WO1979000256A1

WO1979000256A1 - A method of producing a stable preparation having immunoglobulin-binding properties

Info

Publication number: WO1979000256A1
Application number: PCT/SE1978/000066
Authority: WO
Inventors: U Jonsson
Original assignee: U Jonsson
Priority date: 1977-10-31
Filing date: 1978-10-31
Publication date: 1979-05-17
Also published as: DE2857506A1; EP0007953A1; SE7712244L; FR2472612A1; CH649385A5; GB2050386B; GB2050386A

Abstract

A method of producing a stable bacterial preparation having nonspecific immunoglobulinbinding properties. The bacteria, e.g. Staphylococcus aureus or Streptococcus pyogenes, with a nonspecific immunoglobulin-binding component, such as protein A of Staphylococcus aureus, on the surface of the bacteria, are grown in a substrate wherein the bacteria form the immunoglobulinbinding component. Then the bacteria are killed and lyophilized, the lyophilization step being carried out soon after killing of the bacteria. After reconstitution the lyophilized bacteria are used as reagent in immunological reactions.

Description

A METHOD OF PRODUCING A STABLE PREPARATION HAVING IMMUNOGLOBULIN-BINDING PROPERTIES

In most strains of Staphylococcus aureus there is a protein, designated protein A, in part as a sub¬ stance bound to the^'exterior of the cell^" wall, and in part as a soluble substance excreted into the growth medium of the bacteria. Originally, protein A was procured in soluble form after initial heat 0 extraction from washed bacteria. By heating suspended staphylococci close to the boiling point, cell-wall protein A was solubilized. In the middle of the 1960 's it was demonstrated in a number of different ways that antibodies from man and many mammals , which can be shown to react with soluble protein A in such a way that a precipitate appears, take part in the reaction not- because of previous immunization result¬ ing in antibodies directed to structures of protein A - a reasoning that would be sensible considering 0 the general accurence of Staphylococcus aureus in the environment .- but because of protein A having at least one receptor for a structure present on the Fc part of a large fraction of antibody molecules (immunoglobulin) in man and all mammals, irrespective 5^' of what^"these^"protein A-reactive aήtiB HTes^™are^" d re^'c"-^' ted to by virtue of the antigen-binding_, structures of their Fab parts. In man and certain mammals such as

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BAD ORIGINAL . also the capacity to bind antibody molecules very rapidly, should they already have bound antigen or not. It should be made clear that an IgG-antibody molecule, although attached to protein A by its Fc 5 part, may effectively bind antigen by one or both of its Fab parts. -A particularly illustrative way of using protein A-containing staphylococci as a reagent in im unological laboratory work is to apply them as a so-called separation reagent in radio-

10 _ immunoassays (RIA) .

Radioim unoassay is the designation of a nowadays classical radioimmunological method of identification by which it is possible to make quantitative determinations of a given substance

15 (antigen) , also when it is present in very low amounts, by arranging for competitive inhibition of the reaction between fixed amounts of the same antigen, which has been purified and labelled with a radioactive substance such as radioactive iodine,

20 and of antibodies, e.g. from rabbit or guinea pig, directed to the same substance. When this reaction is approaching equilibrium or has been running for a period which according-^'to experience is sufficient - varying from 4 days to less than 15 minutes - the

25 labelled antigen is separated into a free and an antibody-bound fraction, respectively.

For this separation step a great number of methods have been applied, e g. chemical precipitation with reagents such as ammonium sulphate and ethanol,

30 which require a chemical difference between free

(labelled) antigen and antigen-antibody complexes, and the already classical double antibody method, in which the specific antibody, e.g. from rabbit, is precipitated immunologically after the addition of an

35 antiserum directed to rabbit immunoglobulin. Problems

encountered in the latter procedure include the long time required for precipitation, sometimes 24 hours, the relative sensitivity to irrelevant disturbing factors, and the fact that the separation - termin¬ ated with centrifugation and elimination of the supernatant fluid -^"hence may be incomplete.

However, protein A-containing staphylococci, added as a finely dispersed suspension, have been demonstrated to reach the end-point in their reaction with immunoglobulin within one minute. Then, centri¬ fugation is performed, the supernatant fluid is decanted, and the tube is brought to a gamma radiation counter for the determination of the anti¬ body-bound fraction of labelled antigen, associated with the bacteria on the tube bottom.

The preparations of staphylococci first applied for the purposes mentioned usually originated from a broth culture, the bacteria being treated by repeated washing in a centrifuge, resuspension in a 0.5 % (v/v) formalin solution in neutral buffer, where the staphylococci were incubated for 3 hours under constant mixing, and final repeated washing in a centrifuge. The resulting suspension was stored in neutral phosphate-buffered saline with some pre¬ servative added such as sodium azide (NaN , 1 gram per liter. The formalin-treatment of the staphylo¬ cocci, performed as first described by Lind and Mansa, was intended to "fix" the protein A-containing super¬ ficial layers of the bacteria, and in this way the spontaneous extraction of protein A to the surround¬ ing suspension medium was delayed. There was, how¬ ever, no killing of the bacteria, and after two weeks storage in the refrigerator, the bacteria tended to autolyse. Furthermore, just before use the required amount of reagent had to be washed again.

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, S ty_Λ - W1PO The drawbacks mentioned above and also the fact that potentially pathogenic, living bacteria were used, stimulated developmental work, initiated by G. Kronvall and based primarily on the obvious heat stability of protein A, which had been demonstrated by said heat extraction method previously applied for the production of soluble protein A, but also on the possibility of fixing protein A with formalin tp the cell wall during heat treatment, which would tend to liberate protein A from it. The result has been described in the major paper about the first radioimmunoassay with staphylococci as a separation reagent, co-authored by S. Jo^'nsson and G. Kronvall: The use of protein A-containing Staphylococcus aureus as a solid phase anti-IgG reagent in radio- immunoassays as exemplified in the quantitation of alpha-foetoprotein in normal human serum, Eur. J.. Immunology 4:29, 1974. In this paper there is a description of heat treatment, reproducible as to temperature and time and intended to kill the for¬ malin-treated staphylococci (Staphylococcus aureus) strain Cowan I, efficiently, and this method was performed by pumping such a suspension at a given velocity through a relatively narrow metal tubing immersed in a waterbath at a temperature of 80 C, and then directly to a similar metal tubing immersed in an ice water bath or a refrigerated waterbath. In this set-up the volume of-the total tubing in_. which the suspension is kept at 80 C, and the pumping velocity determined the so-called keeping time; 4 - 6 minutes were found to be suitable at this particular temperature. In this way a suspension of killed staphylococci was obtained, which had an IgG-binding capacity of roughly 80 % of the binding capacity of staphylococci that had been formalin-treated only.

" UR O . - W1 This suspension turned out to be stable as to immuno¬ globulin-binding capacity when stored at refrigerator temperature for more than a year, but it had a tendency to fragmentation of part of the bacteria, release of protein A and yellow discoloration of the suspension medium. Also with this preparation of reagent staphylococci it is clearly necessary to wash the desired amount of staphylococci before use. When stored for more than three years in the refrigerator, the bacteria form a ucoid mass which is difficult or impossible to resuspend before use.

The object of the present invention is to simplify the production of preparations having immunoglobulin-binding properties in a drastic manner and to provide such a preparation which is stable and is easy to transport and distribute and also may be stored for a practically unlimited period without significant destruction.

The object referred to is achieved by the method according to the invention which is characterized in that bacteria of species having immunoglobulin-binding capacity beyond that based on a reaction between anti¬ gens of the bacteria and specific antibodies directed against such antigens, e.g. Staphylococcus aureus or Streptococcus pyogenes, are grown in a substrate wherein the bacteria form animmunoglobulin-binding substance, that the bacteria are killed, and that the killed bacteria are lyophilized for producing a bacteria suspension that can be reconstituted with liquid. A preparation produced in this way can be reconstituted at any time during a period of probably several years to form a suspension of killed bacteria which may be used directly without further washing, e.g. in the case of staphylococci applied in radio- immunoassay, as indicated above. As a non-limiting example the method according to the invention for producing preparations of immunoglobulin-binding bacteria belonging to the species Staphylococcus aureus will be described below. _

Staphylococci^"to be used for the purposes referred to herein are best grown in a fluid sub¬ strate, also designated nutrient medium or broth, since it often comprises an extract obtained from meat by boiling. In a laboratory scale there have been used shake flasks of about 1 litre volume and so-called fermentors of about 10 litre volume, i.e. vessels where it is possible by effective mixing, aeration, neutralisation with pH-control etc to optimize the growing conditions. There are corre¬ sponding possibilities on industrial scale by using tanks of a size up to several cubic metres. The method has been successfully applied' to material procured from 250 litres of nutrient medium in a standard fermentor situated at the Department of

Bacteriology at the Karolinska Institute in Stock- " holm, Sweden. At the end of a fermentation extended for about 8 hours after inoculation the mass of bacteria in the substrate has reached levels of about 25 grams per litre (wet weight) ; in the absence of technical failure yields of at least 15 grams per litre may be regularly expected.

In experiments, initiated and performed by the inventor, it has been demonstrated that freshly grown staphylococci can be killed directly after washing with the same pasteurization-like method as that described in the paper cited above and applied on formalin-treated bacteria. Surprisingly it turned out to be a false supposition - although it apparently has geared thoughts and actions for a long time - that formalin-treatment performed before heat-killing be a necessary prerequisite to avoid heat extraction of protein A; in fact, bacteria which had been heat- -killed directly after growth appeared to have largely the same binding capacity as formalin- and heat-treated staphylococci from the same culture or at least 90 % of the binding capacity of the latter.

In addition, the experiments of the inventor have demonstrated that the chemical composition of the substrate may have effects during the subsequent treatment of the material. Thus, it has been shown that the so-called CCY-medium, a particularly rich substrate for growing staphylococci, has a disadvan¬ tage in the case where one tries to heat-kill the bacteria directly after growth while still suspended in this substrate by keeping the bacteria at 80 C for about 4 minutes; apparently the substrate contains substance (s) which tend(s) to precipitate and/or sustain aggregation of the bacteria themselves at 80 C, e.g. proteins which denature at this tempera¬ ture. However, there are some alternative media that may be used for growing staphylococci such as the so-called TSB medium used at advantage by the inventor, just because it allows heat-killing of bacteria suspended in it at 80 C without significant precipitation or aggregation.

On the other hand, the inventor has also found that heat-killing of Staphylococcus aureus may be performed also after growth in CCY-medium avoiding the phenomena of precipitation and/or aggregation observed at heat-killing at 80 C as mentioned above, i.e. if heat-killing is performed as a non-limiting example, at 60 C during a correspondingly longer incubation time, about 30 minutes according to the inventor's experience. The transition to the killing

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Λr- Wipo , temperature should preferably take place rapidly, e.g. in a heat exchanger located in a waterbath of the desired temperature, and should not be calculated as part of the so-called keeping time (incubation time) of about 30 minutes necessary for complete killing. It has also_±>een demonstrated tfrat it is possible to heat-kill Staphylococcus aureus at 56 C without precipitation or aggregation phenomena, but total killing requires about 3 hours' incubation at this temperature, and apparently there are no additional advantages of killing bacteria for the present purposes at this lower temperature.

The inventor has also cultured various immuno¬ globulin-binding strains of Streptococcus pyogenes in so-called Todd-Hewitt broth, an established fluid medium for good yields of streptococci, and performed heat-killing at 80°C according to the same principles as for Staphylococcus aureus according to the above cited paper by Jonsson and Kronvall. Also in the case of streptococci the bulk of immunoglobulin-binding capacity present on living strains remains after direct heat-killing, i.e. without prior formalin treatment. Furthermore, also similar to staphylococci, streptococci grown and left in the rich Todd-Hewitt medium during heat-killing at 80°C form aggregates. Such effects may be minimized, also in the case of streptococci, either by exchanging the Todd-Hewitt medium for fresh buffer before-heat-killing at 80°C or by performing the heat-killing in the used growth medium at about 60 C. The latter procedure is pre¬ ferred since it implies less processing of the bac¬ teria.

Bacteria heat-killed in one of the manners mentioned above without prior formalin treatment have been washed and stored as suspensions in neutral buf- fer at refrigerator temperatures.

However, the inventor has registered a quite significant disadvantage of such bacteria suspensions, e.g. of staphylococci treated with heat only for the purpose of killing the bacteria, viz. that such sus¬ pensions after only_"a few weeks' storage-in the refrigerator, even with a chemical preservative in the buffer, degenerate through fragmentation of the particles constituted by the killed bacteria. The same problem has been observed with formalin- and heat-treated staphylococci, particularly when they have been grown under conditions giving optimal yields, such as maximal aeration and mixing. One possible explanation of such unexpected problems might be that the absolute amount (not concentration) of formalin added to the harvested bacteria may have been too small for sufficient effect on the large bacteria mass.

For various reasons it appears advantageous in some instances to effectively separate the bacteria mass from its substrate before subsequent steps; one reason already mentioned is the possibility of nega¬ tive effects from the substrate on the bacteria during heat treatment, and another may be the desire to collect from the substrate one or a number of sub¬ stances that would be destroyed by the heat killing process. Centrifugation or filtration procedures may be applied for washing of the-bacteria mass. In another situation it may be sufficient and desirable to reduce somewhat the volume of the bacteria sus¬ pension to be heat-treated without need for changing the composition of the substrate.

After heat-killing it is advantageous to separate the bacteria from their present substrate, particular- ly since the latter often contains a certain amount of immunoglobulin-binding substance in solution. Also in this case centrifugation or filtration procedures may be applied. After this, the washed bacteria are found as a concentrated suspension or as a packed, wet sediment.

Suspensions of" heat-killed - however specifi¬ cally not formalin-treated - staphylococci and streptococci were subjected to freeze-drying (lyophilization) , primarily as a means of storage of heat-killed bacteria not suitable to be stored in suspension as such, and with the aim of producing bacteria suspensions of these materials by recon¬ stituting it with a suitable buffer. In our case, after lyophilization, which takes place in vacuum, the lyophilized materials have been in contact with dry air. After the reconstitution one did expect to have suspensions with reproducible properties includ¬ ing the previous tendency of autolysis of the bacterial particles within a few weeks as in the corresponding suspensions of non-lyophilized bacteria. These expectations were substantiated as to the immunoglobulin-binding properties and the appli¬ cability for various purposes of such reconstituted bacteria. However, there was a notable absence of tendency to autolysis, an observation that has been found to be a quite surprising, unexpected and hithertofore not described effect, which may potentially have a great practical importance.

The effect of exposing the lyophilized material to dry air has not been investigated to such extent that it could be concluded that one component or the other of dry air would contribute, to the suspension stability which is a characteristic of the preparation produced according to the invention. The preparation of killed Staphylococcus aureus, grown in TSB-medium JU

O and treated according to the method given above including heat-killing at 80°C for about 4 minutes, has repeatedly been demonstrated to have a binding capacity as to human IgG of more than 1.5 mg IgG per ml of a 10 % (v/v) suspension, corresponding to at least 0.75 mg per.20 mg of lyophiliz.ed staphylo¬ cocci, usually about 2 mg and 1 mg, resp. These figures should be compared with the data given in the cited paper (Jonsson & Kronvall 1974) about formalin- and heat-treated staphylococci, the bind¬ ing capacity of which was reported to be about 1 mg per ml 10 % (v/v) suspension. In this connection it should be stressed, however, that the growing condi¬ tions were rather different from those referred to in the paper. A different substrate was used as well as an improved equipment for oxygenization with the addition of pH control.

Experiments performed by the inventor have demonstrated that equivalent preparations of immunoglobulin-binding staphylococci and streptococci may be obtained also after variations of conditions for the heat" treatment (killing) of the bacteria, the first variation being heat treatment of the bacteria at lower temperatures than 80 C. Thus, it has been found that Staphylococcus aureus of strain Cowan I will be killed after incubation at 60 C for 30 minutes, whether suspended in a nutrient broth or resuspended in a buffer, and JLargely irrespective of the density of the suspension. Correspondingly, of two immunoglobulin-binding strains of streptococci investigated at the laboratory of the inventor, one was killed after 30 minutes at 56 C while the other required up to 60 minutes at 56 C.

Moreover, it has been demonstrated by the inven- tor that heat-killing may be performed at significant- ly lower temperatures, e.g. at 37°C by adding formaldehyde to a final concentration well below the formaldehyde concentration that would be required for killing with formaldehyde at room temperature. It is evident from the experiments that formaldehyde (=formalin) treatment requires very close control of parameters-such as temperature, pH, concentration of bacteria to be killed, aldehyde-reactive material in the buffer (particular- ly primary amines, i.e. proteins, amino acids etc . present in substrates) and, of course, of the amount of active formaldehyde added (considering the tendency of formaldehyde to polymerize or otherwise react dur¬ ing storage) . Since formalin-treatment of immuno- globulin-binding bacteria prior to heat treatment has not been performed so far with a great degree of control except for the calculated final concentra¬ tion of formaldehyde, i.e. without due consideration of bacterial concentration, buffer conditions and temperature, the step of the inventor-to modify heat treatment by the addition of a small amount of formaldehyde - unable to kill per se at so-called room temperature - constitutes a significant step of progress in the processing of immunoglobulin-binding bacteria, a step to be followed by lyophilization for the exploitation of the additive stabilizing effect of such treatment.

Preparations produced according to the invention has been tested as a reagent in various radioimmuno- assays, e.g. for hepatitis B-antigen, antibody to this antigen and for aminoglycoside antibiotics such as gentamicin. In these tests a suspension of staphylo¬ cocci was used which had been derived from lyophilized staphylococci reconstituted in a neutral phosphate- -buffered saline solution with addition of a so-called

IJ _. - _>- R non-ionic detergent, Tween 20, to a final concentra¬ tion of 0.1 % (v/v), just as has been found advantageous with such preparations of reagent staphylococci (Jonsson & Kronvall 1974) as has been available earlier. It has been found that the reconstituted suspension can be used directly without further washing for the purposes mentioned. However, for particular applications one single step of centri- fugation and resuspension in fresh buffer may be recommended for the elimination of traces of soluble protein A or protein A carried on minimal fragments of cellular debris released from the killed bacteria, e.g. as a consequence of freezing prior to freeze drying (lyophilization) . Furthermore, the reagent of reconstituted staphylococci has been successfully tested in an established method for the classification of specific antibodies directed to infectious agents such as rubella virus or toxoplasma parasites. Infections with one of these agents during pregnancy involves a substantial risk for infection and malformation of the fetus. Therefore, various routine procedures have been developed for the study of the immune response towards these antigens and many others. It is an established fact that the primary antibody response of mammals towards foreign substances (antigens) starts with the production of specific antibodies of the IgM class followed by specific antibodies (i.e. antibodies directed to^"the same antigen in question) belonging to the IgA class and later antibodies of the IgG class. The latter tend to remain detectable in the serum/plasma for a long period and to increase permanently at subsequent exposures to the antigen. IgG antibodies constitute the long term protective antibody immunity against infectious agents, which is often boostered by several injections of vaccines where this is possible. On the other hand, IgM and also IgA antibodies tend to disappear within a few weeks after their first appearance. Conversely, the presence of specific IgM antibodies - and to some extent also the presence of specific IgA antibodies - is generally regarded as an indication of recent infection caused by the infectious agent forming the antigen in question. As has been shown by independent investigators, formalin- and heat-treated protein A-containing staphylococci may advantageously be used for the characterization of specific anti¬ bodies in this regard. This is because more than 90 % of serum IgG and not more than about 25 % of IgM and 25 % of IgA are absorbed by protein A, and because one has not been able to demonstrate in the residual IgG belonging to subclass IgG3 the presence of anti¬ bodies to rubella virus. However, experience has shown that non-specific reactions may be encountered with some staphylococcal preparations, apparently partly as a consequence of fragmentation of staphylo¬ cocci in aging suspensions stored as such, in the refrigerator. These fragments appear not to sediment in the normal way during centrifugation of the serum- reagent mixture, and to disturb, e.g., the sedimenta¬ tion of red blood cells in so-called microtitre plates in the so-called haemagglutination inhibition test for rubella virus antibodies^ Without exception, the preparations of staphylococci produced according to the invention have given reliable results in such serological investigations of rubella antibodies, and generally there has been a very good agreement, as expected, with the results obtained with independent control systems, such as the expensive and tedious ultracentrifugation technique. To summarize, through the method according to the invention it has been made possible to produce preparations with immunoglobulin-binding properties in a radically simplified manner. These preparations are of a kind which allows easy distribution and storage while their^"properties are maintained for very long periods - probably for years as may be judged at present - satisfying the intention of yielding, through ordinary reconstitution, a first- -class preparation for application in immunological testing procedures. In this context it should be emphasized in particular that the invention yields a suspension of reconstituted immunoglobulin-binding bacteria, which are notably stable as far as their properties as particles are concerned. The lyophiliza¬ tion process appears, according to what may be con¬ cluded from -the given facts, to explain this particle stability, never obtained before with only heat killing or with classical formalin-treatment before heat killing. The particle-stabilizing effect of formalin treatment before heat killing - although well established - may thus, according to the invention, not only be substituted but be significantly surpassed in efficiency by the effect of lyophilization of just heat-killed bacteria.

Claims

lόCLAIMS

1. A method of producing a stable preparation having immunoglobulin-binding properties, c h a r a c t e r i z e d in that bacteria of species having immunoglobulin-binding capacity beyond that based on a reaction between antigens of the bacteria and specific antibodies directed against such antigens, e.g. Staphylococcus aureus or Streptococcus pyogenes, are grown in a substrate wherein the bacteria form animmunoglobulin-binding substance, that the bacteria are killed, and that the killed bacteria are lyophilized for producing a bacteria suspension that can be reconstituted with liquid.

2. A method as claimed in claim 1, c h a r a c t e r i z e d in that the bacteria are killed by heating.

3. A method as claimed in claim 1 or 2, c h a r a c t e r i z e d in that the bacteria are killed in the presence of a substance having bacteria killing properties.

4. A method as claimed in claim 3, c h a r a c t e r i z e d in that said substance comprises formaldehyde.

5. A method as claimed in any of claims 1 to 4, c h a r a c t e r i z e d in that the bacteria are enriched to a higher concentration before they are killed, e.g. by centrifugation or filtration.

6. A method as claimed in any of claims 1 to 5, c h a r a c t e r i z e d in that the bacteria mass is washed, before it is killed, to be substantially free from substrate.

7. A method as claimed in any of claims 1 to 6, c h a r a c t e r i z e d in that the bacteria preparation after lyophilization is exposed to a mixture of dry gases, e.g. air. 3Ϊ3

S AMENDED CLAIMS (received by the International Bureau on 6 April 1979 (06.04.79)

1. A method of producing a stable preparation having immunoglobulin-binding properties, c h a r a c t e r i z e d in that bacteria of species having immunoglobulin-binding capacity beyond that based on a reaction between antigens of the bacteria and specific antibodies directed against such antigens, e.g. Staphylococcus aureus or Streptococcus pyogenes, are grown in a substrate wherein the bacteria form an immunoglobulin-binding substance, that the bacteria having said immunoglobulin- -binding substance thereon, without any other preceding treatment than washing are killed, and that the killed bacteria are lyophilized for producing a bacteria suspension that can be reconstituted with liquid.

5. A method as claimed in any of claims 1 to 4, c h a r a c t e r i z e d in that the bacteria are enriched to a higher concentration before they are killed, e.g. by centrifugation or filtration. 6. A method as claimed in any of claims 1 to 5, c h a r a c t e r i z e d in that the bacteria mass is washed, before it is killed, to be substantially free from substrate.

7. A method as claimed in any of claims 1 to 6, c h a r a c t e r i z e d in that the bacteria preparation after lyophilization is exposed to a mixture of dry gases, e.g. air.

STATEMENT UNDER ARTICLE 19

Enclosed herewith are new pages 16 and 17 including new clai which should replace the claims as originally filed. The cla have been amended in order to distinguish the invention over the references cited in the international search report.