DK146138B - PROCEDURE FOR THE PREPARATION OF STABLE ERYTHROCYT PREPARATIONS - Google Patents

Description

«9) DENMARK (w

| J | (12) PUBLICATION WRITING <«> 146138 B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Patent Application No. 5109/76 (51) Int.CI.3: G01N 33/54 (22) Filing Date: 12 Nov 1976 (41) Aim. available: 15 May 1977 (44) Submitted: 04 Jul 1983 (86) International Application No :-(30) Priority: 14 Nov 1975 DE 2551200 (71) Applicant. 'BEHRINGWERKE AKTIENGESELLSCHAFT; Marburg / Lahn, DE.

(72) Inventor: Hans' Schweinsberg; DE, Guenter * Zealch; THE.

(74) Plenipotentiary: Budde, Schou & Co__ Engineering Company (54) Procedure for the preparation of stable erythrocyte preparations

The invention relates to a process for the preparation of stable erythrocyte preparations for coating with antigen, by which a lower aliphatic aldehyde is allowed to interact and wash erythrocytes suspended in an aqueous isotonic solution.

Antibodies have long been detected by a passive or indirect hemagglutination test. Hereby, blood cells from various animal species are used, mostly praying, as the carrier for dissolved 00 (V) antigen. Since native erythrocytes are only stable for a short period of time, senere treatment of the cells with aldehydes ^ (formaldehyde, pyruvaldehyde, glutardialdehyde) or sulfosalicylic acid has been undertaken later.

^ Cells thus treated can be preserved for a longer period. They are first loaded with the corresponding antigen prior to use.

Such erythrocytes loaded with antigen are generally only suspension stable in colloids, e.g. serum, albumin and gelatin containing solutions. Depending on the amount of stabilizer present in the ready-to-use reagent, the serum to be tinned may be used in a saline environment or other suitable medium. The duration of use depends on the antigen used from hours to days. An improvement in shelf life can be achieved by freeze drying.

It is furthermore known from DK Publication No. 126,452 and the article by N.R. Ling in Brit. J. Haemat., 7 (1961), pages 299-302, to combine the aldehyde treatment of erythrocytes with a treatment with tanning agent tannin.

It has also been described already to treat erythrocytes simultaneously with chromium (III) chloride and an antigen, whereby the antigen is coupled to the erythrocytes.

The coupling of antigen to native bed erythrocytes by various authors by the simultaneous action of antigen and chromium (III) chloride has significant disadvantages because the chromium salts affect both the erythrocyte surface and the added antigen and thus make it difficult to optimize the reagent.

In the preparation, storage and use of antigen-coated erythrocyte preparations, a number of further problems have not been solved, e.g. the just mentioned optimal setting of the detection sensitivity of hemagglutination antigens. Also, regarding the stability of the erythrocytes under storage conditions and reproducibility, sometimes the haemagglutination studies thus conducted leave something to be desired.

Therefore, there was a need to prepare for coating with antigens suitable erythrocyte preparations which, by the quantitative determination of haemagglutination studies, provide reliable results that can also be reproduced by long-stored preparations.

The present invention relates to a process of the kind set forth in the preamble of the specification, characterized in that the lower aliphatic aldehyde and a water-soluble salt of di- or trivalent chromium have the effect of affecting those in the aqueous solution. isotonic solution suspended erythrocytes and, if desired, lyophilizes.

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The erythrocyte compositions prepared by the method of the invention exhibit the following advantages over the prior art: 1. The optimum antigen concentration for binding an antigen to the erythrocytes prepared by the process can be more simply determined.

2. The erythrocytes prepared by the process of the present invention are better suited to bind smaller, non-dissolved particulate antigens.

3. The erythrocytes prepared and coated with an antigen by the process in question settle more rapidly, so that the time required for reaction with the specific antibodies is shorter.

4. The erythrocytes prepared and sensitized by the method in question exhibit a greater sensitivity, especially to antibodies from immunoglobulin class M.

5. The stability of reagents containing the suspension erythrocytes prepared by the present process is better.

As erythrocytes, nuclear bird erythrocytes are preferably used. The use of cell-nuclear bird erythrocytes promotes sedimentation and at the same time a more simple readable sedimentation pattern. This provides a quick and safe assessment of the reaction.

Above all, the treatment of the cells with a chromium salt significantly improves their storage capacity by approx. 37 ° C. A further advantage of the erythrocytes treated according to the invention lies in the low tendency for nonspecific positive reactions, whereby in most cases the use of a control reagent becomes superfluous. In particular, the treated poultry erythrocytes are characterized by a rapid, sensitive and specific responsiveness by hemagglutination tests and by a stability in liquid and lyophilic dried form.

Those with aldehydes and chromium salts pretreated poultry throocytes are suitable for adsorption of various bacterial, parasitic and viral antigens and also enable the storage of antibodies for detection of antigens.

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For use in the method of the invention, native whole blood erythrocytes are collected from various animals as well as humans, preferably, however, nucleated, such as from birds, e.g. chickens, pigeons and turkeys. To the blood samples are customary anticoagulants added, on which the erythrocytes, e.g. is recovered by centrifugation and washed several times down compatible isotonic buffer solutions to remove the antigens present in the blood. The washed erythrocyte sediment is suspended in not less than 8 volumes of an isotonic neutral buffer solution or an isotonic saline solution. To this suspension is added an aqueous solution of a lower aliphatic aldehyde, preferably an aldehyde having a chain length of from 1 to 6 carbon atoms, e.g. formaldehyde, methylglyoxal or glutaraldehyde, in an amount of 0.2-10% by weight of the aldehyde attributed to the residual volume of the erythrocyte sediment. The reaction time for the effect of the aldehyde on the erythrocytes depends on the reaction temperature and the concentration of the aldehyde used. Conveniently, such reaction conditions are observed which cure the erythrocytes without forming aggregates. The aggregate formation can be observed microscopically.

In particular, reaction times of 1 to 24 hours are used at temperatures of 5 to 45 ° C. According to particularly preferred conditions using formaldehyde, which is commercially available as an approx. 35% 1s solution, bring to 10-15% diluted formaldehyde solution to effect the erythrocyte suspension. In detail, the suggestion may be made by L. Csizmas, Proc. Exp. Biol. With. 103, 157 et seq. (1960). Example 1 described illustrates the principle of procedure.

In addition to the aldehyde treatment, the erythrocytes are washed several times. If they are not to be further processed, an antimicrobial active agent may be added to the thus-treated erythrocytes, e.g. sodium timon phonate at a concentration of 0.01-0.05% and can be stored as 10-30%, preferably 25% suspension at 4 ° C.

Prior to treatment with a chromium-II or chromium-salt, the aldehyde-treated erythrocytes are suspended in an aqueous environment. Suitably water is used for this. The concentration of the erythrocytes is 0.5-25%, preferably 1.25%. To this erythrocyte suspension, an aqueous chromium salt solution is added at a concentration of 0.0001-2.0% and, after mixing, is allowed to stand for 5-30 minutes. at 5-45 ° C.

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Then the erythrocytes are separated by sedimentation or centrifugation. To remove excess chromium salt, wash several times. As chromium salts for use in the process of the invention, all water-soluble chromium-II and chromium-III salts are present, e.g. chromium II or chromium III chloride, sulfate, nitrate, phosphate or acetate.

The order of use of aldehyde and chromium salt can be exchanged without undue influence on the result. Aldehyde and chromium salt can also be used simultaneously with good results.

The formaldehyde and chromium salt-treated erythrocytes thus obtained are suitable for coating with various antigens.

Generally, the stabilized erythrocytes are coated with antigens in an aqueous solution adjusted to a pH below 7, suitably to a slightly acidic pH between 5.5 and 6.5. Among the selected antigens, for example, which can be successfully used to coat the stabilized erythrocyte preparations, in particular, are protein antigens, especially the plasma protein antigens of human or animal origin, microbial antigens, e.g. toxoplasma antigens, as obtained by ultrasound, or Treponema pallidum antigens recovered from infected rabbit testicles. It is a particular advantage of the erythrocyte preparation of the present invention that it is suitable for coating with antigens of different chemical structure.

The stabilized erythrocytes coated with antigens are conveniently suspended in a solution containing a lyophilized colloid, e.g. a 0.1-1% gum arabic solution or in a corresponding solution of a gelatin degradation product or polyvinylpyrrolidone appropriate in the presence of additional stabilizers, e.g. amino acid such as sodium glutaminate, or glycine at a concentration of 1-10% and / or in an animal serum at a concentration of 0.5-20%. In this suspension liquid, the reagent can be stored liquid at refrigerator temperature, ie at approx. 4 ° C or can be freeze-dried after freezing. After dissolving the dried composition in distilled water, the reagent is available for the conduct of haemagglutination tests of the same quality as was the case before the drying.

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The conduct of the haemagglutination studies is known to one skilled in the art. Usually, a serum sample containing the putative antibodies is mixed with the corresponding antigen-coated erythrocyte preparation in an aqueous environment to determine if an agglutination occurs. The agglutination reaction is advantageously carried out on microtiter plates. For this, e.g. 0.05 ml of serum or serum dilution with 0.025 ml of the erythrocyte preparations according to the invention after coating with antigen (1.25%) and shaking for 30 sec. to 1 min. By using the above-described method for preparing coated erythrocytes, the reading of the test results can be carried out already after 30 minutes. progress.

As noted above, the erythrocyte preparations stabilized by the process of the invention can be coated with protein antigens. This means that they can also be coated with a specific antibody, so that in the subsequent haemagglutination study, the erythrocytes agglutinate in the presence of the corresponding specific antigen. The passive hemagglutination test can be used to detect bacterial infections, to detect viral infections, to determine histocompatibility, and to detect autoimmune diseases.

The process according to the invention will be further elucidated by the following examples.

Example 1 (a) Stabilization of the erythrocytes 300 ml of chicken blood is captured in 100 ml of 3.5% citrate solution and processed on the same day.

The erythrocytes are centrifuged at ca. 2000 x g and washed 5 times in 10 volumes of cold (about 10 ° C) 0.85% sodium chloride solution. This avoids foaming. The washed cell sediment is suspended in 8 volumes of 0.85% sodium chloride solution with a pH of 6.8. 1/4 of the volume of a cell suspension of a 14% formaldehyde solution of pH.5.5 is poured out. in a cellophane dialysis tube (width 2.5 cm) and closed at one end, thus keeping about 1/3 of the tube empty. The cup is stirred at room temperature with a mechanical shaker, the speed is adjusted so that a strong mixture is obtained with as little foaming as possible. for 16-18 hours, then clumped cell residues are found on the surface of the liquid and on the cup walls, which can be removed by gentle decanting.

To the homogeneous cell suspension is poured half a volume of isotonic sodium chloride solution and then washed 6 times with 750 ml each time. Then, the washed, formalinized cell sediment is suspended in a phosphate buffered saline solution of pH 7.2 (PBS) with 0.02% sodium timon phonate to form a 25% solution stored at 4 ° C.

For treatment of the aldehyde-treated, washed, stabilized erythrocytes with CrCl 2, they are suspended for approx. 1: 4-diluted buffered saline solution of pH 7.5. To this suspension is added the same volume of an aqueous chromium (III) chloride solution at a concentration of 0.002% and the mixture is allowed to stand for 10 minutes. at 37 ° C. The chromium salt is then removed by washing several times with a physiological boiling salt solution.

b) Coating with antigen.

1. An ultrasonic effect (2x1 min. 22 KHz) from toxoplasm prepared and centrifuged for particulate constituents purified antigen extract is added in suitable dilution determined by a preliminary experiment with the aldehyde chromium (III) chloride treated erythrocyte suspension (1, 25% s) and allowed to operate for 1 hour at 37 ° C. The pH is hereby kept at 6.4.

Then, wash twice in a 0.1-1% gum arabic solution and suspend the sediment im / 15 trishydroxy-methyl-aminomethane-HCl buffer with pH 8.0 to which 5% sodium glutaminate is added. and 15% rabbit serum.

The suspension contains 1.25% erythrocytes.

In this suspension and stabilizer liquid, the reagent can be stored liquid at 4 ° C or freeze-dried. The solution takes place in the original volume of distilled water.

2. Similarly, a Lues reagent can be prepared by using, instead of a toxoplasma ultrasound extract, an extract of Treponema pallidum of infected rabbit testicles and conducting the serum dilution in an absorption medium.

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Example 2

Freshly recovered sheep erythrocytes are suspended in twice the volume of an Alsever's solution of the following composition: 20.5 g dextrose 8 g sodium citrate (Na 2 gH 2 C 2 · 2H 2 O) 0.552 g citric acid (H ^ CgHgO ^ Γ H 2 O) 4.2 g sodium chloride in 1000 ml with distilled water.

Then the erythrocytes are centrifuged at ca. 2000 x g and washed five times in 10 volumes of cold phosphate buffered 0.15 molar saline solution with a pH of 7.2. After washing, the erythrocytes are suspended to a concentration of 8% (v / v) in a 0.15 molar buffered saline solution with a pH of 7.2. To a volume portion of this erythrocyte suspension, the same volume of a 3% glutardialdehyde solution in a buffered boiling salt solution having a pH of 7.2 is added dropwise with slow stirring of the cell suspension. Then, stir for 17 hours at room temperature, wash the suspension five times with a buffered saline of pH 7.2 and resuspend the aldehyde-treated erythrocytes in a saline solution to form a 15% (volume / volume) suspension of the in this way erythrocytes stabilized.

For the treatment of the aldehyde-treated, washed, stabilized erythrocytes with chromium (II) chloride, they are suspended in a ca. 1: 4-diluted buffered saline solution of pH 7.2. To this suspension is added the same volume of an aqueous chromium (II) chloride solution at a concentration of 0.05% and the mixture is allowed to stand at 37 ° C for 30 minutes. The chromium salt is then removed by washing several times with a physiological boiling salt solution.

The coating with antigen can be done in the same manner as described in Example 1b).

Suitable erythrocyte preparations for coating with antigen are also obtained when, instead of glutaraldehyde, as in the present example, methylglyoxal is used.