ARTIFICIAL SUPPORT MATERIAL FOR IMMOBILISATION OF BIOLOGICAL PROTEIN AND PROCESS FOR PRODUCING THE SAME

Abstract: An artficial support material for immobilisation of biological proteins such as antigens, antibodies or enzymes used in serological testing comprises gelatin, water-soluble polysaccharide, alkali metal polymetaphosphate and a ferro-­ magnetic substance.

Description: ARTIFICIAL SUPPORT MATERIAL FOR IMMOBILISATION OF BIOLOGICAL PROTEIN AND PROCESS FOR PRODUCING THE SAME This invention relates to an artificial support material for use in serological testing and to a process for producing the same. The support material of the present invention is useful as a carrier to immobilise biological proteins such as antigens, antibodies and enzymes. Immobilised antigens, antibodies and enzymes are used in assays involving antigen-antibody reactions. Artificial support materials in particulate form which comprise organic polymers as principal ingredients may be of a variety of types. One such material consists of particles comprising gelatin having an isoelectric point of 8 to 9, a water-soluble polysaccharide and an alkali metal polymetaphosphate, which particles are insolubilised with respect to water by cross-linking treatment with an aldehyde. Such particles are described in our European Patent Application No.82301235.6 (0062968). It is an object of the present invention to provide a modification of such artifical support material which will enable one to achieve control of agglutination time in an antigen-antibody reaction and easy recovery of the support material used therein. According to one aspect of this invention there is provided an artificial support for use in serological testing which comprises particles comprising gelatin, a water-soluble polysaccharide and an alkali metal polymetaphosphate, which particles are insolubilised with respect to water by cross-linking treatment with an aldehyde, and are characterised by the additional presence of a ferromagnetic substances. In a second aspect, this i invention provides a process for the production of an artificial support material for use in seroloqical testing which comprises farming an aqueous solution containing gelatin, a water-soluble polysaccharide and an alkali metal polymetaphosphate having a temperature higher than the gelation temperature of said gelatin, adjusting the pH of said solution to a value of from 2.5 to 6.0 to produce a suspension of gelatin-containing particles and contacting said particles with an aldehyde cross-linking agent to render the particles insoluble in water, wherein a ferromagnetic substance in colloidal form is suspended in the solution from which said particles are formed. The magnetic support material of this invention has advantages as shown below when it is used as a carrier for immobilisation of biological protein: (1) Agglutination time in an antigen-antibody reaction can be controlled by a magnet. (2) Magnetic particles can be much more easily separated or recovered from a suspension thereof, compared with conventional support materials not containing magnetic substances, simply by application of magnetic force. The fundamental principle of using magnetic particulate support materials has previously been proposed by K. Widder et al. However such support materials are produced by covering magnetic particles with human serum albumin and then subjecting the particles in turn, to emulsification and heat treatment (see K. Widder, G. Flouret and A. Senyei: J. Harm. Sci. 68, 79 (1979)). Widder's process has the disadvantage that it is difficult to produce homogeneous magnetic particulate materials by this procedure. In contrast, it has now been found that homogeneous magnetic particulate support materials can be obtained by mixing fine particles of a ferromagnetic substance with the materials used to produce a support material of the type described in the aforementioned European patent application. rtse magnetic particles of the support material of this iivgntion preferably comprises from 5-308 by weight of gelatin, 1-5% by weight of water-soluble polysaccharide, 0.01-1% by weight of alkali metal polymetaphosphate, preferably sodium polymetaphosphate, 0.00001-2% by weight of colloidal ferromagnetic substance and 62-94% by weight of water. The particles preferably have a particle size of 0.8-50% um and are water-insoluble and hydrophilic, the gelatin of the particles having been cross-linked for this purpose using aldehyde to form links between amino groups thereof. In the practice of this invention, the gelatin used preferably has a molecular weight of about 100,000. Moreover the gelatin preferably has an isoelectric point of pH 8.4 to 9.1. The most preferred water-soluble polysaccharide is gum arabic, although carboxymethyl cellulose, sodium sodium alginate and agar are also suitable for use in this invention. The alkali metal polymetaphosphate is preferably sodium polymetaphosphate having the formula (NaPO3)n wherein n is an integer of 3 to 6. Ferromagnetic substances which can be used include ferrites having the formula MFe2O4 wherein M represents a divalent metal, preferably Fe++, Mn++, Cu++, Ni++ or ++ Co . When producing the support material of this invention, the ferrites are preferably in the form of an aqueous suspension containing colloidal ferrites in an amount of about 30% w/v. In a first step in producing a support material of this invention, an aqueous suspension colloid of a ferrite needs to be produced. An aqueous suspension colloid of a ferrite, for example MnFe204, can be prepared, for example, by the steps of: mixing 100 ml a 1 M MnCl2 solution and 100 ml of a 2 M FeC13 solution, 3 adding a 6 N NaOH solution dropwise to the mixture until the solution has a pH of 11, heatinq the mixture at 100 C for 30 minutes, washing the precipitate which has formed with hot water and dispersing the precipitate in 3 32tUt 100 ml of an aqueous solution ; about 0.1% w/v) oF a surfactant such as sodium oleate. Aqueous suspension colloids of other ferrites, e.g. CuFe2O4, NiFe2O4 and CoFe2O4, can be prepared by repeating the procedure of preparing the aqueous suspension of MnFe2O4 as shown above, but using CuCl2, NiCl2 and CoCl2 instead of MnCl2. It will generally be necessary the reaction mixture to be heated at higher temperatures than 1000C in an oil bath or heated an autoclave. When an aqueous suspension colloid of magnetite (Fe3O4), is to be used as the ferrite, it can be prepared by the steps of: mixing 100 ml of a 1 M FeCl2 solution and 100 ml of a 2 M FeCl3 solution, adding 200 ml of a 6 N NaOH solution to the mixture to produce a precipitate, washing the precipitate with hot water and dispersing the precipitate in 100--ml of an aqueous solution (about 0.1% w/v) of a surfactant such as sodium oleate. An aqueous suspension colloid of the chosen ferrites is preferably prepared which contains colloidal ferrite in an amount of about 30% w/v. The artificial support material of this invention may then be produced as follows: 0.01 to 2 g of gelatin, 0.01 to 2 g of water soluble polysaccharide, 0.001 to 2 g of alkali metal polymetaphosphate and 0.01 to 5 g of colloidal ferrite (about 36 ul to 18 ml of aqueous suspension colloid of ferrite) are added to 100 ml of an aqueous solution containing about 0.1% (w/v) anionic or nonionic surfactant to produce a colloidal solution, and after the pH of the colloidal solution has been adjusted to 2.5 to 6.0 with an acid such as acetic acid, the gelatin is cross-linked with an aldehyde, for example glutaraldhyde or formaldehyde, at a temperature of from 5 C to 35 C with stirring. An anionic surfactant which is used may be an alkylsulphosuccinate, alkylsulphomaleate, alkylsulphuric acid aster or plyoxyethylene alkyl ether sulphuric acid ester. A nonionic surfactant which is useu may be a polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether or polyethylene glycol fatty acid ester. The alkyl group for both groups or surfactants has from 6 to 25 carbon atoms. The present invention is further illustrated by the following examples. Example 1 One gram of gelatin having an isoelectric point of pH 9 was dissolved in 25 ml of water at 40 C and to this solution was added a 10% by wt. NaoH solution to adjust the pH to 9.0. A solution of 1 g of gum arabic in 20 ml of water at 400C was then added and the mixture obtained was poured into 150 ml of a 30% v/v aqueous ethanol solution, and after stirring, there was added to this mixture, in turn, 0.8 ml of a 10% by wt. sodium hexametaphosphate solution, 1 ml of a 1% by wt. aqueous solution of an alkylsulphomaleate (trade name: Demol Ep, manufactured by Kao Soap Co.) and 60 ul of a suspension colloid of Fe304 prepared as described above, and the mixture obtained was stirred to achieve homogeneity. Subsequently, a 108 by wt. acetic acid solution was added dropwise to the mixture while it was kept at 400C and the pH of the mixture was adjusted to 4.8 to form a particulate suspension. The particulate suspension was cooled to 5 C in an ice bath, and 0.65g of glutaraldehyde was added. After stirring, the suspension was allowed to stand at 5 C overnight. Then, the suspension was centrifuged at 2000 rpm for 10 minutes, and the resulting particles were collected. The particles were suspended in a 0.005 by wt. Dcmol Ep solution, and the suspensior was centrifuged again. This washing procedure was repeated three times, and the particules were suspended in 50 ml of a 4@ v v formaldehyde solution, and this suspension was allowed to stand at 5 C for one week. By working in this manner, a suspension of machetic particles compr@@@@ne ab@@ 20% by wt. gelatin, about 2% by wt. gum arabic about 0.1% by wt. (NaPO3)6, about 0.001% by wt. of Fe3O4 and about 78% by wt. of water, the particles having a particle size of about 3 um, was obtained. Example 2 The procedure of Example 1 was repeated except that sodium trimetaphosphate and 600 l of a suspension colloid of MnFe2O4 prepared by the above indicated procedure were used instead of sodium hexametaphosphate and 60 ul of the suspension colloid of Fe304. As a result of operating in this manner a suspension of magnetic particles containing about 20% by wt. of gelatin, about 2% by wt. of gum arabic, about 0.1% by wt. of (NaPO3)3, about 0.0058 by wt. of MnFe204 and about 78% by wt. of water, and having particle size of about 3.5 um was obtained. Magnetic particles in powder form can be obtained by separating out from suspension the magnetic particles and lyophilizing the wet magnetic particles. In Examples 1 and 2, suspensions of magnetic particles containing 0.0018 and 0.0058 by wt. respectively of ferromagnetic substance and having particle sizes of about 3 um and about 3.5 um respectively were obtained. It is to be understood, however, that the composition of the magnetic particles constituting the support material of this invention can be changed by changing the amount of the ingredients to be used in the production thereof. Furthermore, the particle size of the magnetic particles can be varied by producing the magnetic particles under different conditions of temperature and solution pH or employing different stirring conditions. A support material embodying this invention is suitable for use in the same type or @@rological tests as the support material of European Patent Application No.82301235.6 (0062968) to which reference is directed, but with the aforementioned added advantages of allowing control of agglutination lime and easy recovery of the support material made possible by the presence of the ferrite, coupled with homogeneity of the particulate material

Claims: CLAIMS: 1. An artificial support material for use in serological testing which comprises particles comprising gelatin, a water-soluble polysaccharide and an alkali metal polymetaphosphate, which particles are insolubilised with respect to water by cross-linking treatment with an aldehyde, and are characterised by the additional presence of a ferromagnetic substance. 2. An artificial support material as claimed in Claim 1, wherein said gelatin has an isoelectric point of from pH 8.4 to 9.1. 3. An artificial support material as claimed in Claim 1 or 2, wherein the water-soluble polysaccharide is selected from gum arabic, carboxymethyl cellulose, sodium alginate and agar. 4. An artificial support material as claimed in any preceding claim, wherein said alkali metal polymetaphosphate has the formula (NaPO3)n wherein n is an integer of from 3 to 6. 5. An artificial support material as claimed in any preceding claim, wherein said ferromagnetic substance is a ferrite having the formula MFe2O4 wherein M is a divalent metal selected from Fe++, Mn++, Cu++, Ni++ and Co++. Co 6. An artificial support material as claimed in any preceding claim, which contains from 5 to 30% by wt. gelatin, from 1 to 5% by wt. of the water soluble polysaccharide, from 0.01 to 1% by wt. of sodium polymetaphosphate, from 0.00001 to 24 by wt. of the ferromaqoetic substance and from 62 to 948 by wt. of water. 7. @@ process for the production of an artifical support material for use in serological testing which comprises forming an aqueous solution containing gelatin, a water-soluble polysaccharide and an alkali metal polyùetaphophate having a temperature higher than the aelation temperature of said gelatin adjusting the pH of said solution to a value of from 2.5 to 6.0 to produce a suspension of gelatin-containing particles and contacting said particles with an aldehyde cross-linking agent to render the particles insoluble in water, characterised in that a ferromagnetic substance in colloidal form is suspended in the solution from which said particles are formed. 8. The process of Claim 7, wherein said gelatin has an isoelectric point of from pH 8.4 to 9.1. 9. The process of Claim 7 or 8 wherein the concentration of gelatin in said solution is from 0.01% to 2% by weight. 10. The process of Claim 7, 8 or 9 wherein the concentration of water-soluble polysaccharide in said solution is from 0.01% to 2% by weight. 11. The process of any one of Claims 7 to 10 wherein said water-soluble polysaccharide is selected from gum arabic, carboxymethyl cellulose, sodium alginate and agar. 12. The process of any one of Claims 7 to 11, wherein said alkali metal phosphate is sodium polymetaphosphate present in said solution in a concentration of from 0.001% to 2% by weight. 13. The process of Claim 12, wherein said sodium polymetaphosphate has the formula (NaPO3)n, wherein n is an integer of from 3 to 6. 14. The process of any one of Claims 7 to 13, wherein said ferromagnetic substance is suspendedin said solution in an amount of from 0.01% to 5% by weight. 15. The process of any one of Claims 7 to 14, wherein said ferromagnetic substance is a ferrite having the formula MPe2O4, wherein M is a divalent metal selected from Fe++, Mn++, Cu++, Ni++ and Co++. 16. The process of any on of Claims 7 to 15, wherein said aldehyde is selected from glutaraldehyde and formaldehyde.

Inventors: IKEDA, MIKIO, SAKAMOTO, SHIRO, SUZUKI, KAZUMASA
Applicants: FUJIREBIO KABUSHIKI KAISHA ALSO TRADING AS FUJIREBIO INC.
Application year: 1984
Main classification: G01N33
IPC codes: G01N33, B03C1