CS231660B1 - A method of immobilizing a ligate for saturation analysis - Google Patents

Abstract

The invention consists in immobilizing the ligand by means of a coordination bond to a carrier material activated by metal hydroxides forming coordination bonds. The carrier material used is porous glass, or glass plates or rods with a surface treated by the action of acid or alkali or treated by simple roughening, e.g. grinding. The actual separation of the free and bound ligand consists in the sedimentation of these particles with the bound ligand in the case of a carrier with small particles, and in the case of a carrier of larger dimensions in simply removing the carrier material from the substrate. The invention can be used for highly sensitive and specific analysis of peptides, hormones, vitamins, steroids, alkaloids, pesticides, toxins, etc.

Description

The invention relates to a method for immobilizing a ligate for saturation analysis.

Saturation analysis is an analytical procedure that uses the principle of specific binding of various substances such as hormones, vitamins, steroids, enzymes, alkaloids, pesticides and antigens to a specific protein specific for these substances. The assay is based on the competition of ligands, the analyte present in the sample, and the same additive labeled, for example, with a radionuclide or a fluorescent agent, for binding to a ligate, which is a specific substance, such as a protein. Depending on the configuration of the assay, the ligand or ligand may be proteins, peptides, hormones, vitamins, steroids, alkaloids, pesticides, toxins.

After separating the bound ligand from unbound, the concentration of the withdrawn substance in the sample can be directly determined by measuring the radioactivity, fluorescence or other quantity. The saturation analysis method is highly specific and can be determined in concentrations of the order of nmol to fmol.

A variety of methods are used to separate the free and bound ligand, for example adsorption of the free ligand to a suitable adsorbent (activated carbon, silica gel, etc.), precipitation of the ligand with the bound ligand with organic solvents, inorganic salts, etc. 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 the support. The simplest form of immobilization is adsorption to the walls of the reaction vessel.

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The binding protein is most commonly adsorbed by the lyatyrene assay. However, the adherence forces that hold the binding protein on the surface of the support are relatively weak and therefore immobilixation is often inadequate. Also, a low titer of binding protein may further aggravate the resulting effect. For this reason, the binding protein is sometimes cross-linked on the surface of the support with 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, particles formed by the polymerization of postyrene latex and glycidyl methacrylate and styrene, particles formed by the polymerization of cellulose, acrolein, and iron oxides were used to immobilize the ligate by covalent bonding. also 1A-aminopropyltriethoxysilanene-activated glass or hydroxysuccinimide activated glass. *

The method of immobilization for the saturation analysis according to the invention is characterized in that the ligate is immobilized by means of a coordination bond to a carrier activated with transition metal hydroxides forming the coordination bonds. As carriers, glass with a suitably treated surface which is activated with metal salts, preferably titanium tetrachloride or titanium tetrachloride followed by hydrolysis to form complex transition metal hydroxides may be used. When the activated glass is introduced into the ligate solution, it is immobilized on the carrier surface by means of a coordination bond.

The glass surface is treated by etching with hydrofluoric acid, hydrochloric acid or other acids, sodium hydroxide or other alkali prior to activation with metal salts, or preferably by simply roughening the glass surface, for example by grinding. If porous glass is used, this treatment is not necessary.

The advantage of this method is, besides the experimental simplicity of the immobilization of the ligate, also the simplification of the free-bound ligand separation. When small glass particles are used, they quickly sediment, so that it is not normally necessary for other materials

231 ΒβΟ centrifugation. In the case of glass in the form of platelets or rods, the entire separation consists in simply removing the ligand-bound support from the incubation solution. In both cases, the free-bound ligand disruption which may occur using other techniques is also excluded. It is also possible to work with a low-titer binding protein, since its binding and utilization is effectively achieved by means of coordination binding.

The invention is illustrated by Examples.

Example 1

To a 12.5 / 0 solution of titanium tetrachloride in hydrochloric acid was added porous glass at a ratio of 1 g per 20 ml and the reaction mixture was shaken for 20 minutes. at 40 ° C. After removal of the supernatant, the aspirated solid was dried at 45 ° C for 6 h, washed with water (6 x reaction volume) and dried again at 45 ° C for 6 h.

Antisera against papain were added to the activated porous glass. After 16 h of gentle shaking at 4 ° C, the porous antibody-bound glass was washed with 0.1 L phosphate buffer pH 7.4 and used to determine papain. Papain was detectable in the range of 80 to 2000 ng / ml.

Example 2

10 x 4 x 1 mm glass plates were heated at 10 ° C for 5 h in 20.4% hydrochloric acid, washed with water and activated as described in Example 1. Anti-papain antisera was added to the activated glass plates. After 16 h of gentle shaking at 4 ° C, the plates were washed with 0.1 12 phosphate buffer pH 7.4 and used to determine papain. Papain was detectable in the range of 80 to 2000 ng / ml.

EXAMPLE 5

Glass rods of 10 mm length and 5 mm diameter were heated to boiling for 5 h in a solution of 1 M sodium hydroxide and 1 12 sodium carbonate. The following papain assay procedure was the same as in Example 2. Papain was detectable in the range of 80-200 ng / ml.

Example 4

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Glass rods of 10 mm length and 3 mm diameter were surface treated with 5% hydrofluoric acid at 20 ° C for the time as in Example 2. Papain was detectable in the range of 120 to 2000 ng / ml.

Example 5

The grinding treated surface of 10 x 4 x 1 mm glass plates was activated as described in Example 1. Papain antiserum was added to the activated plates. After 16 h of gentle shaking at 4 ° C followed by washing with 0.1 M phosphate buffer pH 7.4, papain in the range of 80 to 2000 ng / ml was determined.

Example 6

The chitin powder was activated as in Example 1.

Antiserum against papain was added to the activated chitin. The reaction mixture was shaken gently at 4 ° C for 8 h. The chitin powder was then 0.1 M phosphate buffer pH 7.4 and used to determine papain. Papain was detectable in the range of 120 to 2000 ng / ml.

Example 7

The surface of sorsilen was activated as in Example 1.

Antisera against papain were added to the activated sorsilen. The reaction mixture was incubated with gentle shaking for 24 h at 4 ° C. Then sorsilen was washed with 0.1 M phosphate buffer pH 7.4 and used for papain assay. Papain was detectable in the range of 200 to 2000 ng / ml.

Example 8

To 5 g of glass (porous glass, glass plates) was added 50 ml of 12.5% SnCl 2 in concentrated HCl and allowed to shake for 20 minutes at 40 ° C. It is then aspirated and dried in a thermostat at 45 ° C for 6 hours. The dried activated glass is washed 6 times with 100 ml of water and finally dried at 45 ° C. The further procedure is the same as in Examples 1 and 2. Papain was detectable in the range of 200 to 2000 ng / ml.

Claims (6)

A method of immobilizing a ligate for saturation analysis in determining biologically active compounds by separating free and bound lithium by competing for ligands of the analyte and the same labeled, for example, with a radionuclide, fluorescent or enzyme, in binding to the ligate, e.g. and wherein the ligate is immobilized by means of a coordination bond to a carrier activated with transition metal hydroxides forming the coordination bonds. 2. Process according to claim 1, characterized in that glass is used as carrier, the surface of which is treated with acid (preferably hydrochloric acid or hydrofluoric acid) and the surface treated with metal salts such as titanium tetrachloride or titanium tetrachloride followed by hydrolysis. 3. Process according to claim 1, characterized in that glass is used as carrier, the surface of which is treated with sodium hydroxide or other alkali, optionally in combination with alkali metal carbonates, and the surface thus treated is activated with metal salts followed by hydrolysis. 4. Process according to claim 1, characterized in that glass is used as the carrier, the surface of which is treated by roughening, preferably by grinding, and the surface treated in this way is activated by metal salts followed by hydrolysis. 5. A process according to claim 1, wherein the carrier is a porous glass whose surface is activated by metal salts followed by hydrolysis. 6. Process according to claim 1, characterized in that natural or synthetic polymers such as chitin and sorsilen are used as carriers.