IMMUNOSUBSTRATE AND IMMUNOADSORPTIVE SURFACE COATING FOR SOLID-PHASE IMMUNOSUBSTRATE

Abstract: The field of the present invention is immunoassay. Disclosed is a solid-phase immunosubstrate (14), which has a surface coating (12) composed of a water based emulsion of microdiameter sized latex polymer beads (16) and materials which act as light scattering centers (17). The surface coating (12) has an immunoreagent added to it, in order to form an immunoreactive immunosubstrate (27). Also disclosed is a method for manufacturing a composite including the immunosubstrate and coating. The surface coating provides an immunoreagent adsorptive volume; thus permitting adsorption of larger quantities of immunoreagent than possible for prior art solid-phase immunosurfaces, and increasing assay sensitivity. Further, the coating will retain adsorbed immunoreagent against vigorous washing.

Description: Specification B1MUNOSUBSTRATE AND EVfMJNOADSORPTTVE SURFACE COATING FOR SOLID-PHASE IMMUNOSUBSTRATE BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to solid-phase iiranuno substrates, for use ' in immunolog ical assaying and more particularly to a surface coating or film for application to a solid-phase surface and to the immunosubstrate formed by the utilization of the surface coating. Description of the Prior Art Many solid-phase surfaces are presently employed in immunolog ical testing to which a protein, such as an antibody (specific im unoglobul in ) or an antigen (any one of many protein or protein bound substances), may be adsorbed to form an immunosubstrate. The solid-phase may be a metallic surface, such as is employed in polarization change studies, or a glass or smooth plastic surface in which the protein is fixed onto the surface by air drying, heat denaturation or the use of organic fixing agents such as acetone, ethanol , methanol or formaldehy e. In some cases use is made of protein adherence to the polymer chains of certain plastics without a fixing process, such as in the adsorption of viral, bacterial , hormonal or other antigens to polypropylene and/or polystyrene test tube walls, beads or microtiter plates. Cellulose and cellulose derivatives, such as mixtures of cellulose acetates - B Xr and cellulose nitrates, have also been utilized as adsorptive surfaces for protein binding. These prior art solid-phase surfaces are generally utilized without any surface coatings to alter the adsorptive character of the surface. A common problem with such surfaces is that care must be taken when washing the surface during test procedures to prevent the immunoreagents or analytes that have adhered to the surface from being removed therefrom. Additionally, the use of an uncoated surface limits the user to the surface area of that surface for immunoreagent adsorption, whereas an immuno ad so p t i ve coating on the surface has a thickness which creates an immunoreagent adsoptive volume, thus permitting the adsorption of a greater quantity of immunoreagent and increasing the sensitivity of the assay. The surface coating or film of the present invention is applied to solid-phase surfaces to increase adsorption and retention of immunoreagents. Its main constituent is a water based emulsion of microdiameter size polymer beads to which may be added a material which imparts light scattering centers into the coating. No prior art is known to the inventors that utilizes such polymer beads within a surface coating for solid-phase surfaces, nor that combines them with a material providing optical properties that improves the fluorescent testing capability of the immunosubstrate formed through the use of the surface coating. SUMMARY OF THE PRESENT INVENTION It is therefore a primary objective of the present invention to provide a surface coating for a solid-phase surface which aids in the adsorbtion and retention of immunoreagents. It is another object of the present invention to provide a surface coating which may be applied to both porous and non-porous solid-phase surfaces. It is a further object of the present invention to provide a solid-phase immunosubstrate which is specifically useful in optical immunoassay techniques such as fluorescence studies or absorptomet r y or ref lecto etry with enzyme label utilization. The immunoad sorptive surface coating of the present invention includes a water based emulsion of dispersed polymer beads of microdiameter size. Where the coating is to be used in an optical testing situation, such as fluorescence testing, additional materials may be added to the coating to provide light scattering centers within the coating. In the surface coating of the preferred embodiment, the polymer ay be an acrylic, polyvinyl acetate or butadiene-styrene copolymer or a combination thereof, and a source of light scattering centers may be starch, titanium dioxide, silicon dioxide or finely powdered clay or a combination thereof . The coating is applied by standard coating techniques to a solid-phase surface to create the immunosubstrate -of the present invention. For particular applications of the immuno¬ substrate of the present invention, a droplet of ε particular immunoreagent , such as an antibody or antigen, may be placed on the immunosubstrate to penetrate thereinto and bind therewith. The immuno¬ substrate, with the immunoreagent ■ in place, is now ready for instantaneous future use for testing in the immunoassays associated with the particular immuno- ^ r y reagent. T e immunoreagent may be added to the surface coating prior zo z e applica ion ; e*eof on a solid-phase surface to produce similar results. As a further modification of the immunoreagent impregnated immunosubstrate of the present invention, humectants and flow enhancers may be added to the surface coating or as an additional overcoating to aid in the penetration of the immunosubstrate coating by a test serum. A primary advantage of the surface coating of the present invention is that it- is has greater ' adsorptive properties than non-coated solid-phase surfaces . It is another advantage of the surface coating of the present invention that it will retain the adsorbed immunoreagent against vigorous washing. It is a further advantage of the surface coating of the present invention that it contains light scattering centers which provide improved optical properties to the immunosubstrate. It is an advantage of the coated imm no¬ substrate of thε present invention that an immuno¬ reagent may be placed thereon to impregnate the coating, whereupon the immunosubstrate contains thε immunoreagent in a form ready for instantaneous future use. These 'and other objects and advantages of the present invention will no doubt become apparent to those of ordinary skill in the art after having read the following detailed description of the preferred embodiments which are illustrated in the several figures of the drawing. BAD ORIGINA IN THE DRAWING Fig. 1 depicts a magnified cross sectional view of the surface coating of the present invention as applied to a porous solid-phase surface; Fig. 2 depicts a magnified cross sectional view of the surface coating of the present invention as applied to a non-porous solid-phase surface; Fig. 3 depicts the addition of an immunoreagent to the immunosubstrate shown in Fig. ' l; Fig. 4 depicts the i munoreactive immunosub¬ strate shown in Fig. 3 after an over-coating has been applied thereto; and Fig. 5 depicts the utilization of the immuno¬ reactive immunosubstrate of the instant invention in a fluorescent testing situation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The surface coating for a solid-phase surface of the present invention comprises a ' water based emulsion of dispersed polymer beads of mic odiameter size. The utilization of such polymer beads in a water based emulsion produces a superior substance for protein binding, and where the "bound proteins are immunorea ents such- as antibodies or antigens an excellent im unosubstrate is created . In the preferred embodiment, a bead diameter of 0.1 to 1.0 microns has been found to produce excellent results. Larger bead diameters present little problem until their size affects the consistency of the coating or the ability of the beads to penetrate into a porous solid-phase surface as is hereinafter described. It is to be noted, however, that a larger bead size will also reduce the available bead surface area for protein binding. Almost any material having good protein binding properties and having the capabilit of being formulated into microdiameter size particle within a water based emulsion could be utilized i the instant surface coating invention. Material producing superior results have proved to be in th general class of latex type polymers. Particula latex polymers such as acrylics, polyvinyl acetate and butad iene-styrene copolymers have provide excellent results in immunor eac t ions , and thes polymers or combinations of them are utilized in th preferred embodiment. Many emulsifiers are known t those skilled in the art to create an emulsion of th water-bead mixture, a suitable emulsifier being a ammonium soap of a fatty acid. Where the surface coating is to be used in a optical testing situation, such as fluorescen testing, the addition of a substance which specifi cally provides light scattering centers within th coating has proved to increase the fluorescence o the test sample created through use of the surfac coating. Such scattering centers are importan because it is the nature of the instant coating t permit immunoreagents to permeate therethrough, an the scattering centers within the coating aid in th optical detection of such permeating immunoreagent and their immunoreactions . Suitable materials for providing light scat tering centers have been found to be starch, titaniu dioxide, silicon dioxide and finely powdered clay such as kaolin. However , the quantity of suc materials must be controlled as an increase in non specific binding of iπimuno re agents can be noted wit increasing amounts of light scattering centers. Thi effect is particularly pronounced where titaniu dioxide or clay is utilized. The basic surface coating according to the pres¬ ent invention is therefore primarily a water based latex bead emulsion which may contain additional substances that contribute light scattering centers. Its basic formulation varies according to whether light scattering centers are to be added; thus its composition by weight " may vary from approximately 30% latex polymer beads 69% water 1% emulsifying agent to a formulation including light scattering centers which has a composition by weight of approximately: 15% light scattering centers 15% latex polymer beads 1% emulsifying 'agent 69% water Further refinements of these basic formulations involve the addition of preservatives, surfactants, pH buffers, fungicides and defoa ers to provide various properties to the surface coating that are helpful in the manufacture, storage and usage of the coating and immunosubstrate described hereinafter. Thus a formulation of a preferred embodiment of the surface coating of the present invention comprises the following approximate composition by weight: 4% butad iene-styrene copolymer 7% polyvinyl acetate polymer 4.5% titanium dioxide 6.8% silicon dioxide 0.5% preservation 0.1% pH buffer 0.5% fungicide 0.5% defoamer 4.0% surfactants 1.0% e ulsifier 71.1% water The coating may be applied to a solid-phase surface by any of the standard application techniques; thus brushing, spraying, sponging, blade coating, rolling, dipping or droplet depositing, as well as other standard techniques, may be utilized for its application. The coating may be applied to either porous or non-porous solid-phase surfaces. Fig. 1 is a cross sectional. view of a surface coating 12 according to the instant invention as applied to a porous solid-phase surface 14 whereas Fig. 2 is a cros.s sectional view of a surface coating 12 accordinging to the instant invention as applied to a non-porous solid-phase surface 15. The difference between the two coated surfaces of Fig. 1 and Fig. 2 is the penetration 13 of the coating 12 into the surface 13 of the porous solid-phase 14 which is not found where a non-porous solid-phase 15 is utilized. It is noted that the polymer beads 16 are shown as circular elements and light scattering particulates 17 are shown as triangular elements, both of which are sho n disproportionately la ' rge to aid in understanding the invention. A porous surface 14 may consist of a paper-like material composed of matted cellulosic fibers 20. For use in optical analysis, such as by fluorescence, the fiber 20 diameters must be considerably larger than the wavelengths of visible light, such that any light entering the disordered matrix of fibers will bε scattered about by reflections from the fiber-liquid interface. Fiber diameters exceeding 0.4 to 0.7 microns have been found to be quite suitable. Larger fiber diameters present little problem until their size affects the ability of the porous mesh to retain the coating in place. The light scattering centers component 17 of the coating is particularly important where the coating is applied to a porous surface, as good immunoassay results utilizing, optical methods require light penetration to significant depths within the porous material. The light scattering centers aid in obtaining such light penetration. A non-porous solid-phase surface 15 for the coating may be metal, glass or any of the various plastic materials that have been utilized in the prior art. The coating , when utilized on a non- porous surface , provides a thickness to the immunoad so rbant layer in which the immunochem ical reactions occur. This thickness creates a volume in which reactions occur and, as it results in an increased amount of adsorbed immunor e acμen t per unit of surface area over a non-coated solid-phase surface, it thereby generally produces a more sensitive immunoassay than a non-coated solid-phase surface. A coating thickness of approximately 0.1 millimeters has been found to produce good results. After a solid-phase surface has been coated, it is termed an immunosubstrate 19. As shown in Fig. 3, it may then be prepared for a particular i muno chem¬ ical reaction by selecting an immunoreagent, such as a diluted antigen, antibody or enzyme solution, and wet¬ ting surface of the immunosubstrate with a few drop¬ lets 22 thereof. Humectants and wetting agents such as hydroxypropylmethyl cellulose may be added to the immunoreagent solution to speed the wetting process. Where a porous surface has been utilized, the immunoreagent solution penetrates into the coating and wets the body of the porous material, causing fiber swelling 24. The polymer beads 16 and light scattering centers 17 are carred ' into the fibrous mesh of the porous material where they are entrapped 26. The immunoreagent is also carried into the mesh and adsorbed onto the entrapped polymer beads 16. The immunoreagent impregnated porous immunosubstrate is then allowed to dry. As the moisture evaporates during the drying process, the fibers return to their original unswollen size and positions, but now firmly enmesh the dried immunoreagent, thus providing an exceedingly stable immunoreact ive immunosubstrate. An alternative embodiment, that has been utilized for the production of an immunor eact ive immunosubstrate , comprises the addition cf an immunoreagent directly to the surface coating described hereinabove prior to the coating of a solid- phase surface. The addition of the immunoreagent to the surface coatin creates an i munoreactive surface coating. Approxi a ely 0.01% by weight of immunoreagent is added to thε surface coating formulation in this embodiment. However, the amount of immunoreagent will vary depending upon the exact immunoreagent being utilized and the desired sensi¬ tivity of the immunoreactive immunosubstrate being formed. The immunoreactive surface coating may then be applied to a solids-phase surface in any of the above-mentioned standard application methods. The resulting immunoreactive immunosubstrate can be made everywhere reactive on its surface by a uniform coating of the immunoreactive surface coating on thε solid-phase or it can be made reactive in localized areas by placing droplets of immunoreactive surface coating at discrete locations on the solid-phase. As shown in Fig. 4, an immunoreactive immuno¬ substrate 27 may be made even more sensitive by then overcoating the surface thereof with a coating 28 of basically the same ingredients as the instant coating, but containing a higher percentage of the polymer beads 16. A typical overcoating liquid comprises substan- tially the following formulation by weight: Stock emulsion: 10-20% light scattering centers 10-25% latex polymer beads 1% emulsifier plus water vehicle in amount to total 100% by weight of the stock emulsion. To 100 ml of the stock emulsion is added: .5 g of carboxymethyl cellulose 1 ml of polyvinyl alcohol 1 ml of hydroxypropylmethyl cellulose as humectants and flow enhancers. The immunoreactive immunosubstrate 27 may be utilized according to the following description. The immunosubstrate , having been previously impregnated with a specific antigen, is exposed to body fluid containing unknown amounts of an antibody directed at the specific antigen. The body fluid wets the coating and orks its way through the thickness thereof . If the solid-phase is porous the body fluid penetrates into the mesh in the same manner as did the immuno eagent droplets 22. Any antibody present in the body fluid binds immuno- chemically to its specific antigen on the polymer beads. After washing, to remove any fluid and unbound antibody, the immunosubstrate is exposed to an antihuman immunoglobulin made in another species (for example, goat antihuman immunoglobulin IgG or rabbit antihuman immunoglobulin IgM) . If any of the human antibody had been present in the fluid it would be bound to the specific antigen within the coating, and the goat antibody would bind im unochemically to it as it worked its way through the coating thickness. The goat antihuman antibody is labeled with a detectable tracer such as a radioactive tag, a fluorescent dye, or other standard tag. In the case of a radio label, such as iodine (^-^^1) , a scintil¬ lation counter can be employed to quantitate the amount of label and, by calibration, the amount of human antibody bound in the immunolog ical reaction. Where a fluorochrome label such as fluorescein isothiocyanate (FITC) is utilized, any fluorometεr capable of reading solid surfaces will be able to read the strength of fluorescence signal from the immunosubstrate, and thereby quantitate the amount of human antibody bound in the immunolog ical reaction. In one type of fluorescence testing, as depicted in Fig. 5, a blue photon 30 is isolated through the use of an excitation filter 31 and directed at the immunoreactive immunosubstrate 27. It will eventually either be absorbed by a molecule other than FITC, in which case it will raise the temperature of the immunosubstrate minutely by vibrational decay, or it will be absorbed by the FITC molecule, in which case, with a very high probability (greater than 90%) , it will make a singlet-singlet transition and release one green photon. In its emersion from the FITC, however, the green photon will either be lost to absorption by some other molecules or it will come out 32 of the immunosubs rate for detection by a photoelectric transducer 34 through the use of an emission filter 33 which passes only green photons. The advantage that occurs in an immunosubstrate containing many scattering centers , .such as is * described hereinabove, is that the bouncing about of the blue photon from the center increases its chance of eventual absorption by an FITC molecule which may not have been in its direct flight path. Also, the emerging green photon has a greater probability of being detected in a single direction if it makes many flight path changes, as are creatεd by the scattering centers. Thus, the increase in number of scattering centers will tend to increase the fluorescence of the sample . The instant surface coating and the imsiuno- substrates formed therefrom have been studied employing both radioactive and fluorescence labels and utilizing both antibodies and antigens as immuno¬ reagents. The following examples will highlight their properties. EXAMPLE I - Binding Capacity of the Immunosubstrate Studies were carried out with radiolabeled (125 j ) antibody ( goat anti-human IgG) deposited in varying amounts on the surface of both a coated, porous surfaced immunosubstrate, as described herein- above, and, as a control, on commercially available immunosubstrate films of mixed esters of cellulose which are used frequently for protein binding applications. A butad iene-styr ene copolymer was utilized in the surface coating. Both were subjected to counts in a gamma counter prior to and following four vigorous washing steps in buffers for 30 minute periods. From Table I it can be seen that at low protein loading (50 yg ) both surfaces bind comparably, losing about 30% in a first wash and about half by the end of the fourth wash . As heavier protein loadings are employed, the immunosubstrate of the present invention shows consi¬ derably better binding properties. At 200 μgm of protein per unit of surface area, the mixed cellulose esters will lose almost 80% in the first wash, while the immunosubstrate will lose less than 50%. TABLE I RADIOACTIVE COUNTS (% Loss of Protein) CELLULOSE Buffer 50 ug 100 μgm 200 μgm Preassay 174 320 502 966 First Wash 139 197(38%) 189(62%) 217(78%) Second Wash 136 172 (-46%) 161(68%) 173(82%) Third Wash 136 151(53%) 143(72%) 150(89%) Fourth Wash 140 149 53%) 139(72%) 141(85%) IMMUNO- Buffer 50 μgm 100 μgm 200 μgm SURFACE Preassay 141 349 501 915 First Wash 143 254(27%) 282(44%) 488(47%) Second Wash 143 242(31%) 282(44%) 412(55%) Third Wash 140 189(46%) 256(49%) 359(61%) Fourth Wash 136 185(51%) 256(49%) 334(63%) -IS. EXAMPLE II - Mixed Antigens in the Substrate - Detection of a Mixture of Serum Antibodies For many years, rheu atolog ists and clinicians have relied on the ANA test (anti-nuclear antibodies) as a reliable screen for connective tissue diseases such as Systemic Lupus Er ytha atosus (SLE) and Rheumatoid Arthritis (RA). Since these diseases are characterized by the presence of several antibodies directed at different antigens within the nucleus of mammalian cells, all tests to date have employed whole cells as the substrate. These tests are generally performed with sliced tissue sections or cultivated cell lines in monolayers on glass microscope slides for subjective readout in a fluorescence microscope. One manu¬ facturer has deposited whole suspended epithelial cells on a plastic substrate and read out the fluorescence objectively in a specially designed f luorometer . The coating described hereinabove is capable of binding both cellular components and mixtures of solubilized antigens . ANA tests have thus been performed with cellular components consisting of salmon sperm nuclei separated from cells, and with a solubilized mixture of the most significant antigens considered stimuli to the anti-nuclear antibodies, such as double stranded deoxyr ibonucl e ic acid (D- DNA) , nucleohis tones ( DNA-prote ins ) and ribonucleic acid (RNA) . This test is termed an AΞNA ( antibodies to soluble nuclear antigens) test. Results reported with various antigen-substrate combinations are shown in Table II, including both porous and non-porous solid-phase immunosubstr a tes . A butad iene-styrene copolymer was utilized in the surface coating. TABLE II TESTS FOR ANA AND ASNA WITH VARIOUS IMMUNOSUBSTRATES Serum From Cas ;e Number Subj ect #1 #2 #3 #4 5 SI. Healthy neg 33 * 56 55 ' 57 #2. Healthy neg 35 48 41 — #3. With connec 62 76 65 76 tive tissue disease #4. With Systemic 4+P/D 101 102 . 100 102 Lupus #5. With Systemic 3+D/S 114 84 80 Lupus Case 1 - Standard microscope readout on rat liver slices. neg = no fluorescence, 3+ is bright fluorescence, P = peripheral staining, D = diffuse staining, S = speckled staining of the cell nucleus. Case 2 - Suspended human epithelial cells fixed to solid plastic substrates and read in objective fluorometer. Fluorescence greater than 40 is positive for ANA. Case 3 - Disclosed coating, blade coated on a porous cellulose base and wetted with the mix of soluble antigens (ASNA) or whole nuclei (salmon or herring sperm nuclei) . Fluores¬ cence greater than 60 is positive for ANA. Case 4 - Liquid coating material, dropped onto solid plastic base, dried, then wetted with soluble antigens (ASNA) . Fluorescence greater than 60 is positive for ANA. Case 5 - Soluble antigens (ASNA) blended into liquid coating material, then deposited on solid plastic base. Fluorescence greater than 60 is positive for ANA. Comparison of the results for cases 3, 4 and 5 shows that the coating, as opposed to the surface to which it is applied, is a determining factor in the results obtained, and that the immunoreagent can be incorporated into the liquid surface coating prior to coating the solid-phase surface. EXAMPLE III - Determining Antibodies to a Specific Viral Antigen in Body Fluid Whereas Example II demonstrates that the instant coating is usable on non-porous as well as porous surfaces, in this Example a modification of the typical coating formulat n is made. The amount of light scattering centers is reduced , and the polymer beads (emulsion of polyvinyl acetate beads) represents a much greater percentage of the coating. This coating is much like the overcoating stock emulsion formulation described hereinabove. When this modified mixture is deposited in 25 μl droplets on a smooth, hard solid-phase surface, a thick coating is formed. While milky in its liquid state, it dries to clear , hard and glossy textured spots . To make this an immunoreactive immunosubstrate, 25 μl of commercially available rubella antigen is deposited on the film spots. The antigen, in aqueous suspension, penetrates the thick film spots and binds with the polymer beads therein. The immunoreactive immunosubstrate is allowed to dry for storage until it is to be used in an assay. In an assay, upon incubation with diluted serum, plasma, urine , saliva, etc. , the immuno- reaction occurs in the wetted spot and a milkiness is manifested due to the difference in refractive index between the polymer beads and the aqueous medium. Since the test spot is read wet in a fluorometer, this milkiness provides much of the optical light scattering properties for which other materials such as starch, clay or titanium dioxide were previously emplo e . In a study employing this immunoreactive immuno¬ substrate , a panel of 29 serum samples with known rubella hemaggl utination (HAI) titer were tested employing immunof luorescence techniques. A simul¬ taneous test was conducted with a mixed cellulose esters surface. The comparison of these two surfaces when related to HAI is seen in the data presented in Table III. TABLE III IMMUNOSUBSTRATE COMPARISON WITH HAI TITER HAI CELLULOSE IMMUNOREACTIVE (29 samples) IMMUNOSUBSTRATE Negative-11 Negative-9 Negative-11 Positive-2 ?ositive-0 Specificity 82% Specificity 100% Positive-18 Positive-15 Positive-17 Negative-3 Negative-1 Specificity 83% ' Specificity 94% Overall Agree-24 Agree-28 Agreement Non-Agree-5 Non-Agree-1 Specificity 83% Specificity 96.5% In comparing the data in Table III a significant reduction in the number of false positives and false negatives is shown. The immunoreactive immunosub¬ strate thus presents more reliable test results than the standard cellulose immunosubstrate. It can therefore be seen that the i mu no- absorptive surface coating of the present invention is a unique substance comprising a water based emulsion of polymer beads of microdiameter size to which light scattering centers may be added; the concentrations of these constituents being variable according to the above description. The coating has the ability to adsorb immuno eagents within its thickness whereby more sensitive testing is accomplished. The coating may be applied both to porous and non-porous solid-phase surfaces to create an immuno¬ substrate which shows reduced loss of immunoreagent upon washing. Where an immuno eagent is added to the surface coating and allowed to dry on a solid- phase surface, an immunoreactive immunosubst ate is created having greater sensitivity and reliability than existing solid-phase surfaces. hereas, thε preferred embodiments of the present invention has been described above, it . is contemplated that other alterations and modifications may become apparent to those skilled in the art after having read the above disclosure. It is therefore intended that the appended claims be interpreted as covering all such alterations and modifications as fall within the true spirit and scope of the inven¬ tion. What is claimed is:

Claims: CLAIMS 1. An im unoadsorptive surface coating for a solid-phase surface for use in immunological assay testing comprising a water based emulsion of dispersed polymer beads including latex polymer beads of micro¬ diameter size, water and an emulsifying agent to promote the emulsif ication of latex polymer beads within said water . 2- An immunoadsorptive coating as recited in claim 1 wherein said beads are formed with a minimum diameter of 0.1 microns and said coating has a compo¬ sition by weight of approximately 30%, latex polymer beads, 69% water and 1% emulsifying agent. 3. An immunoadsorptive coating as recitεd in claim 2 whεrεin said polymer beads are composed of one or more of the substances from the group consisting of acrylic polymers, polyvinyl acetate polymers and butad iene-styrene copolymers. 4. An immunoadsorptive coating as recitεd in claim 3 wherein a source of light scattering centers is added to said coating whereby the fluorescent properties of said coating are improved. 5. An im unoadsorptive coating as recited in claim 4 wherein said source of light scattering centers is composed of one or more of the substances from the group consisting of starch, titanium dioxide, silicon dioxide and finely ground clay. 6. An immunoadsorptive coating as recited in claim 5 wherein the concentration by weight of the components of said coating is approximately 15% light scattering centers, 15% polymer beads, 69% water and 1% emulsifying agent. 7. An immunoadsorptive coating as recited in claim 5 wherein p eser atives, surfactants , pH buffers, fungicides and defoa ers are added to said coating whereby the concentration by weight of the components of said coating is approximately 11% latex polymer beads, 11.3% light scattering centers, .5% preservatives, .1% pH buffers, .5% fungicides, .5% defoamers, 4.0% surfactants , 1.0% emulsifying agents and 71.1% water. 8. A solid-phase immunosubstrate for use in immunological assay testing comprising: a solid-phase surface; an immunoadsorpti e surface coating being appliεd upon said surface, said coating comprising a water based emulsion of dispersed latex polymer beads including latex polymer beads of microdiameter size, watεr and an emulsifying agent to promote the emulsi- fication of said latex polymer beads within said wa er . 9. An immunosubstrate as recited in claim 8 wherein said beads are formed with a minimum diameter of 0.1 microns, said coating having a composition by weight of approximately 30% latex beads, 69% water and 1% emulsifying agent. - 10. An immunosubstrate as recited in claim 9 wherein said polymer beads are composed of one or more of the substances from the group consisting of acrylic polymer, polyvinyl acetate polymer and butad iene-styrene copolymers. 11. An immunosubstrate as recited in claim 10 wherein a source of light scattering centers is added to said coating whereby ' the fluorescent properties of said coating are improved. 12. An immunosubstrate as recitεd in claim 11 whεrein said source of light scattering centers is composed of one or more of the substances from the group consisting of starch, titanium dioxide and finely ground clay. 13. An immunosubstrate as recitεd in claim 12 whεrein the concentration by weight of the compnents of said coating is approximately 15% light scattering centers, 15% polymer beads, 69% water and 1% emulsi¬ fying agent. 14. An immunoadsorptive coating as recited in claim 13 whεrein preservatives, surfactants, pH buffers, fungicides and defoamers are added to said coating whεreby the concentration by weight of the componεnts of said coating is approximately 11% latex polymer beads, 11.3% light scattering centεrs, .5% preservatives, .1% pH buffers, .5% fungicides, .5% defoamers, 4.0% surfactants, 1.0% emulsifying agents and 71.1% water. 15. An immunoreactive surface coating for a solid-phase immunosubstrate for use in immunological assay testing comprising a water based emulsion of dispersed latex polymer beads, including latex polymer beads of microdiameter size, water, an immunoreagent and an emulsifying agent to promote the emulsif ication of said latex polymer beads within said water, whereby an immunoreactive surface coating is formed for use in specific immunόreactions which require said immunoreagent. 1 * 6. An immunoreacti e surface coating as recited in claim 15 wherein said beads are formed with a minimum diameter of 0.1 microns and said coating has a composition by weight of approximately 30% latex polymer beads, 68.99% water, 1% emulsifying agεnt, and 0.01% immunoreagent. 17. An immunoreactive surface coating as recited in claim 16 wherein said polymer beads are composed of one or more of the substances from the group consisting of acrylic polymers, polyvinyl acetate polymers and butadiene-styrene copolymers. 18. An immunoreactive surface coating as recited in claim 17 wherein a source of ligh scattering centers is added to said coating whereby the fluorescent properties of said coating are improved . 19. An immunoreactive surface coating as recitεd in claim 18 wherein said -source of light scattering centers is composed of one or more of the substances from the group consisting of starch, -- titaniu dioxide and finely ground clay. 20. An immunoreactive surface coating as recited in claim 19 wherein the concentration by weight of the components of said coating is approximately 15% light scattering centers, 15% polymer beads, 68.99% water, 1% emulsifying agent and 0.01% immuno eagent. 21. An immunoreactive surface coating as recited in claim 19 wherein preser atives, surfactants, pH buffers, fungicides and defoamers are added to said coating whereby the concentration by weight of the components of said coating is approximately 11% latex polymer beads, 11.3% light scattering centers, .5% preservatives, .1% pH buffers, .5% fungicides, .5% defoamers, 4.0% surfactants, 1.0% emulsifying agents, 71.09% water and 0.01% immunoreagent .- 22. A solid-phase immunoreactive immunosub¬ strate for use in immunological assay determinations comprising: a solid-phase surface; a water sed e lsion of dispersed latex polymer beads, incl ding latex poly e eads of microdiameter size, water and an emulsifying agent to promote the emulsif ication of said latex polymer beads within said water; and an immunoreagεnt bεing applied to said coating and adsorbεd into said coating whereby an immunoreactive immunosubstrate is formed for use in specific immunoreactions which rεquire said immunoreagent. 23. An immunoreacti e immunosubstra e as recited in claim 22 wherein said beads are formed with a minimum diameter of 0.1 microns, said coating having a composition by weight of approximately 30% latex polymer beads,' 68.99% water, 1% emulsifying agent and 0.01% immunoreagent. 24. An immunoreactive immunosubstrate as recited in claim 23 wherein said polymer beads are composed of one of the substances from the group consisting of acrylic polymers, polyvinyl acetate polymers and buted iene-styrenε copolymers. 25. An immunoreactive immunosubstrate as recited in claim 24 wherεin a source of light scattering centers is added to said coating whereby the fluorescεnt propεrtiεs of said surface coating are improved. 26. An immunoreactive immunosubstrate as recited in claim 25 wherεin said source of light scattering centers is composed of one or more of the substances from the group consisting of starch, titanium dioxide and finely ground clay. 27. An immunoreactive imm-uno subs tr ate as recitεd in claim 26 whεrein the concentration by weight of thε components of said coating is approximately 15% light scattering centers, 15% polymer beads, 68.99% water, 1% emulsifying agent and 0.01% immunoreagent. 28. An immunoreactive im unosubs rate as recited in claim 26 wherein preservatives, surf actants , pH buffers, fungicides and defoamεrs are added to said coating whereby the concentration by weight of the components of said coating is approximately 11% latex polymer beads, 11.3% light scattering centers , .5% pr e s ε r va t i vε s , .1% pH buffers , .5% fungas.ides , .5% defoamers , 4.0% surfactants, 1.0% emulsifying agents, 71.09% water and 0.01% immunoreagent. » 29. A solid-phase immunosubstrate as recitεd in claims 11 or 25 wherein said surface coating is further coated with an overcoating comprising substantially the following formulation by weight: a liquid emulsion of 100 milliliters including 10 through 20% light scattεring centers, 10 through 25% latex polymer beads, 1% emulsifying agεnts , plus a watεr vεhiclε in an amount sufficient to equal 100%; and added thereto is .5 grams of carboxymethyl cεllulosε, 1 millilitεr of polyvinyl alcohol and 1 millilitεr of hydroxypropylmεthyl cεllulosε. 30. A method for manufacturing an immunosub¬ strate comprising selecting a solid-phase surface; applying to said solid-phase surface a surface coating which comprises a water based emulsion of latex polymer beads which includes latex polymer bεad s of microdiameter size, water and an εmulsifying agent to promote the εmulsif ication of said beads within said water; and drying said surface coating. 31. A method for manufacturing an immunosub¬ strate as recitεd in claim 30 wherein said latex beads are formed with a minimum diameter of 0.1 microns and said coating has a composition by wεight of approximately 30% latex polymer beads, 69% water and 1% emulsifying agent. 32. A method for manufacturing an immunosub¬ strate as recited in claim 31 wherein said polymer beads are composed of one or more of the substances from the group consisting of acrylic polymers, polyvinyl acetate polymers and buted iene- s tyr ene copolymers . 33. A method for manufacturing an immunosub¬ strate as recitεd in claim 32 whεrein a source of light scattering centers is added to said coating whereby the fluorescent properties of said coating are improved . 34. A method for manufacturing an immuno¬ substrate as rεcited in claim 33 wherein said source of light scattering centεrs is composed of one or more of the substances from the group consisting of starch, titanium dioxide and finely ground clay. 35. A method 'for. manufacturing an immunosub¬ strate as recited in claim 34 wherein the concen¬ tration by weight of the components of said coating is approximately 15% light scattering centers, 15% polymer beads, 69% water and 1% emulsifying agents. 36. A method for manufacturing an immunosub¬ strate as recited in claim 34 wherein preservatives, surfactants, pH buffers, fungicides and defoamεrs arε add'-.d to said coating whεrεby the concentration by weight of the components of said coating is approxi¬ mately 11% latex polymer beads, 11.3% light scattering centers, .5% preservatives, .1% pH buffers, .5% fungicides, .5% defoamers, 4.0% surfactants, 1.0% emulsifying agents and 71.1% water. 37. A method for manufacturing an immunoreac¬ tive immunosubstrate comprising selecting a solid-phase surface; applying to said solid-phase surface a surface coating which comprises a water based emulsion of latex polymer beads which includes latex polymer beads of microdiameter size, water and an emulsifying agent to promote the emulsif ication of said beads within said water; applying to said surface coating a solution containing an immuno eagent which will beco e adsorbed to said polymer beads; and drying said solution and surface coating. 38. A method for manufacturing an immunor eac- tive immunosubstrate comprising selεcting a solid-phase surface; applying to said solid-phase surface an immuno¬ reactive surface coating whi h comprises an immunoreactive water based emulsion of latex polymer beads which includes latex polymer beads of microdiameter size, water, an immunoreagεnt and an ε ulsifying agent to promote the emulsif ication of said beads within said water; and said surface coating.

Inventors: BART M, HARTE R
Applicants: BART M, HARTE R
Application year: 1981
Main classification: G01N31
IPC codes: G01N33