CS258181B1 - Method of carriers preparation with immobilized antibodies for immunochemical determination - Google Patents

Abstract

Process for preparing immobilized carriers antibodies for immunochemical assays glass based in the form of coarse grit that is tempered between 400 and 900 ° C after for 30 to 45 hours, then leached through the solution inorganic acid, optionally alkaline acid hydroxide is that it is modifies the skio with a composition of 3 to 17% wt. an alkali metal oxide, for example sodium, potassium and lithium alone or in combination, 15 to 37 wt. boron oxide, 55 to 75 wt. silica and 0 to 6 wt. alumina optionally with a content of 0.2 to 5 wt. phosphorus pentoxide and 0.2 to 5 wt. alkaline fluoride metal that is after rinsing the reaction exhausts gently ground in the form of an aqueous suspension in a corundum planetary mill to size particles of 3 to 30 µm, whereupon it is activated and on the activated carrier is immobilized added specific antibody direct reaction with an activated carrier.

Description

The invention relates to carriers with immobilized antibodies for immuno-chemical assays and methods for their preparation.

In immunochemical assays of various anitgens, immunogens or haptenes such as hormones, vitamins, steroids, alkaloids, pesticides, enzymes and other proteins, virtually the most important step in the analytical procedure of separating free and immunochemical interaction of bound ligand. The immunochemical assays are based on the competition of a ligand, mostly an antigen, as the analyte present in the sample with the same substance labeled with, for example, a radionuclide, a fluorophore or an enzyme bound to the ligate, which is most often a specific antibody. After separating the bound ligand from the non-bound ligand by measuring the radioactivity, fluorescence or enzyme activity, it directly determines the concentration of the test substance in the sample.

A variety of methods are used to separate the free and bound ligands, such as adsorption of the free ligand to a suitable adsorbent (silica gel, activated carbon, etc.), precipitation of the ligand with the bound ligand with organic solvents, inorganic salts, polyethylene glycol and the like.

Since ligate-ligand binding is of a reversible nature, there is a risk of free-bound ligand imbalance in these methods.

Considerable simplification of the separation of free and bound ligand can be achieved by immobilizing the ligate onto a suitable carrier. The simplest form of immobilization is adsorption to the walls of the reaction vessel. Binding protein is most often adsorbed to polystyrene tubes or microtiter plates of the same material. However, the adherence forces that hold the binding protein to the surface of the carrier are relatively weak and therefore immobilization is often insufficient. Also, a low titer of binding protein may further aggravate the resulting effect. For this reason, the binding protein is sometimes crosslinked on the surface of the support by various bifunctional agents or, more often, is immobilized by covalent binding directly to the activated support.

A variety of materials such as cyanogen bromide activated cellulose, polymers containing active isothiocyanate groups, polystyrene latex polymerization with glycidyl methacrylate and styrene, particles formed by polymerization of cellulose, acroleins and iron oxides, and the like were used to immobilize the ligate by covalent bonding.

Glass activated with gamma-aminopropyltriethoxysilane, hydroxysuccinimide or transition metal salts was also used to immobilize the ligates by covalent bonding. For most carriers used, including compact or porous glass with immobilized antibodies, the incubation mixture must be agitated or shaken during saturation, reducing the benefits of using the immobilized antibodies.

These drawbacks are overcome by the method of preparing carriers with immobilized antibodies for immunochemical assays according to the invention, consisting in the use of a 3 to 17 wt. % alkali metal oxide, 15 to 37 wt. % boron oxide, 55 to 75 wt. % silica and 6 wt. % alumina, optionally containing 0.2 to 5 wt. % phosphorus pentoxide and 0.2 to 5 wt. alkali metal fluoride, which is tempered in the range of 400 to 900 ° C for 30 to 45 hours in the form of coarse crumb, then leached with a solution of an inorganic acid such as hydrochloric acid, optionally with an alkali metal hydroxide solution such as sodium hydroxide and after rinsing the reaction The exhaust gases are finely ground in the form of an aqueous suspension in a corundum planetary mill to a particle size of 3 to 30 µm, then activated by reaction with a solution of 5 to 50 wt. % gamma-aminopropyltriethoxysilane in toluene followed by treatment with a solution of 5 to 15 wt. % of glutaraldehyde or directly by reaction with a solution of 5 to 15 wt. glutaraldehyde or by treatment with transition metal salts, such as titanium tetrachloride, followed by their hydrolysis with water on the surface of the glass particles and on the activated nocturnal, the added specific antibody is immobilized by direct reaction with the activated carrier.

During the action of a mineral acid or an alkali hydroxide, alkali oxides, alumina, phosphoric acid and boric oxide are eliminated. The porous skeleton obtained contains 90 to 99 wt. % silica, 1 to 9 wt. of boron oxide and other constituents in tenths of 4 wt.

The defined surface character is very advantageous for subsequent activation and then immobilization of specific antibodies, and in view of the low sedimentation rates of given particle sizes, the carriers of the invention are particularly suitable for applications in immunochemical analyzes. These carriers are chemically very resistant to neutral and acidic environments and perfectly stable to all organic substances.

By immobilizing the specific antibody to the activated porous glass, an antibody-coupled support for an immunochemical assay is prepared according to the invention which, by virtue of its surface structure and particle size, ensures a rapid equilibrium of free and bound ligand without shaking or stirring the incubation mixture. Compared to conventional precipitation techniques, separation of the conjugate / centrifugation of the carrier with the immobilized antibody and immunochemically bound antigens / thereof is significantly easier and faster.

The advantage of the carrier with the immobilized antibody according to the invention is, besides the already mentioned suitable sedimentation properties, its ease of preparation and its availability with regard to the raw materials used. A non-negligible advantage of a vehicle with immobilized antibody is its easy pipetting and very good stability, where it can be analyzed for several months to significantly lose the immunochemical activity of the immobilized antibody.

The invention is illustrated by, but not limited to, examples.

Example 1

Glass composition 6.9% wt. ,7β2θ, 25.7% wt. and 67.4 wt. The SiO 2 is melted in a conventional glass-making process and, in the form of coarse pulp, is tempered in a crucible furnace at 604 ° C for 45 hours. The tempered grit is then leached with 4 wt. hydrochloric acid solution at 70 ° C. After thorough washing with distilled water, the leached pulp is milled for 6 hours in a corundum planetary mill in distilled water. Weight a portion of the prepared porous glass having a specific surface area of 112 m / g is heated to reflux after drying with 10 parts by volume of a 20% (v / v) solution of gamma-aminopropyltriethoxysilane in toluene for 24 hours. The modified porous glass is then washed with toluene and dried.

Κ 1 wt. 5 parts by volume of 5 wt. of glutaraldehyde solution and the suspension was shaken for 2 hours at 37 ° C. After washing the activated porous glass with distilled water and 0.1 M phosphate buffer at pH 7.4, 2.5 parts by volume of the papain antiserum are added to 1 part by weight of the moist activated carrier. After standing for 16 hours with occasional shaking, the antibody-bound support is centrifuged, washed with 0.1 M phosphate buffer pH 7.4 and used, after appropriate dilution, for papain immunochemistry.

Example 2

One part by weight of porous glass having a composition of 15.1% by weight. K ₂ O + Na ₂ O, 19.8 wt. SiO 2, 1.1 wt. P ₂ ° 5 »0 <6 * wt. % fluoride and 0.3 wt. The Al ₂ O ₃ prepared according to Example 1 is suspended in 5 parts by volume of 10 wt. solution of glutaraldehyde. The suspension is heated to 37 ° C for 12 hours with occasional shaking. The activated support is then centrifuged, washed with 0.1 M phosphate buffer 7.4 and centrifuged again. Κ 1 wt. 2.5 parts by volume of anti-thyroxine antiserum are added to a portion of the wet carrier. After standing for 16 hours at 4 ° C with occasional shaking, the antibody-bound support is centrifuged, washed with 0.1 M phosphate buffer pH 7.4 and used, after appropriate dilution, for thyroxine immunochemistry.

Example 3

To 20 parts by volume 12.5 wt. titanium tetrachloride in hydrochloric acid is added 1 wt. % porous glass having a composition of 5.7 wt. Li ₂ O + ItajO, _{31 (wt %} , _wt-

S1O2 and 4.7 Al2O4 ° particle sizes of 5-15 prepared according to Example 1. After standing for 20 minutes at 40 ° C, the mixture is centrifuged and the sediment is left at 45 ° C for 6 hours.

It is then washed with water and dried again at 45 ° C. Κ 1 wt. 3 parts by volume of the anti-thyroxine antiserum are added and the mixture is left for 16 hours at 4 ° C with occasional shaking. After centrifugation and washing with 0.1 M phosphate buffer pH 7.4, the antibody-bound carrier is used for immunochemical determination of thyroxine in the range of 20 to 240 pmoles / ml.

Claims (4)

Method for preparing carriers with immobilized antibodies for immunochemical assays based on glass in the form of coarse crumb, which is tempered in the range of 400 to 900 ° C for 30 to 45 hours, then leached with a solution of an inorganic acid, for example hydrochloric acid, optionally % of an alkali hydroxide, for example sodium hydroxide, characterized in that a glass of 3 to 17 wt. % alkali metal oxide, for example sodium, potassium and lithium alone or in combination, 15 to 37 wt. % boron oxide, 55 to 75 wt. and up to 6 l wt. % alumina optionally having a content of 0.2 to 5 wt. % phosphorus pentoxide and 0.2 to 5 wt. an alkali metal fluoride which, after rinsing the reaction products, is finely ground as an aqueous suspension in a corundum planetary mill to a particle size of 3 to 30 µm, then activated and the added specific antibody is immobilized on the activated carrier by direct reaction with the activated carrier. 2. A process according to claim 1, wherein the finely ground glass is activated with a solution of 5 to 15 wt. glutaraldehyde. Method according to claim 1, characterized in that the finely ground glass is activated with a solution 5 to 50 wt. % gamma-animipropyltriethoxysilane followed by a 5-15 wt. glutaraldehyde. 4. The method of claim 1, wherein the finely ground glass is activated by solution 10 to 20 wt. chloride of transition metals such as titanium or zirconium chloride in concentrated hydrochloric acid followed by hydrolysis with water.