CS268052B1 - A method of making a diagnostic composition for determining <X, - antitrypsin - Google Patents

Abstract

A method for producing a diagnostic preparation for determining c< j- antitrypsin in biological material on the known principle of animal immunization, characterized by purification of the immunogen by the action of one to four parts by weight of animal immunoglobulin on one part by weight of lyophilized human o/,- antitrypsin, obtained from human serum by double precipitation of ballast serum proteins with solid ammonium sulfate at a concentration of 400 g of ammonium sulfate per 1 liter of human serum protein solution. Animal immunoglobulin antibody-active against minor components of the human <X globulin fraction is produced by immunizing an animal with crude unpurified human i- antitrypsin and is adjusted by saturation, i.e. by adding purified human p1,- antitrypsin to the antiserum so that the antiserum does not precipitate C< antitrypsin, but rather precipitates the minor ,- globulin fractions. The mixture of both lyophilizates, i.e. αΙ j-antitrypsin and immunoglobulin against minor components, is dissolved in 0.06 M phosphate buffer, filtered through a layer of diethylaminoethylcellulose at pH 6.8 and αζj-antitrypsin is separated from free antibody and from complexes in the molarity range of 0.06 M- 0.3 M, thereby removing minor components that determine the cost-effectiveness of production. Immunization of pigs with purified human αζ-antitrypsin is performed in three to five doses, by injection of 5 ml of aluminum hydroxide suspension, a 0.02% to 1% solution of αζ-antitrypsin diluted five times with the suspension is injected, immunization of goats is performed continuously for longer periods of time in smaller doses, proportional to the weight of the animal. Albumin, orosomucoid, or other fraction of human serum is added to the serum of immunized animals, and the product is isolated.

Description

The present invention provides a method of making a diagnostic composition for the determination of β-antitrypsin β in a biological material, which addresses the desired specificity by purifying diagnostics, modifying the antitrypsin immunogen and treating the diagnostic serum with respect to minor components of the β-globulin fraction. These minor components of human serum contaminate antitrypsin preparations, are difficult to detect and remove, but apply decisively in the economics of the production process and in the quality of antitrypsin diagnostics.

At present, the diagnostic product is produced by several leading global manufacturers. Analyzes of commercial diagnostics performed at the Institute of Serums and Vaccines in Prague (Department of Immunochemistry) show that only one product from one manufacturer (DAKOPATTS, Denmark) meets the monospecificity requirement. The other commercial preparations analyzed, although declared monospecific, give an undesirable non-specific side reaction in an immunelelectrophoresis with an unidentified minor protein visible in the α-fraction as a separate precipitation arc. The issue of removing minor components from antitrypsin is reported in the literature / Piero Musiani and Thomas B. Thomas, 3r .: Isolation, chemical, and physical properties of - antitrypsin. Biochemistry, 15,798 (1976) /.

. In the Czechoslovak Socialist Republic, the method of production of antitrypsin diagnostics was the subject of several years of research, assessed by opponents and put into practice. Soon after the start of production, it became apparent that diagnosticians could no longer produce due to irreversible non-specificities, a very low antibody titer, and a large number of unsuccessfully immunized animals. Therefore, the corporate thematic task No. 3/81 Increasing the economy, productivity and production of porcine antiserum - antitrypsin, which was solved by the author of the present invention, was announced.

The present invention is a method for producing C> ^ - antitrypsinového diagnostics based ^one him. on the known principle of immunization of production animals, characterized in that one part by weight of lyophilized human antitrypsin is treated with one to four parts of animal immunoglobulin antibody active against minor components of the human serum globulin fraction, a solution of both proteins in 0.06 M phosphate buffer pH 6.8 is filtered through a layer of diethylaminoethylcellulose, the effluent fractions are collected, the antitrypsin fraction eluted from the column in the range of 0.06 to 0.3 14 phosphate buffer is then injected into production animals (rabbits, goats, pigs, sheep, or other animals). ) in doses of 0.2 ml to 5 ml at a concentration of 0.02% to 1% antitrypsin, in 3 to 5 injections per animal. Albumin, orosomucoid, or another fraction of human serum is then added to the serum of the immunized animals to remove the concomitant unwanted antibodies that almost always accompany the immunization process. After saturation, the product is either isolated as an antiserum or an immunoglobulin, optionally a specific antibody, is isolated from the antiserum. Animal immunoglobulin antibody active against minor components of the human serum globulin fraction is prepared by injecting crude human antitrypsin with a production animal of the same species used for diagnostic purposes, alternatively a soluble sulfate fraction of human serum precipitated with ammonium sulfate at 40% saturation. , at least in 3 to 5 injections to obtain the highest possible antibody titer. 6 to 10 days after the last injection, an animal serum is obtained to which as little human β-antitrypsin as is sufficient to remove anti-β-antitrypsin antibodies is added, while the other antibodies in the antiserum remain unsaturated. The presence of any antibodies in the antiserum - other than antitrypsin - is not detrimental. Immunoglobulin is prepared from the thus saturated antiserum by any suitable method, for example by precipitation with 2M ammonium sulfate. The isolated antibody immunoglobulin against minor components - serum globulin fractions - is then dialyzed and lyophilized. Separation of the free antibody from the soluble antigen-antibody complexes occurs to the greatest extent in the next purification step in the diethylaminoethylcellulose layer, as already mentioned.

CS 268052 Bl

The invention solves a particularly difficult technological problem, which is of crucial importance for the applicability of antitrypsin diagnostics in both qualitative and quantitative immunodiagnostics. Antitrypsin preparations always contain 3 to 4 trace minor components in the area of <2 “- serum fractions, which are very dangerous for the success of production;

they are difficult to detect by direct immunoprecipitation, but when injected into animals, they induce precipitating antibodies in the animals. These antibodies, unlike benign "antibodies such as albumin, orosomucoid, and most other proteins, are malignant" because they are indelible, preventing the desired specificity of the diagnostic from being achieved. The end result is a product that is not for sale, unusable for specific qualitative and quantitative analyzes, which leads to economic losses. The reason for the irremovability of minor antibodies is the absence of minor proteins in the available fractions of human serum, suitable for adjustment (saturation) of diagnostics. '

The method of production of the diagnostic according to the invention leads to the production of anti-tetrypsin antibodies in immunized animals with good reproducibility of production cycles. The animal antibody against the minor components is used in the purification step of immunogen preparation in a free, unbound state, unaffected by binding to a solid support. It therefore has a larger binding capacity, it can be dosed well and its effectiveness can be controlled. Antibody-bound unwanted contaminants, along with excess unbound antibody, are removed directly in the purification step, which is a significant advantage because the antitrypsin site can be used to prepare the immunogen with a relatively simple soluble human serum sulfate obtained at 40% ammonium sulfate saturation. Although the concomitant orosomucoid and albumin elicit precipitation antibodies in immunized animals, these can be easily removed by saturating the antiserum; in addition, these fractions have a significant stabilizing effect and reduce antitrypsin denaturation, which otherwise occurs to a large extent. Unlike an antibody fixed on a solid support (e.g., CN Br / sepharose), the free antibody is usable on an industrial scale with minimal cost. The invention achieves a new and higher effect in the current stage of the antitrypsin diagnostic production method by simplifying the preparation of the immunogen, high productivity and good reproducibility.

Example 1

Unpurified, crude antitrypsin is prepared by adding 1 liter of 0.15 M sodium chloride to 1 liter of mixed serum (waste, so-called collection serum) and dissolving solid ammonium sulphate in an amount of 800 g by weight. The precipitate is centrifuged. the supernatant fluid is filtered through a microbiological filter, dialyzed against distilled water and lyophilized. This lyophilized fraction serves both as an immunogen to obtain antibodies against minor serum fractions and as a basis for the production of pure immunogen.

Example 2

Partially purified antitrypsin was prepared by precipitating a 2.5% solution of the crude antitrypsin from Example 1 dissolved in 0.05 M phosphate pH 6, B with ammonium sulfate according to Example 1. Lyophilization and dissolution gave a solution of antitrypsin. suitable for the preparation of an immunogen purified with an antibody against minor components.

Example 3

Immunoglobulin antibody active against minor components - serum globulin fractions was obtained by injecting the production animal - pig - 4 times at three week intervals with the unpurified, crude antitrypsin from Example 1, each immunization dose containing 20 mg of the fraction from Example 1. One week after the fourth injection, purified human β-antitrypsin was added to the serum of the immunized animal in an amount sufficient to remove antibodies to β-antitrypsin, and the antiserum was salified with 2M ammonium sulfate immunoglobulin, the precipitate was dissolved, dialyzed, and lyophilized.

CS 268052 Bl

Example 4

Κ 0.5 g of lyophilized human antitrypsin according to Example 2 was added 1 g of lyophilized porcine immunoglobulin against minor components - human serum globulin fraction, the mixture was dissolved in 50 ml of 0.06 M phosphate buffer pH 6.8 and filtered through a layer of diethylaminoethylcellulose, fraction of purified o <χ - antitrypsin eluted from the layer at an increased buffer molarity in the range of 0.06M to 0.3M. The antitrypsin-containing fraction was then used as an immunogen to inject the animals. Example 5

The pigs were injected in three injections 3 weeks after 5 ml of aluminum hydroxide suspension (Al-Span) containing 0.1 mg to 0.3 mg of purified antitrypsin from Example 4, the last injection containing 10 mg of immunogen (fourth injection). After one to 10 days, antisera were obtained, saturated with the addition of a solution of human albumin and, individually according to the immunoelectrophoretic spectrum, with an orosomucoid or other fraction of human serum in order to achieve the desired specificity of the product. Example 6

Goats were injected continuously for long periods of time by injecting (in consultation with a veterinarian) the minimum volume of emulsion from Example 5, which generally did not exceed 2 to 3 ml. Depending on the animal's health, blood samples were taken and the animal was reimmunized with an observable decrease in antibody titer. Antiserum saturation is performed analogously.

Claims (1)

OBJECT OF THE INVENTION Method for the production of a diagnostic preparation for the determination of antitrypsin on the principle of immunization of animals, characterized in that that one part by weight of human antitrypsin is treated with one to four parts of an animal immunoglobulin antibody active against the minor components of tX}. “Human serum globulin fraction, the solution of both proteins is filtered through a layer of diethylaminoethylcellulose, the N 2 -antitrypsin fraction is injected as a 0.02 to 1% solution in doses of 0.2 ml to 5 ml in four or more injections of rabbit, goat, pig, sheep, or another animal, albumin, oroso.mucoid, or another fraction of human serum is added to the serum of the immunized animals, and the product obtained is isolated.