CA1245981A - Solid phase biological diagnostic assay - Google Patents

Abstract

Abstract Methods and kits are set out having improved selectivity and improved sensitivity for use in field and home immunoassays. A specifically binding biomaterial is attached to a macroextensive surface of a plastic strip or the like. A biological substance which is a specific binding partner to a binding site of the specifically binding biomaterial is attached to each of a plurality of synthetic particles. The particles are of a prese-lected size, refractive index, or the like to enhance their visibility in accordance with the Mie scattering phenomenon. Testing is by either contacting the particles with the strips to obtain adherence of the particles to the strips, or by exposing strips having the particles already adhering to them to a solution containing either the specifically binding biomaterial or the biological substance, whereby the particles adherence to the strip is eliminated. A quick and accurate pregnancy test is one result of the invention.

Description

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.

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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 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 ~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 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 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 ~: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 `'\ ~ : ~

~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 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 . ~ .

:~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.

Claims (50)

Claims:

1. A method 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, said specifi-cally 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 suf-ficient 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 plur-ality of synthetic particles of a preselected size, in about the same range as the selected wavelength, and of a preselected refractive index as calculated by Mie scattering for clear visual observation and having particle surfaces bearing said biological sub-stance associated therewith for a time sufficient for said binding site to bind to said biological 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 adherence of said particles to said synthetic polymeric surface in light including said wavelength.

2. A method as set forth in claim 1, includ-ing, after said step (5) rinsing and before said step (6) observing, the added step of:
drying said rinsed support.

3. A method as set forth in claim 1, wherein said particles have an average diameter which falls within a range from about 0.2 micron to about 2.0 microns if the degree of adherence is observed in air and from about 0.47 micron to about 11.1 microns if the degree of adherence is observed in water.

4. A method as set forth in claim 3, wherein said support is transparent.

5. A method as set forth in claim 1, wherein said particles comprise latex particles.

6. A method as set forth in claim 1, wherein said particles include a color imparting material.

7. A method as set out in claim 1, further including:
shielding those portions of said surface of the support which are not bound to said specifically binding biomaterial with a material which prevents attachment of other biomaterials.

8. A method as set forth in claim 7, wherein said shielding step precedes said contacting step (1).

9. A method as set forth in claim 7, wherein said shielding step follows said separating step (2) and precedes said contacting step (3).

10. A method as set forth in claim 9, wherein said shielding is provided by contacting said surface with a shielding material selected from bovine serum albumin and gelatin.

11. A method as set forth in claim 1, wherein said biological substance is selected to have an extremely high affinity for said specifically binding biomaterial.

12. A method as set forth in claim 1, wherein said support comprises a hydrophobic polymer.

13. A method as set forth in claim 1, wherein said specifically binding biomaterial is hCG
or the .beta.-subunit of hCG and said biological substance is a monoclonal antibody for hCG or the .beta.-subunit of hCG.

14. A method as set forth in Claim wherein said macroextensive surface has an intermediate specifically binding biomaterial bound thereto, said intermediate specifically binding biomaterial having the property of binding to said specifically binding biomaterial and of not significantly interfering with binding of said binding site to said biological substance.

15. A method as set forth in claim 1, wherein said biological substance is associated with said particle surfaces via binding to an intermediate specifically binding biomaterial having the property of not significantly interfering with binding of said binding site to said biological substance.

16. 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 range, said specific-ally 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 surface being via binding of said biological substance to said binding sites; and visually observing the degree of release of said particles from said surface in light including said wavelength.

17. A method as set forth in claim 16, wherein said support is transparent.

18. A method as set forth in claim 16, wherein said particles have an average diameter which falls within a range from about 0.2 micron to about 2.0 microns if the degree of release is observed in air and from about 0.47 micron to about 11.1 microns if the degree of release is observed in water.

19. A method as set forth in claim 16, wherein said particles comprise latex particles.

20. A method as set forth in claim 16, wherein said particles include a color imparting material.

21. A method as set forth in claim 16, wherein said biological substance is selected to have an extremely high affinity for said specifically binding biomaterial.

22. A method as set forth in claim 16, wherein said support comprises a hydrophobic polymer.

23. A method as set forth in claim 16, wherein said particles comprise a hydrophobic polymer.

24. A method as set forth in claim 16, wherein said specifically binding biomaterial is hCG
or the .beta.-subunit of hCG and said biological substance is a monoclonal antibody for hCG or the .beta.-subunit of hCG.

25. A method as set forth in claim 16, wherein said macroextensive surface has an intermediate specifically binding biomaterial bound thereto, said intermediate specifically binding biomaterial having the property of binding to said selected one of said specifically binding biomaterial and said biological substance and of not significantly interfereing with binding of said binding site to said biological substance.

26. A method as set forth in claim 16, wherein said selected other of said specifically binding biomaterial and said biological substance is associated with said par-ticle surfaces via binding to an intermediate specifically binding biomaterial having the property of not significantly interfering with binding of said binding site to said bio-logical substance.

27. A kit for assaying an aqueous sample contain-ing 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, comprising:
a solid support having a water insoluble macro-extensive surface capable of associating with said specifically binding biomaterial; and 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 bearing said biological substance.

28. A kit as set forth in claim 27, further including:
a solution for rinsing said support to remove any non-bound particles.

29. A kit as set forth in claim 27, further including:
means for shielding those portions of the macroextensive surface which are not bound to said specifically binding biomaterial with a material which prevents attachment of other biomaterials.

30. A kit as set forth in claim 27, wherein said macroextensive surface has an intermediate specifically binding biomaterial bound thereto, said intermediate specif-ically binding biomaterial having the property of binding to said specifically binding biomaterial and of not signifi-cantly interfering with binding of said binding site to said biological substance.

31. A kit as set forth in claim 27, wherein said biological substance is associated with said particle surfaces via binding to an intermediate specifically binding biomaterial having the property of not significantly inter-fering with binding of said binding site to said biological substance.

32. A kit for assaying an aqueous sample contain-ing 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 specifically binding biomaterial being in association with other bio-materials, with increased speed, ease of assaying, specif-icity and sensitivity, comprising:
a solid support having a water insoluble synthetic polymeric macroextensive surface 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 bind-ing of said biological substance to said binding sites.

33. A kit as set forth in claim 32, further including:
a solution for rinsing said macroextensive surface.

34. A kit as set forth in claim 32, wherein said macroextensive surface has an intermediate specifically binding biomaterial bound thereto, said intermediate specifically binding biomaterial having the property of binding to said selected one of said specifically binding biomaterial and said biological substance and of not significantly interfering with binding of said binding site to said biological substance.

35. A kit as set forth in claim 32, wherein said selected other of said specifically binding biomaterial and said biological substance is associated with said particle surfaces via binding to an intermediate specifically binding biomaterial having the property of not significantly interfering with binding of said binding site to said biological substance.

36. A method as set forth in claim 2, wherein said particles have an average diameter which falls within a range from about 0.2 micron to about 2.0 microns.

37. A method of assaying an aqeuous 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 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 sur-face with an aqueous solution containing a plurality of synthetic particles of a preselected size, conductivity and refractive index for substantially optimal light scattering as calculated by the Mie scattering equa-tions having particle surfaces bearing said biological substance associated 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.

38. A method as set forth in claim 37, includ-ing, after said step (5) rinsing and before step (6), the added step of:
drying said rinsed support.

39. A method as set forth in claim 37, further characterized in that said particles are latex particles

40. A method as set forth in claim 37, further characterized in that said particles are substantially spherical.

41. A method as set forth in claim 1, wherein said particle size and refractive index are preselected so that said biomaterial can be assayed at levels as low as about 10 nanoagrams/ml.

42. A method as set forth in claim 37 wherein said particle size and refractive index are preselected so that said biomaterial can be assayed at levels as low as about 10 nanograms/ml.

43. A mehod as set forth in claim 16 wherein said particle size and refractive index are preselected so that said biomaterial can be assayed at levels as low as about 10 nanograms/ml.

44. A method as set forth in claim 1 wherein said visual observing is from within a cone having an angle from an incident light beam, said angle being substantially that corresponding to substantially the optimal detectivity of Mie scattering.

45. A method as set forth in claim 1, wherein said particles are of a size selected to preferentially Mie scatter light of said selected wavelength, which wavelength corresponds to a selected color.

46. A method as set forth in claim 16, wherein said particles are of a size selected to preferentially Mie scatter light of said selected wavelength, which wavelength corresponds to a selected color.

47. A method as set forth in claim 1, wherein said visual observing is by the naked eye.

48. A method as set forth in claim 16, wherein said visual observing is by the naked eye.

49. In a method of assaying an aqueous sample con-taining a specifically binding biomaterial having a binding site which is a specific binding partner to a biological substance by observation in light includng a selected wavelength in the visual range, said specifically binding biomaterial being in association with other biomaterials, comprising:
(1) contacting a solid support having a water insoluble macroextensive surface capable of associating wit said specifically binding biomaterial with said aqueuous sample for a time sufficient for said specifically binding biomaterial to associate with said macroextensive surface;
(2) separating said support from contact with said aqueous sample;
(3) contacting said macroextensive surface with an aqueous solution containing a plurality of particles having particle surfaces bearing said biological substance associated therewith for a time sufficient for said binding site to bind to said biological substance and to thereby bind said particles to said macroextensive surface;
(4) separating said support from said aqueous solution;
(5) rinsing said support to remove any non-bound particles; and (6) observing the degree of adherence of said particles to said macroextensive surface; the improvement comprising attaining increased speed, ease of assaying, specificity and selectivity, wherein said particles are synthetic polymeric particles and said plurality of synthetic particles are substantially spherical having an average diameter which falls within a range of from about 0.2 micron to about 11.1 microns, are of the substantially the same size within said range and have substantially the same selected refractive index, all as calculated by Mie scattering for clear visual observation of said particles when said particles are adhered to said macroextensive surface and are viewed in light including said wavelength.

50. In 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 range, said specifically binding biomaterial being in association with other bio-materials, comprising:
contacting said aqueous sample with a solid support having a water insoluble macroextensive surface associated with a selected one of said specifically binding biomaterial and said biological substance, said solid support having bound thereto a plurality of particles having particle surfaces associated with a selected other of said specifically binding biomaterial and said biological substance, the binding of said particles to said surface being via binding of said biological substance to said binding sites; and observing the degree of release of said particles from said macroextensive surface; the improvement compris-ing attaining increased speed, ease of assaying, specific-ity and selectivity, wherein said particles are synthetic polymeric particles and said plurality of synthetic particles are substantially spherical having an average diameter which falls within a range of from about 0.2 micron to about 11.1 microns, are of the substantially the same size within said range and have substantially the same selected refractive index, all as calculated by Mie scattering for clear visual observation of said particles when said particles are adhered to said macroextensive surface and are viewed in light including said wavelength.