Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/548—Carbohydrates, e.g. dextran

Definitions

• This invention relates to biological or immunological substances attached to solid carriers for use in diagnostic tests, enzyme processes, affinity purifications, and the like.
• Soluble biological substances attached to solid carriers have many uses in diagnostic tests, enzyme processes, and affinity purifications. For example, attachment of antibodies or antigens to a solid carrier allows their immunological partners to be easily removed from a mixture of many substances. Similarly, attaching enzymes to a solid carrier allows them to be easily removed from the reaction mixture or to be used in a continuous flow process. Heterogeneous radioimmunoassays and enzyme immunoassays rely on attachment of one or more of the reactants to a solid phase to venable separation from the free reactants.
• Agglutination assays utilize indicator or carrier particles (upon which are carried the appropriate 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 or antigens coupled to solids. Biological particles will, for example, specifically adhere to solids coated with appropriate antibodies and antigens so that separation from other particles can be affected. Identification of biological particles can be made through the 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 makes their presence more readily detectable.
• U.S. Patent No. 3,645,852 discloses a process wherein cyanogen halides are used to activate a water insoluble polymer which then couples to a water soluble protein.
• Water soluble carbodiimides can be used as a condensing agent to bind protein to polymeric carrier particles according to U.S. Patent No. 3,857,931.
• Biological substances can be covalently bound to plastic materials whose surfaces have been coated with glutaraldehyde as discussed in U.S. Patent No. 4,001,583.
• preactivated latex-polysaccharide particles cannot be readily stored or shipped to an ultimate user who would then be able to attach any desired immunologically active material.
• the Shiff's bases produced by the reaction of the amino groups of the immunologically active material with the dialdehydes must be stabilized by sodium borohydride. This must be carried out at 0°C after removal of excess immunologically active material to keep denaturation at a minimum.
• the latex particles in U. S. Patent No. 4,264,766 have the surfaces entirely covered with the amino polysaccharide which is bonded to the carboxyl groups on the latex surface. This prevents proteins in solution, other than those that are partners for the attached immunologically active material, from becoming attached to the latex particles.
• a problem with this method is that formation of the dialdehydes, reaction with the dialdehydes, and reduction of the Shiff's bases are reactions which require a good deal of skill and care and thus involve a good deal of expense and time.
• a composition is set out which is useful for specifically binding to a specific binding protein which is a specific binding partner to a biological substance when the protein is associated with other proteins.
• the composition includes a water insoluble support having a surface having the capability of associating with the specific binding protein and with the other proteins.
• the composition also includes a polysaccharide coating covering a first substantial portion of the surface sufficiently to substantially prevent binding of protein to said first substantial surface portion and not covering a second substantial surface portion of the surface, the surface consisting essentially of the first and second substantial surface portions.
• the aforementioned composition further includes a biological substance attached to the second substantial surface portion.
• a method is provided of preparing a water insoluble surface of a solid support for specifically binding to a specific binding protein which is a specific binding partner to a biological substance when the protein is associated with other proteins.
• the method comprises providing a solid support having a water insoluble surface capable of associating with the specific binding protein and with other proteins and covering a first substantial portion of the surface with a polysaccharide coating while not covering a second substantial portion of the surface with a polysaccharide coating, the surface consisting essentially of the first substantial surface portion and the second substantial surface portion.
• a process for assaying an aqueous sample containing a specifically binding protein having a first binding site which is a specific binding partner to a first biological substance, the specifically binding protein being in association with other proteins, with increased specificity and sensitivity.
• the process comprises contacting an aqueous sample with a first solid support having a first water insoluble surface capable of associating with the specific binding protein and with the other proteins, the first surface consisting essentially of a first substantial surface portion shielded by a polysaccharide coating and a second substantial surface portion having the first biological substance attached to it.
• the aqueous sample is separated from the first solid support and the amount of specifically binding protein bound to the attached first biological substance is detected.
• Another embodiment still of the present invention comprises a process for reducing adherence of undesired proteins to a water insoluble surface consisting essentially of a first substantial surface portion and a second substantial surface portion while providing the surface with the capability for binding to a specifically binding protein which is a specific binding partner to a biological substance.
• the process comprises shielding the first substantial surface portion with a polysaccharide coating and attaching the biological substance to the second substantial surface portion.
• the kit comprises a solid support having a first water insoluble surface capable of associating with the specific binding protein and with other proteins, the first surface consisting essentially of a shielded first substantial surface portion and a second substantial surface portion having the first biological substance attached to it.
• the kit further includes a plurality of solid particles, each having a second insoluble surface capable of associating with the specific binding protein and with other proteins, the second surfaces each consisting essentially of a first substantial surface portion shielded by a polysaccharide coating and a second substantial surface portion having the second biological substance attached to it.
• the present invention is based upon discovery that if a polysaccharide coating covers a first portion of the surface of a solid support that is capable of associating with proteins, generally, and if a specific biological substance is attached to the rest of the surface, then proteins which are not specific binding partners for the biological substance will not be able to attach to the surface even in those portions where the surface is only attached to the biological substance.
• Partially coated compositions of the present invention are relatively easy to make and quite easy to attach to biological substances. Generally, they can be made quite inexpensively. And, the compositions of the present invention can be stored or shipped in condition for the attachment of any desired biological substance.
• a composition is provided which is useful for specifically binding to a specific binding protein which is a specific binding partner to a biological substance when the protein is associated with other proteins .
• biological substance is used broadly to indicate any substance which is a specific binding partner to a specific binding protein.
• Illustrative of the biological substance are enzymes, antibodies, natural receptors, e.g., thyroxine binding globulin and avidin, globins , e.g., hemoglobin, ocular lens proteins, surface antigens, histo-compatible antigens and the like.
• a specific binding protein can be any protein which it is desired to link to a water insoluble support. A long list of such substances appears in previously mentioned U. S. Patent No. 4,264,766.
• composition of the invention includes a water insoluble support having a surface having the capability of associating with the specific binding protein and with other proteins as well.
• the solid support may be in the form of micro or macro-particles, or in the form of macroextensive surfaces such as walls, flat plates, wells, and the like, all of which can be used in the separation of proteinaceous mixtures.
• the water insoluble support is in the form of a plurality of particles it is preferred that they have a specific gravity near that of water so as to enable them to be stably suspended in an aqueous medium.
• Such particles will generally be from about 0.2 micron to about 1 cm in diameter.
• the solid support itself must be inert with respect to immunological 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. Glass surfaces which may be used are described in U.S. Patent No. 4,169,138. Other useful polymers may be found in U.S. Patents Nos.
• the solid support be a latex and have active groups which are capable of forming a covalent linkage with a polyhydroxy compound.
• the latex supports can have active groups such as carboxyl groups, amine groups, or groups convertable into them.
• Useful active groups on the latex support are those containing an active hydrogen such as -COOH,-CONH 2 , primary and secondary amine groups, or nitryl groups.
• the preferred latex material is polystyrene for the practice of the present invention.
• the polystyrene will preferably also contain copolymerized therewith a carboxyl containing compound such as acrylic acid, methacrylic acid, or the like.
• a polysaccharide coating is provided covering a first substantial surface portion of the surface of the water insoluble support sufficiently to substantially prevent binding of proteins to the first substantial surface portion of the water insoluble support.
• the polysaccharide coating is also required to not cover a second substantial surface portion of the support.
• the polysaccharide coating will normally be formed of polysaccharides characterized by being water soluble, relatively high molecular weight, normally in excess of 5,000 daltons, more usually in excess of 10,000 daltons, and may be 1,000,000 daltons or higher in molecular weight.
• the polysaccharide may be a polymer or copolymer of glycose(s), e.g., glucose and fructose, a mixture of carbohydrates, such as neuraminic acids, uronic acids, glycosamines, or the like.
• the polysaccharide may be a combination of block or alternating copolymers or combinations thereof of saccharides and condensation monomers, particularly epoxides.
• Polysaccharides of particular interest include dextran, Ficoll (this is a synthetic copolymer of sucrose and epichlorohydron, a trademark of Pharmacia Fine Chemicals, Piscataway, New Jersey), agarose, hyaluronic acid, etc.
• an amino group normally being bonded to a short alkylene chain of from about 2 - 6 carbon atoms, which are bonded to functionalities of the polysaccharide.
• an amino group normally being bonded to a short alkylene chain of from about 2 - 6 carbon atoms, which are bonded to functionalities of the polysaccharide.
• Particularly convenient is the reaction product of diamines with carboxyl functionalities present on the polysaccharide. See, for example, Inman, J. of Immunology 114 , 704-709 (1975) .
• relatively high molecular weight amino polysaccharides such as the previously mentioned Ficoll, generally with molecular weights of 1,000 to 1,000,000 or more. Excellent results have been obtained with an amino Ficoll with a molecular weight of approximately
• the amount of the polysaccharide attached must be controlled to be between about 0.5 x 10 -7 and about 3 x 10 -7 grams per square centimeter of the area of the entire water insoluble surface in order to obtain a covering of only a first substantial surface portion of the water insoluble surface while leaving a second substantial surface portion of the water insoluble surface uncovered with polysaccharide. This corresponds to from about 700 to about 4000 molecules per square micron when the molecular weight of the polysaccharide is about 400,000.
• the biological substance is attached to the second substantial surface portion of the surface.
• This attachment can be by hydrophobic adsorption, electrostatic bonding, covalent bonding, or combinations thereof.
• the biological substance is covalently bonded to the second substantial surface portion.
• An activating agent such as a water soluble carbodiimide can be utilized to form an adduct with carbonyl groups on the latex surface. The carbodiimide adducts is then reacted with amine groups on the biological substance to leave an amide linkage to the latex support.
• Other useful activating agents include the Woodward reagent K (N-ethyl-5-phenyl-isoxazolium-3'-sulfonate) or a water soluble chloroformiate.
• the subject compositions can be used wherever an insoluble material is used for specific binding to a protein present in a mixture. This situation is encountered in competitive protein binding assays, cell sorting, cytology, histology, and the like. Since the procedure can vary very widely, the subject invention generally involves combining the insoluble material, as a particle or surface of a larger structure, with a protein mixture and allowing a sufficient time for binding between the biological substance on the surface and the specific protein binding partner. The solid surface is then washed free of non-specifically bound protein, leaving only specifically bound protein.
• the labels may include radioactive isotopes, fluorescers, magnetic materials, enzymes, enzyme substrates, dyes for producing colors, or the like.
• the labels may be bonded to the water insoluble surface, the polysaccharide, or the biological substance, desirably being bonded to the water insoluble surface or the polysaccharide. If desired, the labels may be uniformly dispersed throughout the particles.
• kits for assaying samples potentially containing a specifically binding protein having a first binding site which is a specific binding partner to a first biological substance and a second binding site which is a binding partner to a second biological substance, the specifically binding protein being in association with other proteins.
• a kit includes a macroextensive surface wall defined on a support such as a slide, a well, or the like.
• the support has a first water insoluble surface which is capable of associating with the specific binding protein and with other proteins.
• the first water insoluble surface consists essentially of a first substantial surface portion shielded by a polysaccharide coating and a second substantial surface portion having the first biological substance attached to it.
• the kit also includes a plurality of solid particles, each of which has a second insoluble surface capable of associating with the specific binding protein and with other proteins.
• the second insoluble surfaces each consist essentially of a first substantial surface portion shielded by a polysaccharide coating and a second substantial surface portion having the second biological substance attached to it.
• the particles can be labelled with a label capable of providing a detectable signal.
• the particles may be colored by a color imparting entity such as a dye and the signal will simply comprise the color itself.
• Alternatively, such other labels as have been previously discussed may be utilized.
• compositions of the present invention can be produced in a number of ways, several of which are disclosed in following:
• compositions of the present invention can be produced by providing a solid support having a water insoluble surface capable of associating with a specific binding protein and with other proteins. A first substantial surface portion of the surface is covered with a polysaccharide coating while a second substantial surface portion of the surface is not covered with a polysaccharide coating. This is accomplished by nitrating the surface to add nitro groups utilizing, for example, a mixture of nitric and sulfuric acids. After the acid mixture has been washed off of the surface, the nitro groups are reduced to amino groups utilizing a convenient reducing agent such as stannous chloride along with hydrochloric acid. The solid support is again washed and cyanuric halide moieties are attached to the amino groups.
• Cyanuric halide is generally added to an aqueous solution in contact with the solid surface with the cyanuric halide itself being in an ethanol solution because of its generally low solubility in water. It is believed that the cyanuric halide is actually in the form of a monoethoxy derivative when added. Any excess cyanuric chloride is washed away. It is believed that the remaining halide on the cyanuric halide moiety then hydrolizes to a hydroxy! group (pKa about 10 -7 ) which dissociates into a hydronium ion and a net negatively charged surface. This serves to promote electrostatic binding of amino polysaccharides to a surface which does not necessarily have carboxyl groups.
• the amino polysaccharide molecules are then attached to the surface.
• the intermediate product thus formed is a solid support having a water insoluble surface having an amino polysaccharide electrostatically attached to at least a first surface portion thereof.
• the intermediate product can then be reacted with a cyanuric halide to cross-link adjacent amino polysaccharide molecules and to form a usable composition.
• the reaction with cyanuric halide also serves, due to its acidity and ionic strength, to free at least the second surface portion from amino polysaccharide coverage.
• a biological substance as described above can be attached to the second substantial surface portion of the water insoluble surface. This can be accomplished by hydrophobic adsorption, by electrostatic bonding, and, more preferably, by covalent bonding as via utilizing an activating agent as set out above.
• Another method of making the composition of the present invention 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 raonomolecular layer of the amino polysaccharide.
• 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 polysaccharide.
• the excess amino polysaccharide is washed off of the solid support with water. Thereafter, a cyanuric halide, in ethanol solution as set out above, is added to the amino polysaccharide coated solid surface.
• the cyanuric halide is used 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.
• a first substantial surface portion of the water insoluble surface is coated with amino polysaccharide while a second substantial surface portion of the water insoluble surface is not coated with amino polysaccharide.
• Another alternative method of forming a composition in accordance with the present invention 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 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 of the surface remains uncovered by amino polysaccharide molecules.
• a biological substance can be attached to the second substantial surface portion by any desired method.
• This method has the advantage that if an excess of carbodiimide is utilized there can still be carbodiimide activated active (e.g. , carboxyl) groups on the second substantial surface portion ready to covalently bond to a desired biological substance.
• carbodiimide activated active e.g. , carboxyl
• a solid support having a water insoluble surface having active (e.g. , carboxyl) groups is reacted with an excess of an activator compound such as a water soluble carbodiimide to form 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 biological substance remains attached to the second substantial surface portion. The surface is then washed to remove any reaction products. Water is again contacted with the surface and an amino polysaccharide is added which then links to the active groups which remain and which have been activated by the activator.
• an activator compound such as a water soluble carbodiimide
• the resulting product has both the amino polysaccharide and the biological substance covalently attached to the water insoluble surface via the active groups and through use of the activating agent. While several of the above described methods have called for the use of a carboxyl active group and a carbodiimide activating agent it should be noted that other active groups and other activating agents, for example those previously set out, may be utilized where appropriate.
• an assaying process is set out for assaying a sample containing a specifically binding protein having a first binding site which is a specific binding partner to a first biological substance, the specifically binding protein being in association with other proteins.
• the process operates by contacting the first support having a first water insoluble surface capable of associating with the specific binding protein and with other proteins, the first surface having a first substantial surface portion shielded by a polysaccharide coating and a second substantial surface portion having the first biological substance attached to it, with an aqueous solution of the sample.
• the aqueous sample solution is separated from the first solid support and from the specifically binding protein bound to the attached first biological substance.
• the support would generally comprise a plurality of particles and they would normally be labelled with a label capable of detection.
• the support would comprise a macroextensive surface such as a plate or a well on a plate having one or more wells.
• the amino polysaccharide, the biological substance, etc., are as defined previously.
• the aforementioned first solid support is a macroextensive surface and the detecting step comprises contacting an aqueous solution having a second solid support in the nature of a plurality of particles, the second solid support having a second water insoluble surface capable of associating with the specific binding protein and other proteins, the second surface having an additional first substantial surface portion shielded by a polysaccharide coating and an additional second substantial surface portion having a second biological substance attached thereto, with the first macroextensive surface, i.e., with the plate or well.
• the specifically binding protein is selected to be of a nature to have a second binding site which is a specific partner to the second biological substance. The degree of adherence of the particles to the macroextensive surface is then observed .
• a process is also set out for reducing adherence of undesirable proteins to a water insoluble surface consisting essentially of a first substantial surface portion and a second substantial surface portion while providing the surface with the capability for binding to a specifically binding protein which is a specific binding partner to a biological substance.
• the first, substantial surface portion is shielded with a polysaccharide coating and the biological substance is attached to the second substantial surface portion. Shielding and attaching steps may be simultaneous or either may precede the other.
• the surface in this instance would generally be a macroscopically extensive surface.
• the adherence reducing process provides an alternative to the utilization of bovine serum albumin on such macroextensive surfaces. This is particularly advantageous because of the expense of bovine serum albumin and because of the lack of complete uniformity of bovine serum albumin from one batch to the next, which nonuniformity is inherent in the biological production of this reactant .
• a kit for assaying samples potentially contanning a specifically binding protein having a first binding site which is a specific binding partner to a first biological substance and a second binding site which is a specific binding partner to a second biological substance, the specifically binding protein being in association with other proteins.
• the kit comprises a solid support having a first water insoluble surface, the support being a. macroextensive wall and the first water insoluble surface being a wall surface.
• the first water insoluble surface is capable of associating with the specific binding protein and with other proteins.
• the first surface consists essentially of a first substantial surface portion shielded by a polysaccharide coating and a second substantial surface portion having the first biological substance attached to it.
• a plurality of solid particles also forms a part of the kit.
• Each particle has a second insoluble surface capable of associating with the specific binding protein and with other proteins.
• the second surfaces each consist essentially of a first substantial surface portion shielded by a polysaccharide coating and a second substantial surface portion having- the second biological substance attached to it.
• the particles would be labelled with a label capable of providing a detectable signal. For most purposes the label would simply comprise a color imparting entity and the signal would comprise the color.
• the kit comprises the plurality of particles as in the kit just described, but the wall support having the first water insoluble surface, while it has the first biological substance attached to it, does not have the polysaccharide coating on a first substantial portion thereof. Instead, bovine serum albumin or other shielding agent is utilized.
• Polystyrene microspheres (Polysciences Inc., Warrington, PA), 5 grams, of one micron diameter, were suspended in 50 ml of ice cold 75% H 2 SO 4 and added to 100 ml of ice cold 1:1 HNO 3 :H 2 SO 4 . The resulting suspension was stirred for thirty minutes in an ice bath, then quenched by pouring into 1L of ice cold water. The thereby nitrated microspheres were washed in water three times utilizing centrifu ⁇ ation for separation.
• the nitrated microspheres were suspended in 100 ml concentrated HCl along with 110 grams of SnCl 2 .2H 2 O and stirred at room temperature for ten hours.
• the amino-microspheres were suspended in 900 ml of cold water and dispersed by sonication in a stainless steel ultrasonic bath cleaner. Cyanuric chloride 0.5 gram, a 2 to 1 mix of water and alcohol (150 ml) was added and the resultant solution was incubated with intermittent sonication for thirty minutes. The amino groups were thereby converted to cyanuric chloride adducts. The suspension was centrifuged to remove the microspheres, which are believed to contain hydroxyl groups formed by hydrolysis of remaining chloride of the cyanuric chloride adducts, and washed twice with cold water by centrifugation.
• AECM-Ficoll Carboxymethylated Ficoll
• microspheres were suspended in 1 liter of cold water by sonication and 1.2 grams of AECM-Ficoll dissolved in 50 ml of water was added. The resultant mixture was incubated with intermittent sonication for twelve hours. The AECM-Ficoll coupled microspheres were washed twice in v/ater by centrifugation. They were vacuum filtered through Whatman #1 paper to remove any clumped microspheres. The AECM-Ficoll utilized had a molecular weight of approximately 400,000 and had about 80 amino groups per molecule. Further Treatment Of Coated Microspheres With Cyanuric Chloride to Cross-link the AECM-Ficoll
• the AECM-Ficoll electrostatically coated microspheres were suspended in 750 ml of water cooled to 0°C. Cyanuric chloride (1 gram) dissolved in 200 ml of cold 1:1 alcohol:water was added to the suspension to cross-link the AeCM-Ficoll. Only a portion of the surface remained covered with AECM-Ficoll because of the acidity caused by the formation of HCl as the cyanuric chloride reacted. The resulting suspension was incubated at 0°C for thirty-five minutes and microspheres were separated from the suspension by centrifugation. They were washed in cold water three times by centrifugation. The microspheres were coupled to proteins by incubation with them at room temperature. It was found that they could be stored, at about 4°C, for over a year without significant deterioration.
• Nitrated spheres were covered with a solution of 110 grams SnCl 2 .2H 2 O dissolved in 100 ml of concentrated hydrochloric acid and incubated with occasional stirring for fifteen hours at room temperature to reduce the nitro groups to amino groups.
• the SnCl 2 solution was decanted and the spheres were washed in turn with water, 0.1N HCl, water, and 0.1N NaOH and water.
• the amino-spheres were covered with a solution of 0.1 gram cyanuric chloride dissolved in 10 ml of alcohol and 200 ml of water and incubated at room temperature for forty minutes.
• the cyanuric chloride solution was decanted off of the spheres and the spheres were washed with several portions of water.
• the spheres were covered with a solution of 0.14 grams AECM-Ficoll dissolved in 120 ml of water and incubated with occasional stirring for twelve hours.
• the AECM-Ficoll solution was decanted off of the spheres and the spheres were washed two times with cold water. This provided an electrostatically bound coating of AECM-Ficoll on the spheres.
• the AECM-Ficoll electrostatically coated spheres were cooled to 0°C and covered with a solution of 150 ml of ice cold water containing 0.18 grams of cyanuric chloride and 18 ml of alcohol. Incubation was continued for thirty minutes at 0°C. The cyanuric chloride solution was decanted off of the spheres and the spheres were washed three times in ice cold water. Only a portion of the surface remained covered with AECM-Ficoll because of the acidity caused by the formation of HCl as the cyanuric chloride reacted. The AECM-Ficoll utilized in the above reaction had about 80 amino groups per molecule of the AECM-Ficoll. The spheres now react with protein macromoiecules such as IgG, enzymes, etc. They can be stored for several months at about 0°C without significant deterioration.
• protein macromoiecules such as IgG, enzymes, etc. They can be stored for several months
• MOPC-21 myeloma protein was added to make the final protein concentration 3 micrograms per ml in a total volume of 1.1 ml normal saline. After one hour of incubation at room temperature the unbound protein was removed by three washes in normal saline via centrifugation. MOPC-21 coupled microspheres were then incubated for one hour at room temperature in 1 ml of saline containing 0.642 milligrams per milliliter I 125 labelled monoclonal antibodies, anti-4a.
• microspheres used were suspension polymerized at 60°C from 95.2 parts by weight of freshly vacuum distilled ⁇ tyrene and 4.8 parts by weight of freshly vacuum distilled methacrylic acid using potassium persulfate as catalyst. After polymerization was complete the microspheres were washed with dilute NaOH, filtered through glass wool, brought to pH 7 with dilute HCl , washed 3 times with distilled water by centrifugation and the concentration of solids adjusted to 0.745% by weight. Scanning electron microscopy was used to determine that the microspheres had a 0.5 micron diameter with less than 3% standard deviation.
• C 14 AECM Ficoll was prepared by reaction of 50 mg CM Ficoll (80 carboxyl groups per 40,000 molecular weight by titration) with 66.5 mg ethylene diamine hydrochloride-C 14 (containing 25 microcuries C 14 ) and 75 mg (3-dimethyl aminopropyl) ethyl carbodiimide hydrochloride (EDAC) at pH 4.7 in 5 ml
• the C 14 AECM Ficoll was separated from the low molecular weight reactants by gel filtration and exhaustive dialysis against distilled water at 4°C.
• Ficoll have weight average molecular weights of 443,000 by HPLC (high performance liquid chromatography) so that the C 14 AECM Ficoll had 1.7 x 10 -11 cpm/molecule under the conditions of measurement.
• HPLC high performance liquid chromatography
• microspheres were treated as were those in Table 1 but the pH of the solutions used for binding and washing were as specified in Table 2.
• AECM-Ficoll is bound at lower pH values, e.g., below about 6, than at higher pH values.
• a method is provided of controlling the amount of AECM-Ficoll attached to the microspheres.
• microspheres were treated as were those in Table 1 but the solutions used for binding and washing were as specified in Table 3.
• PBS phosphate buffered saline, pH 7.3
• Table 4 reports data on covalent binding of AECM-Ficoll using a water soluble carbodiimide activating agent.
• the data of Table 4 were obtained by reaction of C AECM Ficoll (2120 cpm) with microspheres in the presence of various amounts of carbodiimide followed by washing 3 times with pH 7.3 PBS. Incubation of the microspheres of Table 4 with non-radioactive AECM Ficoll demonstrated that, except for the case where no EDAC was used, the AECM Ficoll is not exchangeable. Presumably the amino groups of the AECM Ficoll have been covalently coupled through amide linkages to the carboxyl groups on the surface of the microspheres.
• the PBS appears to inhibit the binding of AECM Ficoll to the microspheres by limiting their approach to one another since it does not interfere with the EDAC activation of the microspheres.
• the usefulness of cyanuric chloride to couple AECM Ficoll microspheres was also investigated as outlined in Table 6.
• the protocol used consisted of reacting 100 microliters of microspheres with either C 14 or non-C 14 AECM Ficoll with or without EDAC in H 2 O, washing 3 times with H 2 O (1st wash) , reacting with cyanuric chloride for 30 minutes, washing 3 times with H 2 O (2nd wash) , reacting with either C 14 or non-C 14 AECM Ficoll for 1 hour, washing 3 times with
• non-exchangeable cpm of AECM Ficoll can be bound by use of cyanuric chloride on microspheres which already have an ionic coating of AECM Ficoll.
• cyanuric chloride treatment of microspheres which already had an EDAC coupled layer of AECM Ficoll could not facilitate uptake of additional AECM Ficoll, e.g., the 3rd wash of sample 4, Table 6, contains 98% of the counts added after the cyanuric chloride treatment.
• the excess counts above 250 on the microspheres for samples 2 and 3 are apparently due to ionic bonding of AECM Ficoll.
• cyanuric chloride non-exchangeably couples the AECM Ficoll is presumably by cross-linking the amino groups on some of the adjacent AECM Ficoll molecules. Those AECM Ficoll molecules thereby cross-linked, to the few such molecules which are so strongly bound that they do not wash off in PBS (e.g. , sample 6, Table 3) , are retained on the microsphere surface. The remainder are washed off.
• PBS e.g. , sample 6, Table 3
• a surface substantially completed covered with AECM Ficoll cannot be used to practice the present invention since proteins, antibodies and the like cannot be bound to it either with cyanuric chloride or with EDAC as shown by some of the other examples.
• U.S. Patent No. 4,264,766 teaches a complicated oxidation, coupling, and reduction procedure to attach proteins to a polysaccharide coating, but this is not simple in the hands of the user and not all proteins retain their biological activity after the borohydride reduction which is necessary to practice that invention.
• the present invention provides proteins, antibodies, etc., attached to the microsphere surface with most or all of the other area of the surface covered by polysaccharide.
• the examples allow for coupling the protein before or after the polysaccharide either covalently or non-covalently.
• the material of the present invention with a partial layer of AECM Ficoll, has been prepared by a number of methods and storage stability at 4°C for longer than a year is routinely observed. This material is readily coupled to protein by simply mixing it with protein, which is a considerable advantage to the user who wishes to couple his own protein and wants long shelf life. Once coupled to portein, the resulting reagent has a shelf life of greater than six months .
• Example V Example V
• T-15 antibody at 1 mg/ml in PBS were added to the tubes thus: Tubes 1, 5, 9, 13, 17 received 50 microliters of T-15. Tubes 2, 6, 10, 14, 18 received 25 microliters of T-15. Tubes 3, 7, 11, 15, 19 received 10 microliters of T-15. Tubes 4, 8, 12, 16, 20 received 5 microliters of T-- 15. All the tubes were sonicated briefly and incubated at room temperature for 1 hour.
• microsphere suspensions in tubes 1-4 were v/ashed twice in PBS by centrifugation and resuspension and resuspended in 500 microliters of PBS with sonication.
• Tubes 5-8 were washed twice in PBS containing 1% bovine serum albumin and 0.1% sodium ozide (protein buffer) and resuspended in 500 microliters of protein buffer with sonication. These were kept for 48 hours at 4°C.
• the microspheres in tubes 9-20 were washed twice in distilled water and suspended in 500 microliters of distilled water with sonication.
• Tubes 9-20 were incubated for 1 hour at room temperature and an additional 48 hours at 4°C.
• Tubes 9-12 were washed 3 times in PBS by centrifugation and resuspension, and the washes were pooled for each tube.
• the pooled washes and the washed microspheres were transferred to scintillation vials, 20 ml of scintillation fluid (a standard fluid which fluoresces when exposed to radiation) was added to each vial and they were counted.
• scintillation fluid a standard fluid which fluoresces when exposed to radiation
• the highest antibody concentration used (tube 9) was estimated to allow a complete monolayer coverage of the microspheres in the sample. That this did not occur, as implied by the similar quantity of binding of C 14 AECM Ficoll, is indicative of the difficulty the negatively changed antibodies have in approaching the negatively changed microspheres.
• microspheres in tubes 13-16 were washed twice in PBS and resuspended in 500 microliters of PBS with brief sonication.
• the microspheres in tubes 17-20 were washed twice in protein buffer, resuspended in 500 microliters of protein buffer and sonicated briefly.
• the PCBGG is the specific binding partner for the T-15 antibody and any attachment of the microspheres (all T-15 coupled in this example) in the absence of PCBGG indicates non-specific sticking. All of the microspheres exhibited specific binding to the PCBGG plate coat. The microspheres which had AECM Ficoll (tubes 13-20) exhibited no detectable non-specific sticking even when the binding was carried out in the absence of protein in the buffer (tubes 13-16) . The microspheres without AECM Ficoll exhibited non-specific sticking in all cases and particularly in the absence of protein in the buffer (tubes 1-4). This example provides clear evidence that the ratio of specific to non-specific binding is enhanced by coating that part of the microsphere surface which is not coupled to antibody with AECM Ficoll. It also demonstrates that the amount of non-antibody coupled surface can be substantial for carboxylated microspheres even when an excess of antibody and covalent coupling is used.
• EDAC/AECM Ficoll/CTC Microspheres Green fluorescent 0.72 micron diameter carboxylated polystyrene microspheres prepared generally as previously described were given partial surface coverings of covalently coupled AECM Ficoll in the following manner. A 44.5 ml suspension containing 3.75 grams of microspheres was added to 706 ml H 2 O and then 39 ml of water containing 0.41 grams of AECM Ficoll was added. The suspension was mixed thoroughly and then 75 mg EDAC dissolved in 30 ml H 2 O was added with swirling and sonication. The pH at this point was less than 5. The suspension was allowed to sit at room temperature for two days then washed 3 times in distilled water.
• microspheres After resuspension in 1 liter of water with sonication the microspheres were filtered through Whatman #1 paper with suction to remove any clumps and centrifuged once more and the supernatant discarded. The microspheres were then resuspended in 186 ml of cold water and sonicated until a single microsphere suspension was obtained. To this suspension v/as added 0.36 grams cyanuric chloride dissolved in 36 ml ETOH and 76 ml cold water. The suspension was sonicated and kept in an ice bath for 30 minutes followed by centrifugation at 4°C. The supernatant was discarded and the microspheres were washed with cold water 3 times by centrifugation. The microspheres were resuspended with sonication in 320 ml H 2 O and contained 1.07% solids by weight.
• Example VI A portion of the microspheres of Example VI were coupled to My-1, a mouse monoclonal antibody IgM (kappa) , by incubating for 75 minutes 0.1 mg My-1 with 10 ml of the microspheres which had been suspended by sonication in 100 ml normal saline at room temperature. The microspheres were then pelleted at 10,000 x g at 4°C for 10 minutes in a refrigerated centrifuge and the supernatant discarded. The pellet was resuspended with sonication in 100 ml RPMI 1640 cell culture medium containing 10% newborn calf serum, pelleted as before and the supernatant discarded.
• IgM mouse monoclonal antibody
• HL-60 and DAUDI cells were resuspended at a concentration of 1 x 10 6 cells/ml in HBSS (Hank's Buffered Salt Solution) containing 0.1% BSA (bovine serum albumin) and 0.1% sodium azide in separate tubes. Aliquots of 0.5 ml of both cell suspensions were placed in separate 2 ml wells of a 24 well tissue culture plate. Twenty microliters of the My-1 antibody reagent prepared above was added to both cell suspensions and mixed gently. The plate was centrifuged at 150 x g for 9 minutes at 4°C, then incubated for 1 hour at 4°C.
• the My-1 antibody recognizes a cell surface marker on the surface of human granulocytes and HL-60 cells. This marker is absent on DAUDI and other human white blood cells. Similar results were obtained on human white blood cells where the cells labelled were granulocytes.
• Microspheres A) 0.99 micron diameter containing fluorescent blue dye B) 0.54 micron diameter containing fluorescent blue dye C) 1.01 micron diameter, undyed
• volumes containing 1.25 grams of microspheres A, B, & C were put into three beakers labelled, respectively, A1 , B1, C1, each containing 220 ml of distilled water, and were sonicated to disperse the microspheres.
• A1 , B1, C1 each containing 220 ml of distilled water
• EDAC carboxyimide
• microspheres were washed three times in distilled water, by centrifugation and resuspension with sonication and were resuspended in a volume of 200 ml of distilled water. The suspensions were then filtered through a Whatman #1 filter paper. The microspheres suspensions A1 , B1 and C1 were brought to 160 ml, 190 ml and 90 ml respectively with distilled water and sonicated well. Aliquots from the treated microspheres A1 , B1 and C1 were then tested for their percentage by weight and the necessary adjustments made to the remaining microspheres. Thus A1 adjusted to 1.47%, B1 to 0.80% and C1 to 1.5%; the total volumes being A 165 ml, B 200 ml, C 100 ml. Treatment II
• microsphere suspensions were each brought to a volume of 70 ml in distilled water and sonicated well. 0.13 grams of cyanuric chloride dissolved in 10 ml of ETOH and diluted in 20 ml of distilled water was added to each of the three suspensions and dispersed by sonicating well. After 1 ⁇ 2 hour incubation the suspensions were washed 3 times in distilled water, by centrifugation and resuspension with sonication, were resuspened in 190 ml distilled water and sonicated well. Aliquots were taken and the volumes and % solids were adjusted (see above in Treatment I) to A 197 ml at 1.47%, B 199 ml at 0.80% and C 193 ml at 1.5%.
• microsphere suspensions A1 , B1, C1 (Treatment I) and A2 , B2 and C2 (Treatment II) were prepared as just described.
• 500 microliters of phosphate buffered saline (PBS) was added, then 50 microliters of one of the six microsphere suspensions (A1 , B1, C1, A2, B2 and C2) was placed in each tube and the tubes were sonicated briefly.
• 25 mililiters of T-15 antibody at 4 mg/ml was added to each tube; the tubes were sonicated and incubated at room temperature for 1 hour.
• microsphere suspensions were then washed 3 times in PBS containing 1% fetal calf serum plus 0.1% azide (protein buffer) by centrifugation and resuspension, and resuspended by sonication in 500 microliters of protein buffer.
• 96 well microliter plate (Dynatech, Inc.) had been prepared by placing 50 microliters of T-15 at 0.1 mg/ml in PBS in each of the first 6 wells. 50 microliters of phosphocholine - bovine gamma globulin conjugate (PCBGG) at 0.1 mg/ml in PBS in each of the next 6 wells, and 50 microliters of protein buffer in each of the next 6 wells. These plate coats were incubated at room temperature for 1 hour then the contents of the wells were removed by suction and the wells were washed 3 times with protein buffer by filling the wells to the top and aspirating the contents.
• PCBGG phosphocholine - bovine gamma globulin conjugate
• Carboxylated polystyrene microsphere suspensions were prepared in bottles of disti lled water with the bottles labelled D1, E1 and F1, as follows :
• each of samples from flasks D2, E2 and F2 were individually placed in tubes labelled, respectively, D5, E5 and F5 , each containing 500 microliters of PBS.
• the tubes were sonicated and 25 microliters of UPC 10 (a mouse myelona protein) at 1 mg/ml in PBS was added to tubes D4, E4 and F4.
• the tubes D4, E4, F4, D5, E5 and F5 were incubated for 1 hour at room temperature.
• the microspheres in these tubes were then washed 3 times in PBS containing 1% fetal calf serum and 0.1% sodium azide (protein buffer) by centrifugation and resuspension. They were each resuspended in 500 microliters of protein buffer with sonication.
• Example X The microspheres in D1, E1 and F1 in PBS did not bind to the protein coats on the plate even after attempted cyanuric chloride treatment thus demonstrating their inability to bind to protein after polysaccharide coating as in Treatment I of Example VIII.
• Example X The microspheres in D1, E1 and F1 in PBS did not bind to the protein coats on the plate even after attempted cyanuric chloride treatment thus demonstrating their inability to bind to protein after polysaccharide coating as in Treatment I of Example VIII.
• Example X Example X
• the T-15 antibody was at three different concentrations. 50 microliters of microspheres were placed in each of tubes labelled nos. 1, 3, 5, 7, 9 each containing 500 microliters of phosphate buffered saline. The tubes were sonicated to disperse the microspheres. 0.05 mg T-15 antibody and 0.05 mg EDAC was added to tube 1, 0.05 mg T-15 antibody and 0.005 mg EDAC to tube 3, 0.005 mg T-15 and 0.05 mg EDAC to tube 5, 0.005 mg T-15 and 0.005 mg EDAC to tube 7, and 0.05 mg T-15 and no EDAC to tube 9.
• Each tube was sonicated briefly after addition of protein and after addition of EDAC.
• the microsphere suspensions were incubated at room temperature for 1 hour, were wached 3 times in PBS and 1% fetal calf serum and .1% sodium azide (protein buffer) by centrifugation and resuspension and resuspended in 500 microliters of protein buffer with sonication.
• 15 wells of a flexible, U-bottom, PVC, 96 well microliter plate had been prepared by placing 50 microliters of T-15 antibody at 0.1 mg/ml in PBS in each of the first 5 wells, 50 microliters of phosphocholine bovine gamma globulin (PCBGG) at 0.1 mg/ml in PBS in each of the second 5 wells, and 50 microliters of protein buffer in each of the last 5 wells. After 1 hours incubation at room temperature the contents of the wells were removed by suction and the wells washed 3 times in protein buffer by filling the wells to the top and aspirating the contents.
• PCBGG phosphocholine bovine gamma globulin
• a flexible, U-bottom polyvinyl chloride, 96 well microliter plate (Dynatech, Inc.) was used. Clear polyvinyl chloride strips have also been substituted for the wells of the microliter plate, the strips being placed in the reagents instead bf the reagents being placed in the wells.
• Microspheres 0.7 micron in diameter were prepared containing fluorescent green dye and were treated with AECM Ficoll/cyanuric chloride to provide a partial coating as in Treatment II, Example VIII. 100 microliters of the microspheres were placed in a tube containing 1 ml PBS and sonicated. 50 microliters of T-15 antibody at 4 mg/ml in PBS was added and mixed by sonication.
• microsphere suspension was washed three times in protein buffer, by centrifugation and resuspension with sonication. The microspheres were resuspended in 1 ml of protein buffer and sonicated well.
• a flexible U-bottomed PVC 96 well microliter plate was prepared: the first 6 wells received 50 mililiter each of T-15 antibody at 0.1 mg/ml in phosphate buffered saline. Well 7 received 50 microliters of phosphocholine bovine gamma globulin (PCBGG) at 0.1 mg/ml in PBS. Well 8 received 50 microliters of PBS containing 1% bovine serum albumin and 0.1% sodium azide (protein buffer). The plate coats were incubated for 1 hour at room temperature; the well contents were aspirated and the wells washed 3 times in protein buffer by filling to the top and aspirating.
• PCBGG phosphocholine bovine gamma globulin
• protein buffer protein buffer
• a clear polyvinyl chloride strip is incubated with antibody #1, then coated with gelatin or bovine serum albumin. It is then placed in the suspect pregnancy urine and incubated for between one and sixty minutes, after which the strip is incubated in microspheres which are coupled to antibody #2. These microspheres are carboxylated polystyrene which have been activated with carbodiimide, washed, reacted with antibody #2, washed, and reacted with AECM Ficoll.
• Antibody #1 and antibody #2 are antibodies which have the property of being able to simultaneously react with human chorionic gonadotrophin or with the beta sub unit of human chorionic gonadotrophin, i.e., a given molecule of human chorionic gonadotrophin or beta sub unit of human chorionic gonadotrophin can have both antibody #1 and antibody #2 attached to it simultaneously. After the strip has been incubated in the microspheres for between one and sixty minutes, it is removed and rinsed. 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. Industrial Applicability
• novel compositions comprising water insoluble surfaces partially coated with water soluble polysaccharides, normally amino functionalized, and partially attached to molecules of a biological substance. Clean accurate and highly selective separation is obtainable between molecules which are binding partners to the biological substance and binding partners which are not binding partners to the biological substance.

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