DE2509895A1 - Proteins immobilization on plastics surfaces - by swelling the surface with a solvent, applying aq-organic protein soln, and evaporating excess solvent - Google Patents

Abstract

Protein cpds. (esp. antigens or antibodies) are immobilised on plastics surfaces by (a) treating the plastics surface with a solvent which does not dissolve but does swell the plastic surface, (b) applying the protein cpd. dissolved in a homogeneous mixt. of water, organic solvent and solubiliser (pref. a surface-active or organic cpd.), (c) subsequently evaporating excess solvent, and (d) drying the plastics surface. The immobilised proteins are useful for diagnostic and other purposes. Thus, when the immobilised protein is human chorionic gonadotrophin antibody or a syphilis-specific antibody the immobilised protein can be used for the immunological diagnosis of pregnancy or syphilis. The need for two liq. phases in diagnostic tests is avoided. Thus tests using antibodies immobilised on plastic foils are carried out by applying a drop of antigen-contg. soln. to the foil; if the test soln. is positive, a white ring appears around the edge of the drop after a short time, and when the test soln. is negative the drop remains clear.

Description

PROCESS FOR FIXING PROTEIN BODIES ON PLASTIC SURFACES =================================================== ================== The present The aim of the process is to fix protein bodies on plastic surfaces using them for diagnostic and other purposes.

Various methods have long been known for performing immunological tests Possibilities for protein bodies to react with one another in a suitable form for this purpose allow. For example, in a rapid pregnancy test, the in the urinary excreted hormone human chorionic gonadotropin (HCG) with close agglutination reaction proven; HCG chemically bound to latex particles is converted into HCG antibodies agglutinates or this reaction takes place in the presence of free HCG (i.e. if Pregnancy).

A laboratory screening test to diagnose syphilis consists of taking a Latex binds a liquor-specific antigen. Brought together with fresh sick serum, agglutination occurs, which is visible as cloudiness or precipitation and appears indicates the presence of syphilis.

The known methods have the disadvantage that you can use two liquid Phases works and that in practice they are associated with other difficulties are.

In contrast, the method according to the invention has many advantages, which are based on the fact that the protein bodies in question are on plastic surfaces are fixed, characterized in that these plastic surfaces are this non-dissolving but swelling organic solvent is treated, after which the protein body, dissolved in a homogeneous mixture of water and organic solvents Solvent and a solubilizer, is applied, with the following Evaporation of excess solvent and drying of the plastic surface.

A plastic surface, usually a plastic film (made of Polyvinyl chloride, polystyrene, polymethacrylate, etc.), is first in an organic Solvent swollen. According to the invention, the solvent may be the plastic do not dissolve it, just fill it up; for example is for polyvinyl chloride the ethyl acetate is a suitable solvent. In special cases it can be a dissolving Solvents cannot be avoided, then you can deal with mixed solvents help; for example, you can use a polystyrene film in a solvent mixture swell from pentane with a small amount of chloroform or toluene added. After a Such inventive treatment is the plastic film in one more or less soft, very elastic to almost gel-like state.

The protein body to be fixed on the plastic surface can be an antibody, antigen, enzyme, hormone, etc. from other substance areas (e.g. vitamins, antibiotics, etc.) are possible.

The particularly interesting group of albuminous bodies is in general insoluble in organic solvents. In order to bring them to a solution anyway, served so-called solubilizers; these are mostly surface-active Substances such as weakly ethoxylated fatty alcohols, ethoxylated arylphenols, alkylphenyl ether disulfonates and other hydrotropes which have the property of being water and having water immiscible organic solvents, as well as substances dissolved in them, in one bring homogeneous solution. Such preparations exist for various purposes in the Trade, for example under the name SCINTISOL-GP (Isolab / USA) or TRITON Xloo (company Röhm & Haas / USA).

After the aqueous solution of the protein body to be fixed by means of a solubilizer and organic solvent into a homogeneous solution has brought, one puts the prepared according to the above description in it Plastic film; in the course of 2 to 24 hours the protein body becomes on the plastic fixed.

The plastic film is then dried in the air; stite can be stored for a long time at room temperature.

If you look at a plastic surface prepared in this way, on which according to the invention, for example, an antibody has been fixed, a drop of a corresponding antigen appears after a short time at the edge of the drop a whitish ring, which indicates a positive reaction; in the negative case the drop remains clear.

The invention is illustrated by the following examples: Example 1 A piece of rigid PVC film (30 x 20 x 0.1 mm) is placed in a sealable glass jar placed in ethyl acetate. After approx. 1 hour the PVC film has entered without any solution has absorbed enough ethyl acetate and appears as more transparent, softer, almost gelatinous film; it is removed from the solvent and from the appended Freed solvent by briefly drying in air.

Now put 0.2 ml SCINTISOL-GP (company Isolab Inc.) and mixes it with 2.0 ml of toluene; then give it drop by drop polyvalent anti-human serum (from the goat) until a clear solution is obtained has become cloudy; a few drops of the above solvent mixture can be used Easily remove the slight cloudiness. The now clear solution is given the PVC film prepared as described above ensures complete coverage the solvent and let stand at room temperature for about 24 hours. Afterward the PVC film is removed from the solution and air-dried for approx. 6 hours, and then washed free of wetting agents, etc., with distilled water. the PVC film prepared in this way can be stored for months at room temperature.

Use in an antibody-antigen reaction is as follows Method: Normal human serum (lo microliters) are mixed with 1.0 ml of distilled water mixed. 1 drop of the serum diluted in this way is applied to the one described above given prepared PVC film; it is best to place them on top of one of the below Glass pane illuminated at an angle of approx. 450. After about 10 minutes you can be on the edge observe a white ring of the drop.

Example 2 Polyvalent human antiserum is made as in Example 1 fixed on a PVC film prepared in the same way: 1. o ml of toluene, mixed with 0.15 g TRITON Xloo (Röhm & Haas), to solubilize 0.16 ml Human antiserum (from goat) used.

A PVC film (40 x 30 x 0.2 mm) is in ethyl acetate for one hour until it has become translucent and flexible like a gel. After taking it out from the ethyl acetate and drying (about 15 minutes) the PVC film is in the above Solution of the human antiserum placed overnight, then 3 - 4 hours on the Air dried and washed with distilled water. The fixation of an antibody and the reaction with an antigen is carried out in a manner similar to Example 1 described.

Claims (3)

PATENT CLAIMS 1. Process for fixing protein bodies on plastic surfaces, characterized in that the plastic surface is covered by a non-releasing but swelling, solvent is treated, after which the protein body is dissolved in a homogeneous solvent mixture of water, organic solvent and a solubilizer, is applied, with subsequent evaporation excess Solvent and drying of the plastic surface. 2. The method according to claim 1, characterized in that the protein compound is an antigen or antibody. 3. The method according to claim 1 and 2, characterized in that the Solubilizer is a surface-active organic compound.