CA1203479A - Process of separating or purifying a sleep promoting factor from a biological medium by an immunological reaction of the antigen-antibody type - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

PROCESS OF SEPARATING OR PURIFYING A SLEEP PROMOTING
FACTOR FROM A BIOLOGICAL MEDIUM BY AN IMMUNOLOGICAL
REACTION OF THE ANTIGEN-ANTIBODY TYPE.

The invention relates to a process of separating or purifying a sleep-promoting factor("S" factor) from a biological medium, such as urine, which contains it.
This process comprises contacting said medium with anti-MDP antibodies and recovering the "S" factor from the "S" factor-anti-MDP antibody complex, by dissociation thereof.

Description

P3'1L~ 3 PROCESS FOR SEPARATING OR PURIFYING A SLEEP-PROMOTING FACTOR
F~OM A BIOI,OGICAL MEDIUM BY AN I~UNOLOGICAL REACTION OF

THE ANTIGEN-ANTIBODY TYPE.

The invention relates to a process for separating or puryfying a sleep-promoting factor from a biological medium, or for enriching said medium in such a factor.
The presence of a sleep-promoting factor, named "S" factor, in biological media such as human urine is known. The process for separating this "S" factor from human urine has been described by J.M.K~UEGER et coll. in an article published under the title "Composiitons of a sleep-promoting factor, isolated from human urine" (J.
Biol. Chem./ 1982, vol.257, 1 665-1 669). This article also indicates that this glycopeptide appears to have a struc-tural analogy with bacterial peptidoglycans. Analysis of the purified S factor has in fact revea]ed that it contains glutamic acid, alanine, diaminopimelic acid and muramic acid, in molar rations of 2 : 2 : 1 : 1, like in pepti-doglycans.
The article also reports results of biological tests obtained by the intraventricular administration of picomolar amounts of the S factor in the animal. The induc-tion by this factor in treated rat, rabbit and cat, of prolonged sleep characterized by the production of slow waves , can be d~tected by electroencephalography. The sleep so induced shows all the characteristics of normal refreshing sleep, denoted as "Slow Wave Sleepl' (SWS).
The process described in this article appears to represent until now the most effective known technique for isolating this S factor. However it involves a considerable number of steps (10 successive steps). This process is all the more difficu1t to employ as it involves the processing of considerable volumes of the biological medium. In fact, to obtain 30,ug of S factor, it was necessary to process an initial volume of 5 000 1. of human urine.
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It is an objec-t of -the invention to provide a process ~oth slmplified and selective for obtaining this S factor, or any factor having its characteristics from any biological medium con-taining it.
The invention arises from the discovery that this factor could be recognized selectively by specific anti-MDP antibodies (abbreviation of N-acetyl~muramyl-L-alanyl-L-isoglutamine).
The process according to the invention for separa-ting or purifying S factor, or more generally, of an analogous factor having the same biological properties, from a liquid biological medium containing it in the dis-solved state, or for enriching this medium in said factor, or analogous factor, comprises contacting said medium with anti-MDP antibodies to form a complex of MDP anti-bodies and of S factor and recovering said S factor from said complex.
Anti-MDP antibodies suitable for the purification process according to the invention ma~ be obtained in any manner known in itself, particularly by immunisation of the animal, for example rahbit, with an MDP or derivative of MDP previously conjugated to a carrier molecule, such as bovine serumalbumin, polylysines or other molecule having a sufficiently high molecular weight to confer on the conjugate o~tained, the required immunogenicity. As regards the general conditions for producing anti-MDP antibodies, reference may be made to the article of REICHERT C.M. et coll. published in Molec. Immun. 17, 357-363 (1980).
Reference may also be made to the article of BAHR.J.M~ et coll published in Molec. Immun. 1~, N 5, 737- 745 (1982).
The antibodies contained in the antiserums obtained may be purified, for example by affinity chromatography on a resin column bearing MDP molecules or derivatives of MDP fixed to this resin. Advantageously, recourse is had to a column of the insoluble carrier material marketed under the name SEPHAROSE 4B on which had been fixed MDP-lysine groups (N-acetyl muramyl-L,-alanyl-D-isogluta-minyl-L-lysine) r after prior ac-~ivation of the carrier ma--terial with cyanogen bromide.
The anti-MDP antibodies tpolyclonal anti-MDP
antibodies) fixed to such a material may then be eluted, for example by acidificaiton of the medium, partic~arly at a pH below 4, or by increasing the ionic concen-tration of the medium, by means of highly water-soluble ionic salts, such as sodium thiocyanate.
According to an important preferred feature of the invention, "selective" anti-MDP antibodies are used, said selective antibodies recogni2ing specifically the N-acetyl-muramyl-L alanyl-D-isoglutamine structure as a whole, to the exclusion particularly either of the isolated N-acetyl-muramic acid structure or of isolated L-alanyl-D-isoglutamine peptide.
The latter selective an-ti-MDP antibodies, preferably monoclonal antibodies, will favor a substan-tially more thorough degree of purification than polyclonal antibodies, since the latter are capable of fixing other constituents of the biological media concerned, for example numerous other glycopeptide structures also present in these media.
Nonetheless polyclonal anti-MDP antibodies may be used with advantage to produce a first enrichment ofs-factor, more t~ough purifications being repeated whenever required by using "selective" monoclonal antibodies. A technique for obtaining hybridomas secreting such monoclonal anti bodies and for recovering the latter from culture media of these hybridomas, as well as the conditions in which said monoclonal antibodies can be used, will be indica-ted hereafter by way of example.
TherefoKe,~the essential features of the process accordingto the invention comprise the formation of co~plexes between the S factor and anti-MDP antibodies, preferably "selective" antibodies, and the recovery of the S factor itself from this complex, after diSsociation thereof.

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Advanta~eous1y the an-ti-MDP antibodies are used in conjugated forr.l with a water-insoluble carrier or support.
To this effect, any carrier, preferably pulverulent, on which the an-ti-MDP an-tibodies can be fixed without loss of their capacity to form complexes with the isolated S
factor, can be used. It is preferred to use selective anti-MDP antibodies fixed to the carrier material marketed under the name SEPHAROSE 4B, after activation of the latter with cyanogen bromide. Of course, other carrier may be envisaged such as glass beads, agarose, polyacrylamides and SEPHADEX resins~ it being naturally understood that it will be up to the specialist to proceed with tests for fixing anti-MDP sleective antibodies and for checking the preserva-tion of the complexing activity of the so-fixed anti-MDP antibodies-with respect to the S-factor.
The operations of recovery of the S factor from the complex previously formed between the latter and anti-MDP
selective antibodies may be conducted in any manner known in itself, for example by acidification of the medium, partic~arlv to a pH below 4 or by increasing the ionic concentra~i~n of the medium, by means of highly water-soluble ion-c'salts, for example such as sodium thiocya-nate, sodium chloride or ammonium thiocyanate. These methods are particularly advantageous when the anti`MDP
antibodies have previously been fixed to a suitable insoluble sllpport. It is preferred to use anti-MDP antibo-dies obtained from a polyclonal anti-MDP antibody solution, previously purified by affinity chromatography on a SEPHAROSE-4B-MDP lysine column, elution of the anti-MDP
antibodies from the column, particularly under the above indicated conditions. Generally any conventional technique can be used, such as for example the technique described in "Affinity chromatography : Principles and Methods", e~ited by Pharmacia.
Preferably, the initial biological medium from which the S fac-tor is to be extracted is constituted by human urine. Of course, other possible media of human or animal ori~in are not excluded, for example goat cerebrospinal fluid.
Na-turally the biological medium to be treated will preferably be concen-trated as much as possible, before its contacting with the anti-MDP an-tibodies. Preferabl.y it will be freed from proteins having high molecular weights, for example higher than about 25 000, or even higher than 1 000 - 2 000. The biological Medium to be treated containing the S factor can be freed previously from glyco-peptides, peptides of proteins, having distinct biochernicalcharacteristics, such as the proteins which can not be fi~ed to anionic exchange resins or which are agglutinated by antibodies which do not cross-react with the S factor.
It is advantageous to use the concentrated and enriched media, obtained from human urine, as obtained at the end of the fourth.of-the ten successive steps of the process described by J.M. KRUEGER et coll. A preferred procedure for separating and purifying the S factor will be disclosed hereafter by way of example~
The invention will be illustrated further by the description of techniques which permit the detection of the capacity of S factor or of similar factors to be recognized immunologically by anti-MDP selective antibodies.

25 1. Production of monoclonal antibodies a) Immunisation of the mice .... _ _ . . . . _ Two groups of 2 month-old female BALB/c mice were immunised with MDP previously fixed to bovine serumalbumine (MDP-BSA). The first group received two intradermal injec-30 tions separated from each other by three-week i~terval, of 100 ~lg of MDP-BSA emulsified with the FREUND complete adjuvant(FCA). The second group underwent the same treatment, intraperiton~ally, and with doses of lOOjug of MDP-BSA in PBS buffer.

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b) Fusion of the cells and culture of cell hybrids ~hybridomas) Nine weeks after the las-t immunisation, the micé
were boosted twice at a one-day interval, with a solution of 100jug of MDP-BSA, in a saline medium, this time intra`
peritoneally . Three days after the last booster, the mice were sacrificed, the spleen cells recovered and cell fusion of these spleen cells was carried out with a line of myeloma cells, for example myeloma NSO,/l. It goes naturally without saying that it is possible to use any other accessible type of myeloma cells capable of forming, by fusion with spleen cells, cell hybrids capable of inducing the production of ascites in the animal, said ascites then being capable of being used as a source of antibodies having the characteristics of those initially produc~d by the spleen cells involved in the fusion.
Cell fusion of 100 million spleen cells of each group of mice with 107 NSO/1 cells in the presence of a 41% poly-ethylene glycol solution 1 500, may be carried out by the 20 technique described by Z. ESHHAR et coll. in J. immunol.l2~, 775, 1980. After fusion, the cellswere distributed in three to four microplates with 96 wells, selected in an EA~LES
medium, modified by DULBECCO, containin~ the characteris-tic constituents of HAT medium, having a content of hypoxan 25 tine, aminopterine and thymidine (HAT-DMEM) and a high content of glucose, said medium being further completed with up to 15% of horse serum, 50 units per ml of penicillin and of streptomycin, 1 mM sodium pyruvate and 2 nM of glutamine.
After two weeks of culture, the cell hybrids were transferred into a HT-DMEM medium. The cells which deve-loped, could then be cultivated routinely in a medium based only on DMEM and horse serum.

Sortin~J the clones_~roducin~ selective anti-MDP antibodies From 10 to l5 days after t:he fusion operation, sorting of the hybrids capable of producing anti-~lDP antibodies was done, by resorting to the technique called "radioimmunoassay"
5 in solid phase, on microplates of poly- (vinyl chloride) whose wells had previously been coated with a solution of MDP-A--L, i.e. with MDP fixed on or conjugated to A--L, that is multi(poly-D-L-alanyl)-(poly-L-lysine) (100 /ul of a solution of 25 ,ug/ml of MDP-A -L per well). After incuba-10 tion at ambiant temperature, for one hour, the microplateswere washed three times with a solution of 196 horse serum in PBS buffer (PBS-HS). 50 ~ul of the supernatant liquors of the hybridoma cultures were then added to each of the wells and incubated for 2 hours at ambiant temperature.
15 After three washings with PBS-HS, 50 jul of goat Ig anti-mouse antibodieslabelled with iodine 125 (125I) (105 counts per minute : cpm) was added. It was left to incubate over-night at 4C. The radioactivity level was determined in a Gamma radiation counter after 4 washings, dryings and 20 cuttings out of the wells.
The table which follows summarizes the "fusion"
efficiencies" of the two fusion tes-ts mentioned above.
.
.... ..
~nber of cultures Group Immunizationof hybrid cells Positive w~iich have grown hybridanas ., . .. , ~. .
1 MDP-B~A in E~ 384/384 72/384 30 ~ 2 MDP-B~ in PBS 288/28829/288 -Those hybridc~nas which led to detection of 5 000 to 15 000 cpm, having regard to a background noise of 500 cpm in tests carried out with supernatants which did not contain anti-MDP antibodies, were considered as providing 35 a positive response.

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A second selection was carried out under similar condltions employiny a second series of tests in microplates whose wells had been coated respectively with ~DP-A--L, with M-A- L and with DP-A--L, ~.e, conjugates of A--L and of MDP's basic constituents: muramic acid (M) and L-alanyl-D-isoglutamine'(DP) respectively~-The monoclonal antibodies which were active against MDP-A--L, yet which had only little or no activity with respect to DP-A--L or M-A~
were selected.
To produce larger amounts of antibodies, the selec-ted hybridomas were administered intraperitoneally atrates of 10 hybrid cells to BALB/c x DBA/2)Fl mice. The anti-bodies secreted were then recovered in known manner from the fluid ascitic tumors induced by these hybridomas in these mice.
Monoclonal antibodies capable of specifically recognizing the carrier molecule, that is to say BSA, were also selected, under the above indicated conditions.
These monoclonal antibodies were used as controls.
To produce larger amounts of antibodies, the selected hybri,domas were administered intraperitoneally in the proportion of 10 x 10 hybrld cells to BALB/c x DBA/2)Fl mice. The antibodies secreted were then recovered in known manner from f]uid ascitic tumors induced by these hybridomas in these mice, particularly by fractionating the ascite on a gel, particularly a SEPHACRYL 200 gel and recovering the band containing the immunoglobulins detectable by cross reaction with MDP (in a zone corres ponding to a molecular weight of approximately 160,000 30 daltons~.

2. Detection of the cd~acity of anti-MDP antibodies to combine with the "S" factor The technique previously described in the article of BAHR and Coll. already of reference has been used, 3S except for'some minor modifications. The essentlal steps of the procedure which has been used are recalled hereafter.

The wells of micro-ELISA plates (marketed ~y the DYNATECH company) were lined with Ig fractions of IgM, IyA
or IgG anti-mouse goa-t an-tibodies, by contactiny their walls with a solu-tion containing 5 ug/ml of the Ig fraction for 3 hours at 4C. After washing the plate with a solu-tion of PsS containing 0.05 % of dispersing agent marketed under the name TWEEN 20, dilution of supernatant liquors of anti-MDP antibody-producing hybridomas were added to the different wells and the plate was incubated for 2 hours at ambiant temperature. The Ig fractions favored the retention of the anti-MDP antibodies. The plates were then washed and a solution of MDP-lysine coupled or conjuga-ted to radish peroxydase diluted to 1/100 was added to the wells. The plates were then left standing overnight at 4C. After washing, the propor-tions of MDP-lysine peroxy-dase fixed in the wells were rated by using ortho phenyl-ene-diamine and hydrogen peroxide as peroxydase substrate.
The reaction was then stopped by the addition of a solution of 12.5% H2S04 and the optical density was read at 492 nm in the TITERTEK MULTISKAN ELISA (FLOW) photometer.
In the following, the value of the optical density obtained as a resul-t of the fixing achieved after con-tac-ting a l/lOOOth MDP-peroxydase dilution with a l/lOOth dilution of anti-MDP monoclonal an-tibodies will be deemed as corresponding to a 100 % activity 47~ `

The capacity of the S fac-tor to be complexed by the an-ti-MDP monoclonal antibodies was e~a]uated by its aptitude to inhibi-t -the fixation of the MDP-peroxydase conjugate, when added to the wells of the abovesaid micro-pla-tes at -the same -time as the MDP-peroxydase conjuyate under -the conditions indicated above.
Thus inhibitions were obtained expressed by activities of :
- 31% when 100~ul of a MDP solution containing 100 ng of S
factor had been added at the same time as the conjugate;
- 24 % when lOO ~1 of a brain extract containing 140 ng of S factor had been added at the same time as the conjugate, - 18 and 15 % respectively when, at the same time as the conjugate, there were added;respec-tively 70 ng and 10 ng of the S factor extracted from a sample of human urine, under the above-mentioned conditions.
The addition under the same conditions of ~-acetyl-muramyl-D-alanyl-D-isoslutamine (MDP-DD) does not produce any inhibition. From all of the results which have been indicated, it follows that the S factor exhibits a great fixing efficiency to the anti-MDP selective monoclonal antibodies.
It follows that this selective complexation reaction of the S factor can be used for separating it from a medium containing it in admixture with other peptides, glycopep-tides and in particular fragments of peptidoglycans. It is remarkable in this respect that the anti-MDP antibodies do not lead to any immunological reaction with peptido-gIycans, unless they contain the MDP structure suitablyexposed.
The S faetor ean be reeovered from the S factor-anti-MDP antibody complexes by the dissociation thereof.
~lowever, as indieated above, this dissociation is produced mueh more simply when the anti-MDP antibodies are used in a form supported on an insoluble earrier.

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A preferred procedure for separatlng larger amounts of S factor from human urine is described here~
after.
a) A cyanogen bromide-activated column of SEPHAROSE 4B carrying anti-MDP monoclonal antibodies fixed thereto is used.
b) Purification of urine batches.
Starting from urine batches containing the con-ventional preservation agents, they are subjected to the four steps A, B, C and D indicated in the article of J.N. KRUEGER et al. These steps consis-t of:
A - extraction of the urine on carboxymethyl-SEPHADEX

B - gel-filtration on SEPHADEX G-10 C - anion exchange chromatography on DEAE-SEPHADEX and D - gel-filtration again on SEPHADEX G-10.
Of course, any other technique for separating proteins having high molecular weigh-ts can be used too.
One of such techniques comprises filtering and de-salting the starting solu-tion on a SEPHADEX G-25 column.
c) Immuno-absorption of the S factor.
The partially purified and concentrated urine, obtained at the end of the preceding steps, is con-tacted with an immunoabsorbant column with anti-MDP
antibodies fixed theretoO After sufficient contact to permit selective fixation of the S factor, the immunoabsorbant material is washed to remove the non-specific products retained. Advantageously, this step is carried out on a column, and the rinsings are carried out by the passage of -the rinsing liquid through the column. The molecules retained on the antibodies of the column, that is to say the S factor and possibly other monokins or mediators capable of containing the MDP s-tructure in a form accessible to the antibodies, are eluted by a buffer with a low pH for example a solution based on glycine/HCl, pH 3.2 or by a solution ,.

~2~3~7g with high salt concentration, for example lM sodium thiocya-nate.
d) In case where other molecules are eluted at thesame -time as the S fac-tor, they may be separated by chromatography on a molecular sieve enabling separation of molecules accordinq ~o their molecular weights.
Among the molecules possibly present are men-tioned pyrogenic endogenous factors which would have a generally higher molecular weight. Consequently, it may be desirable to separate particualrly molecules having a molecular weight higher than 5000 from those having lower molecular weithts.
This separation can also be carried out by contacting the eluants with other immuno-absorbant materials parti-cularly on a column, comprising the antibodies fixed theretospecifically directed against these other molecules, par-ticularly against pyrogen-endogens.
These procedures are particualrly favorable, since the elutions simultaneous1y enable the regeneration of the immuno-absorbant supports. A column of 3 ml of SEPHAROSE
4B anti-MDP permits , as a result of successive alternate fixati.ons and~regenerations the separation of several mg of S factor.
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33~7g As is self-evident and as emerges moreover already from the foregoi.ng, the invention is in no way limited -to the examples con-templated hereabove, it encompasses on the contrary all modifications, particularl.y those emplo-ying monoclonal antibodies obtained from hybridomasresulting from the fusion oE myeloma cells with splenocytes from the mouse or other animals immunised with any MDP
homolog, such as for example,-~ -ester ~-amide derivatives of N-acetyl-muramyl-L-alanyl-D-glutamic acid, to the extent that these antibodies permit, like those~which have been mentioned above, of proceeding with separations which have been described, PE, PL or LAF or of factors having similar biological properties. It is again self-evident that the polyclonal or monoclonal antibodies so obtained, and more particularly their use for effec-ting such such separations constitute equivalents of those which have been more particularly described and, whereby the use of said antibodies would not depart from the scope of the invention as claimed.
In general,the monoclonal antibodies capable of being used in the process according to the invention may be constituted by any monoclonal antibody selectively recognising MDP homologs, characterised by the presence of a peptide chain linked to the N-acetyl-muramic acid group, the first a~inoacyl o~ the ~eptide chain, that is to say the aminoacyl ensur;.ng the linkage with the N-acetyl muramic group, being selec-ted from among L-alanyl, L-seryl or L-valyl. The second aminoacyl of the peptide chain hardly intervenes in the specificity of the impor-tant antigen determinant to be taken into consideration, as regards suitable monoclonal antibodies for use in the process of this invention. Particularly suitable mono-clonal antibodies are those which do not recognise the 4~3~

N-acetyl muramic acid structure not linked to the above~
indicated peptide group, even at concentrations 100 times grea-ter than the minimum concentra-tion necessary for the formation of complex between the MDP derivative (or MDP) and the corresponding monoclonal antibody.
Conversely, suitable monoclonal antibodies may also be obtained by employing ini-tially in the reali-sation of the cell fusions for the production of hybrid-omas producing monoclonal antibodies, spleen cells from animals previously immunized against MDP homologs in which the ~ and ~-carboxyl functions can bear different substituents. In particular, it is possible to resort to monoclonal antibodies obtained starting from animals initially immunized against N-acetyl-muramyl-L-alanyl-D-glutamic acid diesters or again N-acetyl-muramyl-L-alanyl-D-glutamine ~-esters, as described in European patent application N 79 400357, published Dec. 12, 1979.
Any type of myeloma suitable for production of hybridomas can be used. In particular, recourse may be had to any myeloma described in the technical literature or in published patent applications or patents. There may be mentioned, by way of example, the myelomas de-scribed in European patent applications N 0 014 529, 25 published Aug. 20, 1980, N 0 018 794, published Jan.
12, 1980, N 0 018 795, published Nov. 12, 1980 and the published French patent 2 394 607.

Claims (10)

1 - Process for the separation enrichment or purification of S factor from a liquid biological medium containing it in the dissolved state which comprises contac-ting said medium with anti-MDP antibodies to form a complex of the S factor and the MDP-antibodies and recovering the S factor from said complex

2 - Process of Claim 1, wherein the anti-MDP
antibodies essentially consist of antibodies specifically recognizing the N-acetyl-muramyl-L-alanyl-D-isoglutamine structure as a whole, to the exclusion of the isolated N-acetyl-muramic acid structure or of the isolated L-alanyl-D-isoglutamine peptide.

3 - Process of Claim 1, wherein the anti-MDP
antibodies are fixed to a water-insoluble support.

4 - The process of claim 2 wherein the anti-MDP
antibodies are fixed to a water insoluble support.

5 - The process of claim 4 which comprises eluting said factor S from said complex formed on said support with an aqueous medium acidified at a pH below 4.

6 - The process of claim 4 which comprises eluting said factor S from said complex formed on said support with a concentrated solution of anionic salt.

7 - The process of claim 1 wherein the biological medium containing said S
factor consists of natural or pre-concentrated human urine.

8 - The process of claim 4 wherein the biological medium containing said S factor consists of natural or pre-concentrated human urine.

9 - The process of claim 8, characterized in that the human urine concentrate is free of proteins having a molecular weight higher than about 25 000.

10 - The process of claim 8 wherein the human urine concentrate is free of proteins not fixable to anion exchange resins.