IE44471B1

IE44471B1 - Stable preparation of erythrocytes process for preparing it and its use

Info

Publication number: IE44471B1
Application number: IE2494/76A
Authority: IE
Original assignee: Behringwerke Ag
Priority date: 1975-11-14
Filing date: 1976-11-12
Publication date: 1981-12-16
Also published as: NL189133B; AU1958876A; BR7607564A; DE2551208C3; JPS616941B2; MX3937E; BE848377A; AR213420A1; IT1063992B; DK146138C; JPS5261218A; AU511427B2; DK146138B; CH626999A5; SE7612622L; ATA843476A; DE2551208A1; ES453136A1; SE431800B; AT356812B

Abstract

Stable lyophilisable erythrocyte preparations are obtained by treating erythrocytes which are suspended in aqueous isotonic solutions with a lower aliphatic aldehyde and a water-soluble salt of 2- or 3-valent chromium. The treatment with aldehyde and the chromium salts can take place successively in any desired sequence or simultaneously. The treated erythrocytes are washed and, if required, lyophilised. The resulting stabilised erythrocyte preparations can be coated with antigen and antibodies too. The coated erythrocytes can be employed for haemagglutination tests.

Description

This invention relates to stabilized erythrocytes, suitable for being coated with an antigen or an antibody.

The passive or indirect haemagglutination test has been used for a long time for visualising antibodies. For this purpose, blood corpuscles of various animal species, mostly of sheep, have been used as carriers for the antigens, which are themselves soluble. Since native erythrocytes are stable for a short period of time only, attempts have been made to prolong the useful life of the cells by treating them with an aldehyde, for example, formaldehyde, pyruvaldehyde, or glutardialdehyde, or with sulphosalicylic acid. Cells created thus may be conserved for a rather longer time than can native erythrocytes, but must be charged with the antigen prior to use.

Such erythrocytes charged with an antigen are generally stable only when suspended in a solution containing a colloid, for example, serum albumin, or gelatin. Depending on the amount of the stabilizing substance already present in the reagent ready for use, the serum to be tested can be used either in a sodium chloride medium or in other suitable media. The reagent may be used for several hours to several days, depending on the antigen. The stability may be improved by lyophilization.

It has also been proposed to treat erythrocytes simultaneously with chromium(III)chloride and an antigen, whereupon the antigen is coupled to the erythrocytes. Several authors have carried out this method using native ovine ί 4 4 ’? 'ΐ erythrocytes, but this method has considerable disadvantages since the chromium salts act on the surface of the erythrocyte3 and on the antigen, thus rendering difficult an optimum standardization of the reagent.

The difficulty of standardisation is one of a series of problems not yet solved, which include the stability of the erythrocytes during storage and the reproducibility of the haemagglutination tests, which has not always been satisfactory.

The present invention provides a process for preparing a stabilised preparation of erythrocytes, wherein a suspension of erythrocytes in an aqueous isotonic solution is reacted with, simultaneously or successively in any sequence, an aliphatic aldehyde and a water-soluble salt of 2- or 3- valent chromium, the erythrocytes are washed and, if desired, lyophilized. After washing, an antigen may be applied, if desired, to the resulting composition of erythrocytes.

Preferred erythrocytes are avian erythrocytes, which contain a cell nucleus. The use of these avian erythrocytes accelerates sedimentation and also enables the sedimentation patterns to be read more easily, thus permitting the reaction to be followed more rapidly and reliably.

The treatment of the erythrocytes with a chromium salt considerably improves, in particular, their storability at about 37°C. A further advantage of the erythrocytes treated according to the invention is the very low tendency to give unspecifically positive reactions, which makes superflous in many cases the use of a control reagent. Avian erythrocytes treated according to the invention in particular have a rapid, sensitive and specific reactivity in haemagglutination tests and an improved stability in a liquid or lyophilized form. - 4 Erythrocytes pre-treated with an aldehyde and a chromium salt are suitable for the adsorption of antigens of all origins, for example, bacterial, parasitic and viral antigens and also, conversely, the addition of antibodies for the identification of antigens.

For the process of the invention, the erythrocytes are generally prepared as follows: Native erythrocytes are collected from whole blood of animals, including humans. Nucleus-containing erythrocytes of birds, for example, chickens, pigeon and IO turkeys are preferred. The sample of blood is mixed with a conventional coagulation inhibitor, and the erythrocytes are then separated from the other constituents of the blood, for example, by centrifugation, and are then washed several times with a compatible isotonic buffer solution in order fco remove the antigens present in the blood. The washed sediment of erythrocytes is suspended in not less than S parts by volume of an isotonic neutral buffer solution, or an isotonic salt solution.

The process of the invention is then preferably carried out as follows:- To the above suspension an aqueous solution of an aliphatic aldehyde preferably one having from 1 to 6 carbon atoms, for example, formaldehyde, methylglyoxal and glutaraldehyde, is added in an amount of from 0.1 to 10 percent by weight, calculated on the volume of the sediment of erythro25 oytes. The time required for tha reaction of the aldehyde with the erythrocytes depends on the reaction temperature and the concentration of the aldehyde. Suitable reaction conditions are those under which the erythrocytes are hardened without aggregates being formed. (The formation of aggregates can be observed microscopically). The reaction times generally range from 1 to 24 hours at temperatures of from 5° to 45°C. 4 4 7 -i Formaldehyde is readily commercially available as an about 35% solution, so for this reason it is generally used. Such a formaldehyde solution is preferably diluted to about 10 to 15% and then reacted with the suspension of erythrocytes. (For details of a suitable procedure see L, Csizmas, Proc, Exp. Biol. lied, 103, 157 et seq. (I960)).

After treatment with the aldehyde the erythrocytes ars generally washed several times. If they are not to be further processed immediately, the erythrocytes thus pretreated may be mixed with an antimicrobially active agent, for example.- sodium timerfonate in a concentration of from 0.01 to 0.05% and stored at 4°C as a 10 to 30%, preferably 25% suspension.

For the treatment with a chromium-II or ehroraium-III salt the erythrocytes treated with aldehyde are suspended in an aqueous medium, generally water, and preferably in a concentration of from 0.5 to 25%, advantageously 1.25% (v/v). To this suspension of erythrocytes an aqueous solution of a chromium salt is added, preferably to give a concentration thereof in the suspension of from 0.001 to 2,0% (w/v), and after mixing, the reaction mixture is allowed to stand for 5—30 minutes at 5°C to 45°C. The erythrocytes are then separated, generally by sedimentation or centrifugation. To remove an excess of chromium salt they are washed several times. The chromium salts which may be used according to the invention are all the watersoluble chromium-II and chromium-XII salts, for example, chromium-II or chromium-III, chloride, -sulphate, -nitrate, -phosphate or -acetate.

The order of treatment with the aldehyde and the chromium salt may be reversed without any detrimental effects on the result. Furthermore, the aldehyde and the chromium salt may be used simultaneously with the same good result. 44473 - 6 The erythrocytes treated according to the invention are suitable for coating with various antigens.

The stabilized erythrocytes are generally coated with an antigen in an aqueous solution of pH 7 or less, preferably a slightly acidic pH of from 5.5 to 5.5. The antigens used are, in particular, those substances whose identification in blood or urine is important, for example, protein antigens, especially the plasma-protein antigens of human, or animal origin, microbial antigens, for example, toxoplasma antigens obtained, for example, by ultrasonics, and treponema pallidum antigens obtained from infected rabbit testicles. The special advantage of the preparation of erythrocytes of the invention is that the erythrocytes are suitable for coating with substances having various chemical structures.

The stabilized erythrocytes coated with antigens, if not intended for immediate use, are preferably suspended in a solution containing a lyophilic colloid, for example, gum arabic, a gelatine degradation product, or polyvinylpyrrolidone, for example, a 0.1 to 1% solution of gum arabic or a corresponding solution of a gelatine degradation product or polyvinylpyrrolidone, preferably in the presence of a further stabilizing substance, for example, an amino acid or a salt thereof for example, sodium glutamate or glycine, preferably in a concentration of 1 to 10%, and/or an animal serum, preferably in a con25 centration of 0.5 to 20% (v/v). In this form, the coated erythrocytes can be stored at refrigerator temperature (about 4°C). If desired, a suspension of the coated erythrocytes can be lyophilized. After dissolving the lyophilized preparation in distilled vzater the coated erythrocytes are ready for haemagglutination tests, and have the same quality as before lyophilization. 4 4 '7 i - 7 The performance of the haemagglutination test is known to the expert: Generally, a serum sample which may contain the antibody under investigation is mixed in an aqueous medium with a preparation of erythrocytes coated with the corresponding antigen to see if agglutination takes place. The agglutination reaction is generally carried out on micro-titre plates, for example, 0.05 ml of serum or a diluted serum, or urine if appropriate, is mixed with 0.025 ml of the erythrocytes coated with antigens (1.25% v/v) and shaken for 30 seconds to 1 minute. When using the coated erythrocytes cf the invention, tha test results may be read off after 30 minutes.

As described above, the preparations of erythrocytes stabilized according to the invention may be coated with protein antigens. This means that they may be coated with a specific antibody, so that in the haemagglutination test the erythrocytes agglutinate in the presence of the corresponding specific antigen. The passive haemagglutination test may be used, for example, to make evident bacterial infections, to detect virus infections, to determine histocompatibility, to detect auto immunity diseases, and in pregnancy testing. In the latter case the erythrocytes are coated with an antibody directed against a substance whose appearance or change in concentration in blood or urine is characteristic of pregnancy, for example, pregnancy-specific β^-glycoprotein, which is described and claimed in British Patent Specification No. 1,410,338.

The invention'also provides a kit which comprises a sample of stabilised, antibody-or antigen-coated erythrocytes of the invention together with a vessel in which to carry out the test, generally a micro-titre plate, and instructions for the use of the kit. The sample of erythrocytes is preferably 0.025 ml of a 1.25% (v/v) suspension, optionally in lyophilized form. This - 8 amount of erythrocytes is to be mixed with 0.05 ml of serum or a suitably diluted serum, or urine, as described above.

The kit is particularly useful as a pregnancy-testing kit i.e. when the erythrocytes are coated with an antibody fco a substance characteristic of pregnancy, for example, the β^-glycoprotein mentioned above.

The following Examples illustrate the invention.

In them, and throughout the specification, percentages are calculated as weight by volume, unless stated otherwise.

Example 1: a) Stabilization of the erythrocytes 300 ml of chicken blood were collected in 100 ml of a 3.5% citrate solution. The further processing was carried out on the same day.

The erythrocytes were centrifuged at about 2,000 g and washed five times in 10 volumes of a cold 0.85% sodium chloride solution at about 10°C, avoiding the formation of foam. The washed cell sediment was suspended in 8 volumes of a 0.85% sodium chloride solution having a pH value of 6.8. A quarter of the volume of the cell suspension of a 14% formaldehyde solution of pH 5.5 was poured into a Cellophane dialysis tube (width 2.5 cm) and sealed at one end. (Cellophane is a Trade Mark). A third of the tube was left empty. The air was pressed out, the open end tied up, the tube was placed on the bottom of a beaker and the suspension of erythrocytes poured over it.

The beaker was rotated at room' temperature on an orbital shaker at such a speed that there is achieved a thorough mixing effect with the least possible formation of foam. After three hours the cellophane tube was removed, the contents poured into the beaker and shaken for another 16 to 18 hours, whereupon clotted cell debris was found on the surface of the liguid and on the - 9 inner walls of the beaker. This debris was removed continuously by decantation.

An isotonic sodium chloride solution was poured onto the homogeneous cell suspension, the volume of the sodium chloride solution being half that of the suspension, and the cells x-rsrs washed 6 times with 750 ml of isotonic sodium chloride each time. Finally, the washed formalinized cell sediment was suspended in a sodium chloride solution (pH 7.2) buffered wiuh phosphate, and containing 0.02% of sodium timerfonate to give a 25% (v/v) suspension, which was stored at 4°C.

The aldehyde-treated, washed and stabilized erythrocytes were subsequently suspended in a buffered sodium chloride solution of pH 7.5 that had been diluted in a ratio of «bout 1:4. To this suspension an equal volume of ar. aqueous chromiumIII chloride solution was added to give a concentration of 0.002% and the mixture was allowed to stand for 10 Kiiuutes at 37°C. The chromium salt was then removed by washing several times with a physiological sodium chloride solution, b) Coating with antigen 1. An antigen extract obtained by the action of ultrasonics (2x1 min. 22 kHz) from toxoplasmae and purified by centrifugation of particle constituents was mixed in a suitable dilution which had been determined in a preliminary test, with a 1,25% (v/v) suspension of erythrocytes treated with an aldehyde and chromium-IIX chloride as described above, and it was allowed to stand for one hour at 37°c. The pH value was maintained at 6.4.

The mixture was then washed twice in a 0,1 to 1% solution of gum arabic and the sediment was suspended in M/15 trishydroxymethyl - aminomethane - HCl - buffer of pH 8.0 to which 5% of sodium glutaminate and 15% (v/v) of rabbit serum had been addad.

The suspension contained 1.25% (v/v) of erythrocytes.

In this suspending and stabilizing liquid the coated erythrocytes may be stored at +4°C, or the suspension may be lyophilized. Dissolution in the latter case is effected in the original volume of distilled water. 2, In a corresponding manner a Lues reagent was prepared by using instead of an ultrasonics extract of toxoplasmae an extract of Treponema pallidum of infected rabbit testicles and by carrying out the serum dilution in an absorption medium.

Example 2: Freshly obtained sheep erythrocytes were suspended in twice their volume of Alsever's solution having the following composition: .5 g of dextrose g of sodium citrate (Ife2C6H5°7 · 2H2°> 0.552 g of citric acid (H,C_H,0_ . H,0) b i> 7 i 4.2 g of sodium chloride ad 1000 ml with distilled water.

The erythrocytes were then centrifuged at about 2,000 g and washed five times in 10 parts by volume of a cold 0.15 molar sodium chloride solution (pH 7.2) buffered with phosphate.

After washing, the erythrocytes were suspended to give a concentration of 8% (v/v) in a 0.15 molar buffered sodium chloride solution of pH 7.2. To one part by volume of this suspension of erythrocytes the same volume of a 3% glutardialdehyde solution in a buffered sodium chloride solution of pH 7.2 was added dropwise while slowly stirring the cell suspension.

The mixture was then stirred for 17 hours at room temperature, the suspension was subsequently washed five times with buffered -5 d G 7 Λ sodium chloride solution of pK 7.2, and the -arythroc/fee treated with aldehyde were resuspended in a a odium ch 1 ί d : solution, so that a 15% (v/v) suspension cf the thus stab’1’sad erythrocytes is obtained.

The washed, stabilized erythrocytes treated with thi aldehyde were treated with chromium-II chloride by suspending them in a buffered sodium chloride solution of pH 7.5 dilutad in a ratio of about 1:4. To this suspension an equal volume of an aqueous chromium-II chloride solution was added vc gi-.s a concentration of 0.05%, and the mixture was allowed to stand for 30 minutes at 37°C. The chromium salt was then removed by washing several times in a physiological sodium chloride solution.

The coating with antigen may be effected in the same way as described in Example lb.

Example 3: The procedure of Example 2 was carried out except cia; methylglyoxal was used instead of glutardialdehyde.

Claims

1. CIAIMSs1. A process for preparing stabilized erythrocytes, wherein a suspension of erythrocytes in an aqueous isotonic solution is reacted With, simultaneously or successively in any sequence, an aliphatic aldehyde and a water-soluble salt of 2- or 3- valent chromium, and the erythrocytes are subsequently washed.

2. A process as claimed in claim 1, wherein the lower aliphatic aldehyde is an aldehyde having from 1 to 6 carbon atoms.

3. A process as claimed in claim 2, wherein the aldehyde is formaldehyde, methylglyoxal, or glutaraldehyde.

4. A process as claimed in any one of olaims 1 to 3, wherein the aldehyde is used in a concentration of 0.1 to 10% by weight, calculated on the volume of the erythrocytes when sedimented.

5. A process as claimed in any one of olaims 1 to 4, wherein the aldehyde and the erythrocytes are reacted at a temperature within the range of from 5° to 45°C.

6. A process as claimed in any one of olaims 1 to 5, wherein the chromium salt is used in a concentration of from 0.001 to 2% (w/v).

7. A process as claimed in claim 6, wherein the chromium salt is chromium-II chloride or chromium-III chloride.

8. A process as claimed in claim 6 or claim 7, wherein the reaction is carried out at a temperature of from 5 to 45°C.

9. A process as claimed in claim 1 or any one of claims 6 to 8, wherein the erythrocytes are first treated with the aldehyde, then washed, and are then suspended in an aqueous medium in a concentration of from 0.5 to 25% (v/v) prior to treatment with the chromium salt.

10. A process as claimed in any one of claims 1 to 9, wherein the erythrocytes are avian erythrocytes. 5

11. A process as claimed in claim 1, wherein the resulting stabilized erythrocytes are lyophilized.

12. A process as claimed in claim 1, carried out substantially as described in any one of the Examples herein.

13. Stabilized erythrocytes whenever prepared by a 10 process as claimed in any one of claims 1 to 12.

14. Stabilized erythrocytes as claimed in claim 13 and which have been coated with an antigen or an antibody.

15. A process for producing coated, stabilized erythrocytes as claimed in claim 14, which comprises coating a suspen15 sion of stabilized erythrocytes as claimed in claim 13 in an aqueous medium having a pH of 7 or less with an antigen or an antibody and washing the coated erythrocytes.

16. A process as claimed in claim 15, wherein the coated erythrocytes are suspended in a. medium comprising a lyophilic 20 colloid.

17. A process as claimed in claim 16, wherein the colloid is gum arabic, a gelatine degradation product, or polyvinylpyrrolidone.

18. A process as claimed in claim 16 or claim 17, 25 wherein the suspension medium also comprises one or more further stabilizing substances selected from amino acids and salts thereof, and animal sera,

19. A process as claimed in claim 18, wherein the concentration of an amino acid or a salt thereof is from 1 to 10% and/or the concentration of an animal serum is from 0.5 to 20'% (v/v).

20. A process as claimed in any one of claims 15 to 19, wherein the concentration of coated erythrocytes in the suspension is from 0.5 to 25% (v/v).

21. A process as claimed in claim 20, wherein the concentration of coated erythrocytes is 1.25% (v/v).

22. A process as claimed in any one of claims 15 to 21, wherein the antigen is a toxoplasma antigen or a Trepona pallidum antigen.

23. A process as claimed in any one of claims 15 to 21, wherein the antibody is specific against a bacterial or a viral antigen.

24. A process as claimed in any one of claims 15 to 21, wherein the antibody is suitable for determining histocompatibility or autoimmunity.

25. A process as claimed in any one of claims 15 to 21, wherein the antibody is directed against any substance that appears in the blood or urine during pregnancy or whose concentration in the blood or urine changes characteristically during pregnancy.

26. A process as claimed in claim 25, wherein the antibody is directed against pregnancy-specific β^-glycoprotein.

27. A process as claimed in any one of claims 15 to 26, wherein the coated erythrocytes are lyophilized.

28. A process as claimed in claim 15, carried out substantially as described in Example 1 herein.

29. Antibody- or antigen- coated eryfchr·- y1=’ prepared by a process as claimed in any one of claiv;, λ

30. A kit for performing a passive haemagylutics. test, which comprises a sample of coated eryth.rooyr»ij 5 claimed in claim 14 or claim 29, together with R which to carry out the test, and instructions for the u kit.

31. A kit as claimed in claim 30, wherein the να is a micro-titre plate. 10

32. A kit as claimed in claim 30 or claim 31, the sample of coated erythrocytes is 0.025 ml o£ a 1.1-. suspension of erythrocytes.

33. A kit as claimed in any one of claims 30 to wherein the erythrocytes are coated with an antibody 3.15 in claim 25 or claim 26.

34. A kit as claimed in claim 32 or claim 33,. the erythrocytes are lyophilized.