid support having a water insoluble
synthetic polymeric, macroextensive surface capable of
associating with said specifically binding biomaterial
with said aqueous sample for a time sufficient for said
specifically binding biomaterial to associate with said
synthetic polymeric surface; (2) separating said synthetic
polymeric support from contact with said aqueous sample;
(3) contacting said synthetic polymeric surface with
an aqueous solution containing a plurality of synthetic
:~2~5~8~
particles of a preselected size, in about the same range
as the selected wavelength, and of a preselected refrac-
tive index as calculated by Mie scattering for clear
visual observation and having particle surfaces bearing
said biological substance associated therewith for a time
sufficient for said binding site to bind to said biolog-
ical substance and to thereby bind said particles to said
synthetic polymeric surface; (4) separating said synthetic
polymeric support from said aqueous solution; (5) rinsing
said synthetic polymeric support to remove any non-bound
particles; and (6) visually observing the degree of adher-
ence of said particles to said synthetic polymeric surface
in light including said wavelength.
In accordance with another aspect of the present
invention there is provided a method for assaying an
aqueous sample containing a quantity of a specifically
binding biomaterial having a binding site which is a
specific binding partner to a biological substance by
observation in light including a selected wavelength in the
visual ranger said specifically binding biomaterial being
in association with other biomaterials, with increased
speed, ease of assaying, specificity and sensitivity,
comprising: contacting said aqueous sample with a solid
support having a water insoluble synthetic polymeric macro-
extensive surface associated with either said specifically
binding biomaterial or said biological substance, said
solid support having bound thereto a plurality of synthetic
particles of a preselected size, in about the same range as
the selected wavelength, and of a preselected refractive
index, both as calculated by Mie scattering for clear
visual observation and having particle surfaces associated
with said specifically binding biomaterial when said solid
support is associated with said biological substance or
with said biological substance when said solid support is
associated with said specifically binding biomaterial, the
binding of said particles to said surEace being via binding
of said biological substance to said binding sites; and
visually observing the degree of release oE said particles
from said surface in light including said wavelength.
J
~ ~ Lq~ 5~
In accordance with still another embodiment of the
present invention there is provided a ki~ for assaying an
aqueous sample containing a specifically binding biomaterial
having a binding site which is a specific binding partner
to a biological substance by observation in light including
a selected wavelength in the visual range with increased
speed, ease of assaying, specificity and sensitivity, com-
prising: a solid support having a water insoluble macro-
extensive sur~ace capable of associating with said specific-
ally binding biomaterial; and a plurality of syntheticparticles of a preselected size, in about the same range as
the selected wavelength, and of a preselected refractive
index, both as calculated by Mie scattering for clear visual
observation and having particle surfaces bearing said
biological substance.
In accordance with yet another aspect of the
present invention a kit for assaying an aqueous sample
containing a quantity of a specifically binding biomaterial
having a binding site which is a specific binding partner
to a biological substance by observation in light including
a selected wavelength in the visible range, said specific-
ally binding biomaterial being in association with other
biomaterials, with increased speed, ease of assaying,
specificity and sensi-tivity, comprising: a solid support
having a water insoluble synthetic polymeric macroextensive
surEace associated with either said specifically binding
biomaterial or said biological substance, said solid
support having bound thereto a plurality of synthetic
particles having particle surfaces associated with said
specifically binding biomaterial when the solid support is
associated with said biological substance or with said
biological substance when the solid support is associated
with said specifically binding biomaterial, the binding of
said particles to said surface being via binding of said
biological substance to said binding sites.
According to yet another aspect of the invention
there is provided a method of assaying an aqeuous sample
containing a specifically-binding biomaterial having
a binding site which is a specific binding partner to
S a biological substance by observation in light includ-
ing a selected wavelength in the visible range, said
specifically-binding biomaterial being in association
with other biomaterials, with increased speed, ease
of assaying, specificity and sensitivity, comprising:
(1) contacting a solid support having a water insoluble
synthetic polymeric, macroextensive surface capable of
associating with said specifically-binding biomaterial
with said aqueous sample for a time sufficient for said
specifically-binding biomaterial to associate directly
with said synthetic polymeric surface; (2) separating said
synthetic polymeric surface from contact with .said aqueous
sample; (3) contacting said synthetic polymeric surface
with an aqueous solution containing a plurality of syn-
thetic particles of a preselected size, conductivity and
refractive index for substantially optimal light scatter-
ing as calculated by the Mie scattering equations having
particle surfaces bearing said biological substance asso-
ciated therewith for a time sufficient for said binding
site to bind directly to said biological substance and to
thereby bind said particles to said synthetic polymeric
surface; (4) separating said synthetic polymeric surface
from said aqueous solution; (5) rinsing said synthetic
polymeric surface to remove any non-bound particles; and
(6) observing the degree of adherence of said particles to
said support surface.
When operating in accordance with the various
embodiments of the present invention, field or home
tests for biomaterials can be performed accurately by
6a
~z~
an untrained person who simply reads some
accompanying instructions. The test can not only be
performed quickly, it can be performed easily and
with great specificity and increased sensitivity over
prior ar~ tests. The methods and kits of the present
6b
rs
invention are not sensitive to vibration and can be
very easily interpreted by an untrained user.
secause of the increased sensitivity of the test, the
biomaterial can be determined in very low
concentrations. For example, pregnancy testing (for
human chorionic gonadotrophin [hCG)) can be
accurately carried out before a period is missed.
Furthermore, the timing during the incubation of the
macroextensiv~ surface with the aqueous sample is not
at all critical. And, the test components can be
moved during the reaction steps with no adverse
effects on the test results. Still further, in
accordance with certain aspects of the invention the
macroextensive surface, with the analyte and
particles bound thereto, can be washed and thereafter
dried and storPd to give a permanent record of the
test results.
,
Brief Description Of The Drawings
The invention will be better understood by
reference to the figures of the drawings wherein:
Figure 1 illustrates, graphically scattering
phenomenon as a function of wavelength;
Figure 2 illustrates, graphically, scattering
by a sphere;
; Figure 3 illustrates, graphically, the
scattering cross-section of dielectric spheres with
1.33 refractive index in air;
Figure 4 illustrates, graphically, extinction
; 30 curves for spheres of six different refractive
indexes; and
Figure 5 illustrates, graphically, extinction
curves calculated from Mie's formula.
est Mode_Por Carryin~ Out The Invention
In accordance with on~ aspect of the present
invention a method is provided for assaying an
aqueous ~ample containing a specifically binding
biomaterial having a binding site which is a specific
binding partne~ to a biologi~al su~stance when the
specifically binding biomaterial is in association
with other biomaterials. The ~erm "biomaterial~ is
used broadly~to indicate any substance which is
biologically active, The *erm ~biological substance"
is used broadly to indicate any ~ubstance whi~h is a
specific binding partner to a specific biomaterial.
Illustrative of the biomatexials and of ~he
biological substances are enzymes, antibodies,
hormones, natural receptors, e.q., thyroxine binding
globulin and avidin, globins, e.g., hemoglobin,
ocular l~ns proteins, surace antigens,
his~o-compatibility antiyens, and ~he like.. The
specifically binding biomaterial and the biological
substance can be any such materials which are
lin~able ~o either a water insoluble support or a
plurality of particles. A long list of such
substances appears in ~.S0 Patent No. 4,264,766,
issued April 28, 1981.
The method and kit of the present invention uti-
lize a water insoluble synthetic polymeric support having
a macroextensive surface having the capability of asso-
ciating with the specifically binding biomaterial~ The
solid support must be macroextensive and must define a
macroex~ensive surface. For example, the solid support
may be in the form of a strip of a suitable size for dip-
ping into an aqueous sample and for being viewed. The
strip may be of any convenient size, for example 2 to
10 millimeters wide by 30 to 80 millimeters long by
perhaps 0.3 to 1 millimeter in thickness. The
macroextensive surface will generally be hydrophobic
thus giving it the ability to adsorb hydrophobic
molecules such as the specifically binding
biomaterial and other biomaterials. In some
instances it may bear a biological substance which is
a specific binding partner to the specifically
binding biomaterial.
The solid support itself must be inert with
respect to immunolcgical diagnostic tests. A large
number of materials can be used as the water
insoluble support. Of particular interest are
latexes as described in U.S. Patents Nos. 4,046,723;
4,118,349; 4,140,662 and 4,264,766. Other useful
polymers may be found in U.S. Patents Nos. 3,619,371;
3,700,609; 3,853,987; ~,108,972 and 4,201,763. The
polymer or latex supports can have active groups such
as carboxyl groups, amine groups or groups
convertible into them. Such is not, however,
necessary. Polyvinylchloride is one particularly
preferred material for the support. Another
particularly preferred material for the support is
polystyrene. The polystyrene may contain
copolymerized therewith a carboxyl containing
compound such as acrylic acid, methacrylic acid, or
the like.
The particles of the present invention may be
made of a like material as the support. That ls, the
latexes, polymers, and the like which can serve as
the support can also serve as the particles.
However, the particles are not so limited. Indeed,
~5~
the particles can be in the nature of cells, inrluding
blood cells, yeast cells and bacterialicells having the
biological substance attached to their membranesO The
exact nature of the particles is thus, not critical if
they are of a preselected size and refractive index. If
the particles are pol~meric in nature it is desired that
the polymers be hydrophobic so that the biological sub-
stance can be adsorbed thereon.
For easy observation of ~ticking of the
particles to the solid support it is desired that
they be of a size so as to provide a cleax visual
clouding appearance on the solid support~ which would
in such ins~ance preferably ~e transparent. It has
been found that if the particles are selected to have
lS an average diameter which ~alls about the range of
the wavelength of visible light, i.e., from about 0.2
misron to about 2.0 microns, the clouding effect in
air will be enhanced thus making ~isual observation
of s~ic~ing of ~he particles ~o the transparent solid
support clearer. In wa~er the ~loudi~g effect is
enhanced when the particle~ have an a~erage particle
diameter between abou~ 0.47 and a~ou~ 11.1 microns.
The visibility of a microsphere when viewed
in visible light depends upon many actors: the siæe,
the refractive index, the color, and the conductivity
of the sphere. The most easily visible case~ that is
where the smalles~ ~uantity of ma~erial can be
detected by it~ effect on light, excluding the case
of fluorescence, is when the sphere resonantly
scatters the light. This phenomenon was first
explained in a paper published in 19n8 by G~ Mie,
Ann. d. Physik, Volume 25 (1908), page 377. Mie
presented his theory ~iving the rigorous solution for
the scattering of a plane monochromatic wave by a
homogenous sphere of any diameter and of any
composition situated in a homogenous medium. The
scattering that Mie described is commonly called Mie
scattering. Mie scattering occurs when the size of
the scattering particle is in the same range as the
wavelength of the light being sca~tered. The
scattering is strongly directed in a ~orward
direction, th~t is, in a narrow cone with its apex at
the center of the particle, in the same direction as
the incident light. Other discussions of such
scattering may be found in Light Scattering by Small
Particles, ~.C. Van de ~ulst, John Wiley & Sons,
Inc., New York, 1957; Principles of Optics, M. Boxn
and E. Wolr, J. Springer, Berlin, 1933 and Particle
Clouds, Dusts, Smokes and Mists, H.L. Green and W.R.
Lane, E. & F.N. Spoon, Ltd., London, 1957.
A bit of basics first: A parallel beam of
light traversing empty space is not attenuated. When
one places an object or objects in the beam of light,
the beam is attenuated. The term extinction is used
for a quantitative measure of this reduction in the
intensity of the light in the beam. The extinction
generally consists of two parts: the scattering part
and the absorbtion part. For the present purposes,
only the effect of the former on the light beam will
be considered. Figure 1 shows the energy that is
scattered out oE a light beam by a particle of radius
r as a function of the wavelength of the incident
light. For light of wavelength much less than the
radius of the particles doing the scattering, the
scattered energy is nearly independent of the
wavelength. For wavelengths much longer than the
radius of the particles, the scattered energy falls
off as the inverse four~h power of the wavelength.
This latter is ~alled Rayleigh scattering. The
Rayleigh scattering follows a 1 ~ Cos2 ~ dependence
5 50 that the light scattered at 90 to the incident
beam has one-half o~ the intensity of the light
scattered in ~he forward direction or in the reverse
direction.
For the case where the diameter of the particles
and the wavelength are nearly the same, the scattered
energy is at maximum. Thi~ is the region o Mie
scattering. Figure 2 shows the coordinate system
which defines the angle ~ through which the light is
scattered~ The calculation of Mie scattering is
rather complicated but res~lts ha~e been obtained ~or
several systems a~d are easy to understand in a
general way. Figure 3 shows the scattering
cross-section, ~, for dielectric spheres of
refractive index lo 33 as ~ function of the parameter
; ~0 x.- 2~a Where "a" is the radius of the sphere and
~is the wavelength of light, this corresponds ~o the
scattering in air by droplets of water, for example,
which have thas refractive index. The first maximum
in the scattering peak occurs near an "x" value of
6Ø The second maximum occurs at an "x" value of
about 16. For a fixed radius "a", then, the "x"
value plotted can be considered to be a plot of
1(wavelength). ~herefore, the extreme left of the
diagram, where the wavelength is very large compare~
to the size of the particle, corresponds to the
Rayleigh scattering regime: the intensity falls off
as the inverse fourth power of the wavelength. As
one goes well past the right hand end of the diagram,
the oscillating curve asymptotically approaches the
value of 2. This corresponds to the fact that the
light is scattered out of the parallel beam by two
effects: 1) the geometric blocking of the light by
the cross-sectional area of the sphere and 2) the
edge effect, which results in Fraunhofer diffraction.
This latter produces another factor of 1.0 in the
cross-section for scattering particles.
The meaning of the scattering cross-section,
in this case with a maximum value of 4 for x = 6, is
that the light scattered out of a peam of wavelength
~ by a spherical particle of radius "a" and an index
of refraction of 1.33, where 6 = 21a, is equal to 4
times the cross-sectional area t~a ~ of that particle
times the incident light intensity.
Figure 4 shows the extinction curves for
spheres with different values of refractive index
(m). It is seen that the maximum obtained value of Q
increases slightly as the refractive index is
increased. The curves indicate that the wavelength
for the phenomenon of Mie scattering is dependent
upon the refractive index of the particle. If,
however, one plots the scattering cross-section as a
function of the parameter p = 2x ¦m-l¦ one obtains
curves that are extremely similar and in fact
essentially superimposable if one neglects the minor
wiggles. This is shown in Figure 5. These curves in
Figure 5 are only accurate for index of refraction
less than about 1.6, which covers almost any
transparent material. The extra bump associated with
the index of refraction of 2.0 in Figure 4
corresponds to an optical resonance in the particle
itself. As a generalization, the scattering curves
~ f~
~2~
in Figure 5 are all calculated for particles in
vacuum. If one is working with particles in a medium
with refractive index other than 1.0 the difference
in refractive index between the particles and the
medium must be used as the refractive index for
calculating the Mie scattering.
For large values of p the maxima occur at
o = (k~ 3/4)2~and the minima at p = (k~ 1/4)2~, where
k is an integ~er. For a refractive index very near to
1.0 the first maximum occurs at p = 4.09, for a
refractive index of 1.5 the first maximum occurs at
p = 4.2, and for a refractive index of 2.0 the first
maximum occurs at p = ~.4. For particles such as
polystyrene microspheres, having an index of
refraction of about 1.59, the first maximum for a 0.7
micron diameter sphere should occur at 620 nanometers
which corresponds to red light. This behavior has
been confirmed experimentally.
Table 1 shows some experimental results
obtained using an antibody which detects the
B-subunit of hCG. The number of spheres per square
millimeter attached to the surface at the end of the
test is shown in column 1. The fractional area
covered by these 0.7 micron diameter spheres is shown
in column 2. The theoretical scattering is showm in
column 3. This theoretical scattering allows for
scattering by the particles on both sides of the thin
plastic strip that was used. The scattering expected
from the 1.0 nanogram per milliliter sample is -thus
about four times background scatterinq from the
negative sample and is easily observed by placing the
strip in a beam of light and observing the light
scattered from the microspheres on the strip. This
14
test was done with an àntibody which was specific to
the detached B-subunit of HCG. Therefore, the
results for the pregnancy urine of 4.4% corresponds
to only about 1% of the total HCG expected in
pregnancy urine. Use of an antibody to the intact
hCG molecule does result in much larger binding oE
microspheres to the plastic strips for pregnancy
urine. Experiments in which strips were artificially
coated with ~icrospheres to higher levels gave the
expected results. For example, a strip coated with
fractional area 0.084 on each side is expected to
scatter 67% of the light out of the incident beam.
It was obvious by eye that this was the approximate
level of scattering that such a strip did exhibit.
TABLE I
Theoretical
Fractional Scattering
HCG inSpheres/ Area Covered (Total soth
test urinemm2 _ Per Side Sides)
negative4,500 .0017 1.3%
1.0 ngm/ml17,000 .0063 5-0%
10.0 ngms/ml 30,000 .0113 9.1%
pregnancy15,000 .0055 q.4%
All of the foregoing has concerned spherical
particles which do not absorb light. The effect of
including an absorber in the particle is to dampen
the oscillation (lower the peaks and raise the
valleys) and ultimately shift the curve to lower
~5
~ ~ r~
~2~
wavelength can either reduce the scatterinq
cross-section or increase it, depending on the
position of the absorbing line relative to the peaks
and valleys of the Mie scattering for that particle.
The equation for finding maxima and minima is
2a p
The particles of interest (plastics, cells, etc.)
have an index of refraction m in the range from 1.4
to 1.6. For m in this range, with air as the medium,
the maximum occurs at p = 4.2. Therefore, 2Aa, at
maximum scattering, varies from 1.67 to 1.11.
For light of visible wavelength, A = 0.4
micron to 0.7 micron, 2a varies from 0.4 micron to
1.2 microns tusing maximum and minimum products).
Looking at Figure 5 one can see that the
maximum scattering falls off by at least half by
p - 2.0 and by p = 7.0, so we extend the size range
to these values and get >0.2 micron to 2.0 micron
diameter as the appropriate preferred ran~e for
particles in an air medium.
For particles in water (refractive index =
1.33) ¦m-1.33¦ should be used in place of ¦m~
Therefore, ¦m-1.33¦ varies from 0.07 to 0.27 and for
particles in this range the maximum occurs at
p = 4.09. Therefore, A at maximum scattering varies
from 9.3 to 2.4. As before, for ~ = 0.4 micron to
0.7 micron, 2a varies from 0.96 micron to 6.5
16
~s~
microns. Again picking p = 2.0 and p = 7.0 as outer
limits one gets )0.47 micron to 11.1 microns diameter
as the appropriate range for particles in a water
medium.
To summarize, particles will be more easily
visually detected in air if their average diameters
are between about 0.2 and 2.0 microns, more
preferably between about 0.4 and 1.2 microns. In
water these ranges are changed to bekween about 0.47
and 11.1 microns and 0.96 and 6.5 microns,
respectively.
It is also possible to add a color imparting
- agent to the particles so as to make clearer the
observation of stic~ing of particles to a solid
support which may or may not be transparent.
~: Fluorescing agents or other tracers can also be added
to the particles, if desired, although a major
advantage of the test is that such are not required.
In accordance with the present invention the
; 20 solid support as just described is contacted with an
aqueous sample for a time sufficient for the
specifically binding biomaterial to associate with
the macroextensive surface of the support. This can
be accomplished, for example, by simply placing a
strip of plastic, for example polyvinylchloride
plastic, in a sample such as the urine of a woman
suspected of being pregnant. The macroextensive
surface will then adsorb not only the sp~ciEic
binding biomaterial but other biomaterials as well.
In this case, the specifically binding biomaterial
might be ~-hCG. The strip would then be separated
from contact with the aqueous sample as by removing
it from the urine sample. ~sually the surface would
then be rinsed off, for example with phosphate buffered
saline.
In those instances wherein the strip has not been in
any way treated to prevent additional binding oE further
biomaterials, the strip would normally be con-tacted with a
material which would shield those portions of the surface
which are not bound to the specifically binding biomaterial
with a material which prevents attachment of other bio-
materials. For example, the strip could be contacted with
bovine serum albumin, gelatin, or the like.
The surface would next be contacted with an aqueous
solution containing a plurality of particles having particle
surfaces having the biological substance associated therewith
for a time sufficient for the binding site to bind to the
biological substance to thereby bind the particles to the
macroextensive surface of the support. This can be
accomplished by placing the strip in a solution having the
plurality of particles therein and agitating or mildly
stirring, i~ necessary, to make sure that the particles
physically contact the macroextensive surface. The bio-
logical substance can be associated with the particles by
any method, including, for example, physical adsorption,
covalent bonding, or the like.
Methods of accomplishing such associating are known.
One useful method is to cover a first substantial surface
portion of the particles with a polysaccharide coating while
not covering a second substantial surface portion with such
a coating. For example, the surface can be nitrated using,
e.g., a mixture of nitric and sulfuric acids, the resulting
nitro groups reduced to amino groups using, e.g., stannous
chloride and hydrochloric acid and attaching cyanuric halide~
moieties to the amino groups. An amino polysaccharide is
then electrostatically attached to the first surface portion.
A cyanuric halide can be used to cross-link ad~acent amino
polysaccharide molecules to form a usable composition. The
cyanuric halide, due to its acidity and ionic strength, also
serves to free at least the second surface portion from amino
polysaccharide coverage. A biological substance can be
attached to the second surface portion by hydrophobic
- 18 -
adsorption, electrostatic bonding, or covalent bonding via a
conventional activating agent.
A second useful method is to provide a water
insoluble support which has carboxyl groups on its surface.
The surface is contacted with an amino polysaccharide in an
amount more than sufficient to cover the surface with a mono-
molecular layer of the amino polysaccharide. For example,
an excess of amino Ficoll can be contacted with the surface
in a water solution. The amino polysaccharide is held to
the surface by electrostatic bonding. This is known since
acid and high salt solutions lead to removal of the poly-
saccharide. The excess amino polysaccharide is washed off
of the solid support with water. Thereafter, a cyanuric
halide, in ethanol solution, is added to -the amino poly-
saccharide coated solid surface. The cyanuric halide isused in a sufficient quantity to convert at least a
significant portion of the amino groups to cyanuric halide
adducts and to thereby cross-link the various amino groups
with one another. At the end of this reaction a first sub-
stantial surface portion of the water insoluble surface iscoated with amino polysaccharide while a second substantial
surface portion of the water insoluble surface is not coated
with amino polysaccharide. While it is believed that the
surface was originally covered with electrostatically bound
amino polysaccharide it has been experimentally shown that
the surface, after the reaction with the cyanuric halide
moiety, is no longer completely covered with an amino poly-
saccharide. Instead, portions of the surface are available for
bonding to biological substances. Further, acid and/or high
salt solutions no longer remove the amino polysaccharide after
it has been reacted with the cyanuric halide moiety. Generally,
the amount of the amino polysaccharide which remains on the
water insoluble surface is between about 0.5 x 10 7 and about
3 x 10 7 grams per square centimeter of khe area of the water
insoluble surface. The biological substance can be attached
to the second substantial surface portion via hydrophobic
adsorption or covalent bonding, all as previously described.
i
-- 19 -
A third method comprises reacting water insoluble
surfaces having active groups such as carboxyl groups with
less than enough amino polysaccharide to cover the entire
water insoluble surface with amino polysaccharide, and with
5 a water soluble carbodiimide, all in a single reaction. The
resultant product includes the amino polysaccharide covalently
bonded via the carbodiimide to a first substantial surface
portion of the water soluble surface through, e.g., the
carboxyl groups. A second substantial portion oE the surface
10 remains uncovered by amino polysaccharide molecules. Once
again, a biological substance can be attached to the second
substantial surfaee portion by any desired method. This
method has the advantage that if an excess of carbodiimide
is utilized there can still be ca.rbodiimide activated aetive
15 (e.g., carboxyl) groups on the second substantial surface
portion ready to covalently bond to a desired biologieal sub-
stance.
In a fourth method, a solid support having a water
insoluble surface having aetive (e.g., earboxyl) groups is
20 reaeted with an excess of an activator compound such as a
water soluble carbodiimide to forrn an adduct, e.g., a
carbodiimide adduct. Any excess activator is washed off. Less
than enough of the biological substance is added to react with
all activated sites. After the reaction is completed the
25 biologieal subs-tance remains attached to the second sub-
stantial surface portion. The surface is then washed to re-
move any reaction products. Water is again contacted with
the surface and an amino polysaccharide is added which then
links to the aetive groups which remain and whieh have been
30 aetivated by the aetivator. The resulting produet has both
the amino polysaccharide and the biological substance
covalently attached to the water insoluble sur~aee via the
aetive groups and through use of the activating agent.
While several of the above described methods have
35 called for the use of a earboxyl active group and a carbodii-
mide activating agent it should be noted that other active
groups and other activating agents may be utilized where
appropriate.
Preferably, those portions of the surface of
- 20 -
~:45~
the particles which are not associated with the
biological substance will be shielded against
attachmen-t of the other biomaterials in the same
manner as is the macroextensive surface of the strip.
Next, the support or strip is separated from the
aqueous sample and rinsed to remove any non-bound
particles. At this point the strip can be dried to
enhance the ease of observation of the degree of
adherence of the particles. The degree of adherence
of the partic~es to the surface is then observed. If
no particles adhere, the surface appears clear and
unclouded. If particles have adhered to the surface
it appears to be quite cloudy. This is particularly
easy to observe when the strip is transparent. ~f
the particles are colored, then the resulting
coloring of the strip can be observed.
It should be noted that timing and
temperature during exposure to the aqueous sample are
not critical limitations in the above set out method
so long as the time of exposure is sufficiently long
for adherence of the biological substance to occur.
For example, one hour or more exposure at room
temperature is sufficient. Handling is also not a
critical limitation since the method is really quite
simple to carry out. That is, the method simply
requires placing a strip in the suspect urine or
other test material and leaving it there for a
desired period of time, generally for an hour or
more, removing the strip from the suspect urine or
other test material, rinsing it, placing the strip in
a solution having a plurality of appropriate
particles for approximately 10 minutes, removing the
strip from such solution, rinsing the strip to remove
any non-bound particles, generally drying it, and
8~
looking at the strip. The time during ~hich the strip is in
the solution with the appropriate particles can be rather
critical if false results are to ~e avoided. Generally,
exposure for 9 to 12 minutes has given xeproducible and
accurate results when an antibody for the HCG has not been
previously adsorbed to the strip. If such an antibody has
been adsorbed to the strip, the time of exposure to the
appropriate particles is not critical in that the minimum
exposure time is increased to at least about one hour, but
exposure for longer periods of time will not deleteriously
effect the test results.
It is desirable in the practice of the invention that
the biological su~stance generally be selected ~o have an
extremely high affinity for the speci~ically binding bio-
ma~erial. This provides very high specificity and sensitivityfor the test.
In some instances it may ~e desirable ~o initially
coat a portion of the surface of the strip with a poly-
saccharide as taugh~ above. This help~ to prevent undesired
biomaterials from attaching to the macroextensive surface.
Also, it provides shielding of those por~ions of the surface
which are not bound to the specifically binding biomaterial,
the shielding-~eing with a ma~erial, the polysaccharide,
which prevents attachment of other biomaterials.
In accor~ance with another aspect of the present
invention a kit is provided for carrying out the assay just
described. The ki~ includes a solid support having a water
insoluble macroextensive surface capa~le of associating with
the specifically
~.~
~Lb - 22 -
binding biomaterial and with other biomaterials when
contacted with an aqueous sample containing the
specifically binding biomaterial and other
biomaterials for a sufficient period of time. The
; 5 kit also includes a plurality of particles having
particle surfaces bearing the biological substance
associated therewith. Such particles would generally
be suspended in an aqueous solution. The kit may
also contain~appropriate rinse solutions, such as
phosphate buffered saline, anionic detergent in PsS,
and/or solutions for providing shielding of portions
of the macroextensive surface. The anionic detergent
- in PBS serves to reduce false positive test results.
The shielding solutions would include, for example,
bovine serum albumin, gelatin or the like.
In accordance with yet another aspect of the
present invention a method is set out for assaying an
aqueous sample by contacting the aqueous sample with
a solid support having a water insoluble
macroextensive surface already associated with a
selected one of the specifically binding biomaterial
and the biological substance, the solid support
having bound thereto a plurality of particles having
particle surfaces associated with a selected other of
! 25 the specifically binding biomaterial and the
biological substance. The particles are bound to the
surface via binding of the biological substance to
the binding sites. When such a solid support having
particles already attached thereto is contacted with
the aqueous sample, -the paxticles are released from
contact with the surface if the aqueous sample
contains a significant amount of either the
specifically binding biomaterial or the biological
- 23 -
`'\ ~ : ~
~z~
; substance. While the theory behind such action is
not completely understood it is believed that this
may be due to competitive reaction by the dissolved
specifically binding biomaterial or biologica]
substance in the aqueous sample for the binding site.
In any event, what i9 observed is the release of the
particles from the macroextensive surface. This is
an extremely straightforward and simple test and is
very easy to. run. For pregnancy testing, for
example, one need only place a plastic strip having a
plurality of particles attached to it into the urine
of the suspected pregnant woman and observe whether
the particles are released from the surface. This
takes only a few minutes, generally about ten
; 15 minutes, although it may be desirable to run the test
somewhat longer for certainty. Generally, the strip
would be removed from the urine and rinsed to
observed whether or not the particles are washed off.
The particles can be of the same nature as the
particles discussed for the previous method. That
is, they may be made of the same materials, may
include a color imparting agent, if desired, may be
of a selected size to increase the amount of clouding
seen on the strip prior to its insertion in the
aqueous sample, etc. Similarly, the strip may be of
any of the materials previously disclosed for the
strip utilized in the first method discussed above.
Also in accordance with the present invention
there is provided a kit which simply comprises a
solid support having a water insoluble macroextensive
surface associated with a selected one of the
specifically binding biomaterial and the biological
substance, the solid support havinq bound thereto a
~æ4~
plurality of particles having particle surfaces
associated with a selected other of the specifically
binding biomaterial and the biological substance, the
binding of the particles to the surface being via
binding of the biological substance to the binding
sites. For comparison purposes it may be desirable
to provide two such strips with only one of the two
strips being inserted in the suspect pregnancy urine
and the othe~ strip being inserted in, fcr example,
male urine. ~owever, the test is so clear that this
is not believed to be normally necessary. i
Appropriate rinsing solukion may also form a part of
this kit.
In all of the embodiments previously
described it is possible, and in some instances may
be desirable, to have an intermediate specifically
binding biomaterial bound as a bridge to the
macroextensive surface and/or the particle surfaces
and to, respectively, the specifically binding
biomaterial and the ~iological substance. For
example, the macroextensive surface can have a first
antigen bound to it while the biological substance
can be a second antigen. In this instance, the
specifically binding biomaterial can have a first
site which is a specific binding partner to the first
antigen (allowing the specifically binding
biomaterial to be bound via the first antigen to the
macroextensive surface) and a second site which is
the binding site to the biological substance (the
second antigen~. It is necessary in such instances
that the intermediate specifically binding
biomaterial not significantly interfere with binding
of the binding site to the biological substances.
~2~
biomaterial not significantly interfere with binding
of the binding site to the biological substances.
The invention will be better understood by
reference to the following lllustrative examples.
Example I
Identical strips of polyvinylchloride plastic
approximately~ 5 millimeters by approximately 40
millimeters and approximately ~ millimeter thick were
placed into respective containers and positioned so
; lO that approximately 20% of their lengths were beneath
the surfaces of respective urine samples, 3 of which
were obtained from women who were pregnant and 1 of
which was obtained from a non-pregnant woman. The
, samples were then allowed to stand without being
disturbed for approximately one hour. The strips
were then removed from the urine samples and rinsed
with a phosphate buffered saline (PsS) solution
containing 1~ bovine serum albumin (sSA) and 0.1%
azide.
The strips were next placed into aliquots of
a reagent solution containing carboxylated
polystyrene particles (MX Covasphereso, registered
trademark of Covalent Technology Corporation Lot
#11~-82, 0.7 micron, fluorescent green) onto which a
monoclonal antibody ko hCG (10 mg/ml in ascites
ka = 3 3 x 101) had been adsorbed. The carboxylated
polystyrene particles were associated with the
monoclonal antibody by contacting 0.2 ml of a 1.05~
suspension of the MX Covasphereso with 0.05 ml of the
monoclonal antibody to ~-hCG and 0.1 ml phosphate
buffered saline ~pH 7.4, made with concentrations of
~2q~
6.8 gms/l NaCl, 1.48 gms/l Na2HP04, 0.43 gm/l KH2C03
and containing 0.1% sodium azide) and incubating at
room temperature for 1 hour. The particles were then
centrifuged down, resuspended by sonication in 0.2 ml
PBS containing 1~ BSA, and recentri~uged. The
washing was repeated two more times. (It has since
been found that vortexing works better than
sonication). The particles were then resuspended in
0.2 ml PBS cOntaining 1% BSA to provide the reagent
solution aliquots. The strips were allowed to stand
in the reagent solution aliquots for approximately 10
minutes. The strips were then removed from the
reagent solution aliquots and were rinsed with water
to remove any non-bound particles. The strips were
then dried and it was noted that the previously clear
and transparent strips which had been incubated with
the urine from pregnant women now exhibited an
observable haze indicating a positive test for
3-hCG. This constituted a positive test for
pregnancy. The strip which had been incubated with
the urine from a non-pregnant woman remained clear.
Example II
One plastic strip (polyvinylchloride) was
incubated for lZ hours in a closed vial containing
monoclonal antibody to the -strand of hCG in a
concentration of 1.27 x 10 2 milligrams per
milliliter. A second plastic strip
(polyvinylchloride) was incubated for 12 hours in a
solution containing monoclonal antibody to the
~-strand of hCG in a concentration of l x 10
milligram per milliliter. The rest of the solution
- 27 -
5~8~
was PBS as in Exampla I~ Each strip was washed for
fifteen seconds with PBS containing l~ BSA. Each
strip was placed in an iodine 125 labelled hCG
solution with an iodine activity o 0.9 nanogram per
100,000 cpm in 2% gelatin solution in PBS. ~he
strips were periodically rinsed and counted on a
gamm~ counter. The strip which had been soa~ed for
12 hours in anti -hCG showed 488 counts per minutes
after 40 minu~es. The strip which had been incubated
for 12 hours in anti B-hCG showed 449 cpm after 260
minutesO Uncoated strips tested in the same way
showed 2325 cpm after 35 minu~es. This indicated
that ~he physiadsorption of the hCG on the uncoated
plastic strip is about 50 times as effective as
bindin~ of the hCG to the strips by means of anti hCG
antibodles which have been adsorped onto the strip.
Example III
.
A pair of strips made of polyvinylchlori~e
were both incubated in 50 nanogram per milliliter
B-hCG in PBS. As a result, they physiadsorbed ~he
~-hCG. Each strip was rinsed in 1% gelatln in PBS.
Each strip was placed in an anti B-hCG microsphere
suspension prepared as described in Example I. Each
of the strips was then rinsed in PBS. Each of the
strips was khen stored for 1 hour in PBS. The
microspheres remained in adherence with the strips.
One of the strips was plaaed in a sample of male
urine for 10 minutes and it was noted that the
microspheres remained bound thereto. The second of
the strips was placed in a sample of male urine to
which 2 nanograms per milliliter of B-hCG had been
added. At the end of ten minutes the microspheres had
28
. ~ .
:~2~5~8~
come off of the strip in the second sample of male
urine.
This experiment indicates the applicability
of a substantially one-step process for determining
possible pregnancy through determining the possible
occurrence of B-hCG in urine.
Example IV
.,
A clear polyvinylchloride strip is incubated
with antibody No. 1, then coated with gelatin or sSA.
It is then placed in a suspect pregnancy urine and
incubated for between 1 and 60 minutes, after which
the strip is incubated in microspheres which are
coupled to antibody No. 2. These microspheres are
carboxylated polystyrene which have been activated
with carbodiimide, washed, reacted with antibody No.
2, washed, and reacted with AECM Ficoll. Antibody
No. 1 and antibody No. 2 are antibodies which have
the property of being able to simultaneously react
with human chorionic gonadotrophin or with the
B-subunit of human chorionic gonadotrophin, i.e., a
given molecule of human chorionic gonadotrophin or
B-subunit of human chorionic gonadotrophin can have
both antibody No. 1 and antibody No. 2 attached to it
simultaneously. After the strip has been incubated
in the microspheres for between 1 and 60 minutes, it
is removed, rinsed and dried. A positive test is
indicated by the plastic strip having a cloudy
appearance caused by adherence of the microspheres.
This assay is also applicable for chorionic
gonadotrophin in the urine of species other than
humans~
_ ~9 _
~5~
gonadotrophin in the urine o species other than
humans.
~x~
Polyvinyl chloride str~ps wexe allowed ~o sit
for 12 hours with the lower Pnds of the strips
immersed in a solution containing PBS (previously
described in Example I) and an antibody to ~-hCG.
The concentxation of the antibody was .01 mg/ml. Its
binding constant was 3.3 x 101. The strips with
their adsorbed antibo~y were removed from the
solution, rinsed with P~S containing 1% bo~ine serum
albumin, rinsed briefly with distilled water and
allowed to dry.
~he following day, 0.1 ml aliquots of
carboxylated polys~yrene particles (CX Covaspheres~ a
registered trademark o~ Co~alen~ Technology
Corporation, Lot #llF82-CX, .7 micron fluorescent
green) were incubated for one hour with 0.1 ml
aliquots of non-pregnant female urine, three of which
had been doped with 50 ngms/ml B-hCG and three of
wh~ch served as controls. The incubation took place
in small glass $2.0 ml) serum vials to prevent
binding of the ~-hCG to the plastic of the Eppindorf
tubes in which the second s~age of the ~es would be
carried out. The samples were transferred ~o
Eppindorf tubes and the antibody-treated strips were
placed in the samples.
First~ a pair of strips were removed after
ten minutes and rinsed with distilled water. Both
the control and 50 ngms B-hCG doped samples yielded
clear strips ~a negative test3. Second, a paix of
strips were removed after 20 minutes and rinsed
with distilled water. These exhibited a clear
control strip and a light haze on the ~-hCG sample
strip. Third, a pair of strips were removed after
thirty minutes and rinsed with dis-tilled water.
These exhibited a haze on both the control and the
~-hCG sample strips.
These results indicate that it is possible to
; attach the antibody to the polyvinyl chloride strip
and adsorb the sample B-hCG onto the polystyrene
particles; however, the timing is critical at this
stage of deve~opment - a time of ten minutes yielding
a false negative result and a time of thirty minutes
yielding a false positive result. A timing of twenty
minutes yielded a visible but not dramatic positive
i test and a clear negative test.
i While the above examples describe the
invention in terms of a test for the presence of
~-hCG in urine it should be noted that the test is
- really much broader in applicability. ~hat is,
through proper choice of antigen and antibod~ the
test can be used for determining the presence of
~ 20 possible diseases such a typhoid fever, diptheria,
; other bacterial infections, and other virus
infections and the like. Also, the test can be
utilizçd for testing for biological and chemical
warfare agents, drinking water contamination,
epideminological studies, mass field screening and
the presence of ovulation. Other tests are also
possible including tests for the presence of drugs,
etc.
Industrial Applicability
In accordance with the present invention test
kits and methods are provided for analyzing ~or any
9~
of a number of analytes including specifically hCG.
The tests are very easy to run and require little or
no training for the operator. They are fast and
provide great specificity and sensitivity.
Although the foregoing invention has been
described in some detail by way of illustration and
example for the purposes of clarity and
understanding, it should be recognized that certain
changes and m~difications may be practiced within the
scope of the appended claims.
