BIOCHEMICAL REAGENT

Abstract: A biochemical reagent comprises an oligosaccharide, preferably one which has been liberated from an immunogenic glycoprotein or proteoglycan, which is immobilised on a carrier via an intermediate spacer molecule such as a lipid. The lipid molecule should preferably have at least two long lipid tails so that the oligosaccharide is held in spaced relationship to the carrier where it exhibits antibody-binding ability which is almost indistinguishable from that of the original glycoprotein or proteoglycan. The reagent has its application in biochemical treating of oligosaccharides and systems which bind to them.

Description: BIOCHEMICAL REAGENT This invention relates to a biochemical reagent, i preparation and its uses. More particularly, the reage of this invention is an immobilised oligosaccharide and h its principal application in the biochemical investigati of oligosaccharides which occur in combination wi proteins, as glycoproteins and proteoglycans, and which m be immunologically active. Production of antibodies by the body is triggered an encounter between a foreign molecule Can "antigen") a cells of the adaptive immune system. The biochemic mechanisms of antibody production are complex and have n been fully elucidated. What is known is that antige trigger the immune system to produce antigen-specif antibodies which on subsequent encounters will bind to t antigen resulting in its inactivation. Regions on t antigen to which antibodies bind are known as "antigen determinants" or, synonymously, "epitopes". In t interests of simplicity, the term "antigenicity" is us herein and in the claims to indicate the ability of molecule to bind to an antibody. It is known that when antibodies bind to proteinaceo antigens such as glycoproteins and proteoglycans, t epitope to which the antibody is directed may be a sequen of amino acids on the protein or a sequence of sugar unit on one of the glyco group oligosaccharide chains. Investigation of the antigenicity of the oligosaccharides i of significant biochemical and medical interest. Oligosaccharides, when isolated as such fro immunogenic glcoproteins and proteoglycans, do not bind t their known antibodies _in vitro; for example, their simpl solutions do not bind to antibodies. The probable reaso for the absence of antibody-binding ability is that epitope are only recognised by antibodies when they are presented t the antibodies in specific orientation and concentration. In Analytical Biochemistry, 139, 168-177, Raja, R. e_t_.aJL_. describe a method of bonding oligosaccharides obtained by enzyme degradation of hyaluronic acid, t immobilising supports to give, for example, affinit chromatography media and polyacrylamide cell cultur surfaces. Hyaluronic acid is a polysaccharide, occurrin in connective tissue, with a molecular weight of severa million and composed of repeating units o N-acetylglucosamine and glucuronic acid. Partial digestio of hyaluronic acid with the enzyme hyaluronidase, under th conditions descibed by Raja produces a mixture of fragments of which about fifty perce have an average size of from six to eight disaccharide uni (i.e. from twelve to sixteen monosaccharide units). Th oligosaccharide mixture is bonded to the immobilisi support by a method which involves the following sequenti steps: (a) reduction of the oligosaccharide with sodi borohydride, which results in cleavage of the terminal sug ring and formation of a methylol group at the site of t ring opening, (b) selective oxidation of the methylol gro with sodium periodate to form an aldehyde group, ( coupling of the aldehydo-oligosaccharide to an alpha, ome alkyldiamine such as hexane diamine in the presence sodium cyanoborohydride resulting in a derivative which h the oligosaccharide residue at one end of a polymethyle chain and an active amino group at the remote termin (omega postion) ; and, (d) bonding of the derivatiz oligosaccharide, via the omega amino group, to chromatography support, such as Sepharose (Trade Mark under the action of cyanogen bromide or, alternatively, to polyacrylamide cell culture surface under the action sodium cyanoborohydride. It is known that reducing disaccharides (i.e. tho having active aldehyde groups) may be coupled to proteins to aminoethyl polyacrylamide gels by selective reduction a Schiff base, formed between the reducing (aldehyde) gro of the disaccharide and the amino group of the protein the aminoethyl gel using sodium cyanoborohydride [Gra G.R., (1974), Arch. Biochem. Biophys.; 163, 426-428]. It the reduction method of Gray which is utilised by Wood a Kabat to produce conjugates of isomaltosyl oligosaccharid with lipids [J. Exp. Med., 154, 432-449, (1981)]. In Wo and Kabat 's immunological study of isomaltos oligosaccharides, the oligosaccharides are conjugated t stearylamine (octadecylamine ). The stearyl isomaltos oligosaccharide conjugate was found to trigger production o immunoglobulins (i.e. the conjugate was immunogenic) whe injected into test rabbits, provided it was first emulsifie with complete Freund's adjuvant (CFA) in which, it is said the conjugate forms miscelles. The conjugate was als found to induce antibody production in rabbits when it w incorporated into liposomes. It is reported that th larger oligosaccharides are rather more active than th shorter, the suggestion being that the shorter chains do no protrude sufficiently from the liposome surface to be active An object of the present invention is to enable th analysis of the interaction of oligosaccharides an molecules to which they bind by providing materials an methods directed to this end. According to the present invention there is provided biochemical reagent comprising an antigenic conjugat between an oligosaccharide and an immobilising carrier, sai oligosaccharide being bound in spaced relationship to th carrier by means of an interposed non-antigenic space molecule whereby the oligosaccharide is presented i antigenically active steric configuration for binding o encountering its antibody. The term "antibody" is used herein is to be constru as including all carbohydrate-binding moieties. The invention also provides a method for the screeni of antibodies which comprises contacting a soluti containing an antibody with the immobilised antigenical active conjugate aforesaid and thereafter detecting t occurrence (or non-occurrence) of binding. The spacer molecule is preferably a lipid molecu and, more preferably, a glyceride or phosphatide whi possesses at least two hydrophobic polyalkylene chains. The combination of the oligosaccharide and the lipid hereafter referred to as a "neoglycolipid". The spacer molecule may be a long chain fatty acid from 5 to 50, preferably 5 to 30 and more preferably 10 25, carbon atom length, an example being stearylamine a similar amino derivatives of lauric, myristic and palmit acids. Simple amino hydrocarbons may also be employed. The amino group is necessary for chemical bonding to t oligosaccharide to form the antigenic conjugate. Neoglycolipids may be formed by reacting oligosaccharide having at least one aldehyde group with amine having one or more hydrophobic groups. Preferably the neoglycolipid has the following formul where R represents a carbohydrate group, 2 R represents an alkyl, or alkoyl group, R represents an alkyl, or alkoyl group, R represents an alkyl, or alkoyl group, or hydrogen R , R and " R being the same or different groups, z is zero or 1,2 or 3 and X represents a linking group which may be selected from: 1 ^ / / / OH O O 0 II CM,L - o - P - c — Ctt -Ctf —£ -> ( ( I i o o 0 μ Neoglycolipids of particular interest, have th general formula: R- L -NH CH 2 — CH 2 — O - P - O - CH 2 — CH CH 2 - OH CO CO (CH 2 ) n (CH 2 ) CH CH where the integers n, and m have a value within the range to 50, preferably 5 to 30, and desirably 10 to 25. The carbohydrate group R may be derived from monosaccharide, oligosaccharide, or even a polysaccharid which contains an aldehyde group. A carbohydrate grou R , which does not in itself contain the necessar aldehyde group, may be subjected to oxidation, for exampl using periodate oxidation. Of particular biochemica interest are carboyhdrate groups which can be isolated fro glycoproteins and proteoglycans by a reduction treatmen resulting in a reduced oligosaccharide which contains methylol group, in which case, to form the necessar aldehyde they may be subjected to a periodate oxidatio treatment as described by Raja et al, in Anal. Biochem 1984, Vol 139, pages 168-177. The most preferred spacer compounds are represented b phosphatidyl ethanolamine dipalmitoyl (PPEADP) and it analogues, L-alpha- phosphatidyl-L-serine L-alpha-phosphatidyl- ethanolamine (PPEA) dilauroyl, PPE dimyristoyl, PPEA distearoyl, PPE beta-o1eoyl-gamma-palmitoyl. The immobilising carrier may be a plate of plastic material such as polyvinyl chloride, a chromatographi support material such as silica gel or a liposome. In preferred embodiments of the invention th oligosaccharide is obtained from a naturally-occurrin glycoprotein or proteoglycan. Oligosaccharides may be bound to proteins via oxygen atom (0-linked) or via a nitrogen atom (N-linked Two different approaches are necessary for the liberation the oligosaccharides from their bound protein by chemic means. Treatment with alkaline sodium borohydride wi break the 0-link and simultaneously open the termin saccharide ring with formation of a methylol group at t ring cleavage site (Iyer § Carlson, Arch. Biochem. Biophys 1971, Vol 142, pages 101 to 105).. Similarly, N-link oligosaccharides may be obtained and purified in the reduc state (Fukuda et al J Biochem. 1976, Vol 80 pages 1223-12 and Takasaki et al. Methods Enzymol 1982, vol 83, pag 263-268) and suffer from the limitation described above that, being fully reduced they are not reactive per se a require oxidation to introduce an aldehyde group. Alternatively, the oligosaccharides may be liberated fr their bound protein by enzyme action. Reference is made to Fig.l of the accompanyi drawings, which shows a reaction scheme for the liberati of 0- and N-linked oligosaccharides from glycoproteins a proteoglycans and their reaction with lipids to fo neoglycolipids. The pyranose ring structure shown in Fig is shown as N-acetylglucosamine for the purpose illustration only and is to be construed as representati of a terminal saccharide ring of any oligosaccharide, t remainder of the oligosaccharide molecule being represent by the symbol [Oligo], An N-linked protein bound oligosaccharide (structu I) is subjected to hydrazinolysis which breaks the link between the saccharide and its bound protein (represented [Protein]) with formation of an hyrdazido group at the si of the original saccharide/ protein link (structure II). Acetylation with acetic anhydride a neutralisation converts the hydrazido group to a hydrox group and re-N-acetylates the amino group (structure III). Treatment of the hydroxy compound shown in structu III with sodium borohydride cleaves the terminal sacchari ring structure with formation of a methylol group (-C^O at the site of ring cleavage (structure IV). An oligosaccharide bound to a protein via an 0-li (structure V) is treated with sodium hydroxide/sodi borohydride which cleaves the terminal saccharide ring wi formation of a methylol group at the cleavage si (structure IV). The methylol compound shown as structure IV is ful reduced and is unreactive with amines. Selective oxidati of the methylol group using sodium periodate under strict controlled conditions, of which details will be giv hereinafter, results in the reducing (aldehyd oligosaccharide shown as structure VI. The reducing oligosaccharide (structure VI) is react with an lipid amine such as is represented by t phosphatide indicated in Fig.l, in the presence of sodi cyanoborohydride to give the neoglycolipid (structu VII). The R groups of the phosphatide are as hereinbefo described.. It is possible to react the sugar alcohol, shown structure III, directly with the cyanoborohydride and lip to obtain the neoglycolipid VII but this route (indicated the broken line in Fig.l), although removing several ste of the multi-step reaction scheme, results is a produ which may contain isomeric forms of the neoglycolipid a its utility is thus reduced. Therefore, according to the present invention, the is provided a method for preparing a biochemical reage comprising liberating an oligosaccharide moiety of immunogenic glycoprotein or a proteoglycan under reducti conditions to form thereby a reduced oligosaccharid selectively oxidising the reduced oligosaccharide wi periodic acid or a salt thereof to form an aldehyd oligosaccharide, and conjugating the aldehyd oligosaccharide with an immobilising carrier via a spac molecule containing an amino group. While not wishing to be bound by this particul theory, the following is given by way of explanation of t invention. It is known that when antibodies are generat in vivo in response to glycoprotein and proteoglyc immunogens they often display specificity for epitopes the oligosaccharides which make up the glyco group. T binding of antibodies is a stereospecific mechanism whi depends on the ability of an antibody to recognise complementary molecular configuration on an epitope. Both of the sho arms (Fab) of the antibody seek epitopes and bind there and so it is evident that correct three-dimension presentation of the epitopes with respect to orientation a spacing between pairs of epitopes, and with respect to t physical separation of each epitope from the carrier matri is essential to ensure efficient and stable binding of t antibody via both of its arms. It is probable, then, th regeneration of the antigenicity of oligosaccharid released from glycoproteins and proteoglycans requires th the epitopes be presented in a stereospecific configuratio and, it may be postulated, in a manner which allows so molecular flexibility to enable the antibody arms to loca two epitopes and draw them into the required latera displacement. Oligosaccharides appear to lose thei antigenicity when released from glycoproteins a proteoglycans, i.e. they are in the free state, nor ar short chain oligosaccharides rendered antigenic by mer direct binding to an immobilising carrier. When bound to carrier via short chain spacer molecules the antigenicity o short chain oligosaccharides is present but may be weak. Long chain oligosaccharides do display some wea antigenicity when bound to a carrier via short chain space molecules. There does, therefore, appear to be som relationship between antigenicity and the spacing betwee the epitope and the surface of the carrier. Th antigenicity of the conjugate of the invention may b optimised b experimentation with spacer molecules of various chai lengths. However, in practical terms, the oligosaccharide of biochemical interest have normally no more than abou seven or eight sugar units and for molecules of this siz spacers with twelve to eighteen carbon chains allow th epitope of the sugars to be presented antigenically. Th second feature which is thought to participate , in th binding mechanism is the lateral spacing between th epitopes. It is well known that the hydrophobic tails o lipid molecules tend to aggregate in solution . It ha been found tnat antigenicity can be optimised by using lipi spacer molecules which have at least two hydrophobic lipi tails. This is believed to induce localised clustering o the tails on the carrier which should affect the latera spacing of the epitopes at the remote end of th lipid/oligosaccharide conjugate. This postulate i supported by the observation that double-tailed conjugate have greater antigenicity than single-tailed conjugates o the same oligosaccharide, presumably because of. an enhance clustering effect induced by the multiple hydrophobic tails. This invention also relates to a method for evaluatin the antigenicities and/or receptor functions σ oligosaccharides released from glycoproteins an proteoglycans. There is an increasing awareness of the importance o carbohydrate chains of glycoproteins and proteoglycans o animal cells as onco-developmental antigens recognised by monoclonal antibodies, and as receptors for infective agents " as well as endogenous ligands. Consequently there is a ne for a micromethod for evaluating the antigenicities and/ receptor functions of oligosaccharides released from t peptide moieties of glycoproteins or proteoglycans by th methods described above. Although several hundreds milligrams of immunogenic proteins may be available starting material, the amount of individual purifie oligosaccharides, released from the glycoproteins, is ver sma11. The method of this invention may be performed usin minute amounts of oligosaccharides by converting them int neoglycolipids prior to assay. Therefore, according to one aspect of the presen invention there is provided a micromethod for evaluating th antigenicities and/or receptor functions of a oligosaccharide comprising, (a) reacting a reducing oligosaccharide with an amin spacer molecule having the formula /!/- X IS - where R 1 represents an alkyl or alkoyl group, R represents an alkyl or alkoyl group or hydrogen, R represents an alkyl or alkoyl group or hydrogen,R R 2 and R3 being the same or different, and represents a linking group to form a neoglycolipid, (b) conjugating the neoglycolipid on to an immobilisin carrier; and, (c) contacting the conjugate with an antibody or othe carbohydrate binding system and observing th occurrence (or non-occurrence) of binding of th conjugate. Preferably the said micromethod includes the step o liberating protein-bound oligosaccharide from the protei and selectively oxidising the oligosaccharide with a salt o periodic acid under controlled conditions to form a reactiv reducing oligosaccharide. Particularly preferred spacers molecules are the derivativ where the R groups are alkoyl groups derived from the fat acids lauric, myristic, palmitic and stearic acids. Long or shorter chain lengths may be appropriate in particul circumstances but chains of 12 to 18 carbon atoms a appropriate for most purposes, being a compromise betwe the shorter chains which may only produce antigenicity wi longer chain oligosaccharides, and longer chain spac molecules which impart no significant enhancement of t antigenicity and which are not readily available. As wi be later illustrated in the Examples, it is apparent th there is a correlation between the chain length of t spacer and the induction of antigenicity. The extent of binding of the conjugat oligosaccharide of the invention with an antibody may determined by any of the well known methods. For exampl radioactively labelled antibodies or carbohydrate recept system may be used and the amount bound to t oligosaccharide detected by means of the radioactive labe Where unlabelled antibodies are bound, radio-labell secondary antibodies specific to the relevant class unlabelled antibody (e.g. anti-mouse IgG antibody) may added, and after incubation the degree of radioactivi measured. Especially when the neoglycolipids are derived fr PPEADP, their antigenic activity is virtual indistinguishable from its activity on the glycoprotein proteoglycan from which the oligosaccharide is isolate Thus the conjugate of the invention enables t oligosaccharide moiety to: (a) be recognised by antibodies or carbohydrate bindi systems, or (b) compete with other molecules having antibody carbohydrate binding activity. Thus the test method may be used for characterisi physiological status, or for the typing of tissues, cells infective agents. An alternative assay enables the identification oligosaccharides by contacting same with an immobilis antibody so that oligosaccharides containing an epito recognised by the antibody will be bound to the immobilis antibody thereby confirming its identity. In general, the assay method of the invention enabl the study of the interaction between specif oligosaccharide structures and antibodies. It therefo provides a valuable tool for: 1. basic research, for characterising antibodies; 2. basic research, for characterising oligosacchari structures, that is, using specific antibodies structural reagents; 3. diagnosis, for measurement of quantitative qualitative changes in oligosaccharides or antibodies; 4. purification of antibodies; and 5. therapy. The following are specific examples of the assa procedures which may be performed using the reagent of thi invention: _I Determination of Antibody binding characteristics Oligosaccharides are immobilised in a conjugate of th invention and the binding of antibodies thereto ar measured. The oligosaccharides may be homogeneous o derived from a particular immunogenic glycoprotein o proteoglycan, cell fraction, cell, tissue or body fluid. For example, the assay method of the invention may i particular be used to diagnose the nature or status of disease in a living organism. This may be achieved fo example when antibodies with specific oligosaccharide binding characteristics are produced in the organism as result of a disease state, and are hence diagnostic for tha state. In this procedure, antibodies would be isolate from, for example, plasma, and their specificity determine by performing binding assays to conjugates of the inventio which contain a range of oligosaccharides. II Determination of the characteristics o oligosaccharides Using the procedure described in paragraph I, specifi antibody/conjugate complexes are selected. Then a tes preparation of conjugate, in which the carrier is a liposom and the identity of the oligosaccharide is unknown, i allowed to compete with plate-bonded conjugate for the boun antibody. Removal of the antibody by the test antige indicates the similarity between the structures o plate-bound and liposome-bound oligosaccharides. Thi procedure allows characterisation of oligosaccharides an typing of cells according to their oligosaccharide-bindin ability. III Determination of the characteristics of cells an microorganisms A conjugate containing a specific oligosaccharide i bound to a PVC plate and cells or microorganisms are teste directly for their ability to bind to the plate. Thi method enables the typing of cells or strains o microorganisms. IV Isolation of Antibodies By passing a solution of antibodies over a conjugat of this invention containing an oligosaccharide of interest the antibody to the oligosaccharide will bind, allowing th remainder to be washed away and the linked antibod thereafter released in purified form. V Effector Function A conjugate of the invention may be administered i vivo to produce an immune response, for example, as antagonist, the conjugate may compete with t oligosaccharides on the surface of infective microorganis for binding sites on receptor cells, and vice versa, th preventing infection. Also, as an agonist or antagonist modify the function of glycoprotein hormones or glycoprote receptors whose functions are mediated by carbohydra structures. The invention will now be described, by way illustration in the following Examples. EXAMPLE 1 Thin Layer Chromatography The following experiments were conducted to illustra the binding of the neoglycolipid to silica gel and elucidate the relationship between the chain lengths of t spacer molecule and the oligosaccharide. At the same ti optimum conditions for the binding were investigated. Eight different amines as spacer molecules we investigated in three experiments. Oligosaccharides we chromatographed on high performance silica gel TLC plat using standard methods and the plates were then oxidis with periodate followed by reaction with the a ine a sodium cyanoborohydride to bind the oligosaccharid fractions to the plate. The method employed for the periodate oxidation was follows: 1. From 3 to 5 micrograms of the oligosaccharide a applied to appropriate lanes of a HP-TLC plate. 2. The chromatogram is developed in an appropria solvent system, for example butan-1-ol: acetone: wat 6:5:4 by volume, and the plate then dried and cut in lanes. 3. A solution of 0.02% of sodium periodate in a mixtu of 40mM imidazole hydrochloride (pH 6.5) :chlorofor mmeetthhaannooll 11::55::55 bbyy vvoollume is prepared, cooled to 4 and stored in the dark. 4. Each lane of the TLC is immersed in the perioda solution for ten seconds. The oragnic solvent allowed to evaporate in air for a few seconds. 5. The strips are immediately placed in a humid box 4 C in the dark and maintained therein for minutes. 6. Each lane is then immersed in a 0.5% w/v solution the coupling amine (usually PPEADP) in chlorofor methanol 1:1 v/v for ten seconds 7. The lanes are then placed in an air oven at 60°C f a period of one hour. 8. Each lane is then immersed into a Q..2% w/v solution sodium cyanoborohydride in chloroform: methanol 1 for ten seconds. 9. The lanes are replaced in the 60 C air oven for further two hours. It was the protocol described above which was employ in all the chromatograms discussed in this Example, vario amines being employed to determine the extent of the bindi and the antigenicities of the various amines as spac moleculed for the conjugates. In addition, a further test was undertaken investigate the influence of the periodate oxidation step the binding properties of the conjugate. Dextr hydolysate was used as the test oligosaccharide which w oxidised with periodate by the protocol described, t concentration of the periodate was varied to attempt ascertain an optimum value. The chromatogram was stain with orcinol and then washed. The density of the stain bands was assessed. As a result of this test it was found that the low limit for periodate concentration was 0.005% and the upp 0.05% with the optimum at 0.02% which was confirmed in t practical use of the invention in TLC binding assays. The results are tabulated below in Tables I to III. In the experiments leading to the results shown in Tables and II, the conjugated oligosaccharides were exposed t anti-Ma antibody or- anti-5D4 antibody followed by radiolabelled second antibody then washed a autoradiographed. The results presented in Table III wer produced by direct orcinol staining of the oligosaccharid moiety of the conjugate to determine the level of binding t the TLC plate. An assessment of the density of the band was made (on a scale of 0 to 1, the maximum being indicate by 1), giving an indication of the binding efficiency. Table I shows the effect of using various space amines on the antigenicities of a trisaccharide. Table I gives the results using a dodeca-saccharide, and, the dat in Table III demonstrates the level of binding to TLC plate of a mixture of oligosaccharides derived from dextra hydrolysate, as a function of the type of spacer molecule. TABLE I Immunostaining of trisaccharide v Ma antibody AMINE DENSITY PPEAD myristoyl 1.0 PPEAD stearoyl 1.0 PPEADP 1.0 PPEA oleoyl palmitoyl 0.5 PPEAD lauroyl 0.1 Sphingosine not detectable Stearylamine not detectable PPS not detectable TABLE II Immunostaining of Jl Dodecasaccharide v 5D4 Antibody AMINE DENSITY PPEAD myristoyl 1. .0 PPEADP 0. .9 PPEAD stearoyl 0. ,8 PPEA oleoyl palmitoyl 0. .5 PPS 0. 5 Stearylamine 0. 2 PPEAD lauroyl 0. 2 Sphingosine trace TABLE III REDUCED DEXTRAN HYDROLYSATE with ORCINOL STAIN- AMINE DENSITY PPEAD lauroyl 1.0 Stearylamine 0.8 PPEAD myristoyl 0.8 PPEAD oleoyl palmitoyl 0.8 PPS 0.5 PPEAD stearoyl 0.5 PPEADP 0.5 Sphingosine 0.2 The various amines used in generating the data given Tables 1 to 3 are characterised as follows: AMINE M.W. NOTES L-alpha-phosphatidyl-L-serine (PPS) 313 no lipid tail D-sphingosine 300 single C-- tail L-alpha-phosphatidylethanolamine (PPEA) dilauroyl 580 two C,, tails PPEA dimyristoyl 636 two C,- tails PPEA dipalmitoyl 692 two C-- tails PPEA distearoyl 748 two C ιg tails PPEA-beta-oleoyl-gamma-palmitoyl 718 one C__ tail, and C- Q tail with double bon By comparing the results given in Tables 1 to 3 abo the following conclusions can be drawn: (a) The antigenicity of a short chain oligosacchari (tri¬ saccharide: Table 1) bound with short chain spa amines such as PPS, is low; (b) The antigenicity of a short chain saccharide, Table bound with spacer amines with double lipid tails, s as PPEADP myristoyl and stearoyl, is high; (c) The antigenicity of a long chain oligosacchari (dodecasaccharide, Table 2) is relatively strong ev with short spacer amines such as PPS; (d) The antigenicity of a long chain oligosacchari (Table 2), bound with spacer amines with single lip tails, such as sphingosine, is weak; (e) The strength of binding of the neoglycolipid to t silica gel, as opposed to the antigenicity of t conjugate, (Table 3) is not of the same order indicated for the antigenicity. For example, where PPEAD lauroyl binds the oligosaccharide more strong than, for example, PPEADP (Table 3), the antigenici of the PPEAD lauroyl complex is much lower for bo tri- and dodeca-saccharides (Tables 1 and 2). (f) The binding strength of neoglycolipids with sing tails may be high (stearylamine in Table 3) but t antigenicity is low in comparison with double tail molecules (Tables 1 and 2). EXAMPLE 2 Materials (a) Oligosaccharides The oligosaccharides used were: lactose, maltos tetrasaccharide N,, obtained from human meconi glycoproteins [Hounsell E. F. e_t a_l , Eur. J. Biochem 14 (1985) 367-377] and, chemically synthesised disacchari N-acetyllactosamine, trisaccharide (O4.) a pentasaccharide (0 g ) [Auge, C. , David,S. , ξ Veyrieres, A. (1979), Nouveau J. de Chimie, 3, 491-497], and chemicall synthesised 3-fucosyl-N-acetyllactosamine (3F). T synthesised saccharides were reduced with sodi - 27- borohydride. Lacto-N-fucopentaitol III (LNFP III aldito was derived from a fraction of human milk pentasaccharides. (b) Monoclonal Antibodies: (i) human serum, Ma, containing a monoclonal anti- antibody, at a dilution of 1:1000, and, (ii) mouse hybridoma antibody, anti-SSEA-1, at a dilutio of 1:100. (c) Lectin The plant lectin Ricinus communis agglutinin, which contai a carbohydrate binding system. Controlled Oxidation The reduced oligosaccharides (10 micrograms) we oxidised with sodium periodate (4 equivalents) in 10 microlitres of 40mM imidazole hydrochloride (pH 6.5) 0°C for one hour. The resultant solutions were us immediately for combination with L-alpha-phosphatidy ethanolamine dipalmitoyl (PPEADP). Combination with PPEADP Lactose and maltose (10 micrograms) or 50 microlitr of the prepared solutions of the periodate oxidis oligosaccharides were added to 150 microlitres of chloroform/methanol (1:2 v/v) solution containing 1 micrograms of PPEADP. The solutions were allowed to rea at 50°C for two hours. .Sodium cyanoborohydride ( micrograms in 10 microlitres of methanol) were then add and the solutions again maintained at 50°C for a furth 116 hours. Thereafter the solvents were evaporated fr the solutions under nitrogen and the residues redissolved 40% v/v aqueous methanol. Dilutions of 10, 1.0, and 0. micrograms of carbohydrate per millilitre in 40% v/v aqueo methanol were prepared for PVC plate-binding assays. Binding to Carrier Matrices (a) Binding to Plates Aliquots of 50 microlitres of the solutions neoglycolipids, prepared as described by combination of the disaccharides , oligosaccharides with PPEADP, were added t the wells of round-bottomed flexible PVC microtitr plates. The plates were held at 4°C for 90 minutes a then the wells were washed five times with phospha buffered saline (PBS) of pH 7.4, and treated with a 3% w/ solution of bovine serum albumin (BSA) in PBS at 37°C f one hour. The wells were then washed five times with PBS. (b) Chromatographic Carrier For chromatogram binding assays , the reduce oligosaccharide 0. and lactose (3 micrograms per lane were chromatographed on HP-TLC plates ( aluminium shee with silica gel 60 from E. Merck) using a solvent syst butan-1-ol/acetone/water 6:5:4 v/v. The chromatograms we dipped into a chloroform/methanol/40mM imidazo hydrochloride solution (pH 6.5) containing 0.02% w/v sodium periodate and left at 4 C for for 45 minutes a then dipped into a 1:1 v/v chloroform/methanol solution 0.5% w/v PPEADP. They were then maintained at 60 C for o hour and then dipped into a solution of 0.2% w/v of sodi cyanoborohydride in chloroform/methanol 1:1 v/v a incubated at 60°C for a further two hours. Chomatogram Binding Assay The chromatograms were then either stained with orcin stain or were overlaid with 5% BSA in PBS for two hours room temperature and immunostained with anti-I(Ma) 1:1 125 dilution or control serum (Ns) followed by I-labell anti-human IgM, as described in Uemura, K. et.al. (198 Biosci. Reps., 3, 577-588, and autoradiographed usi Singul-X RP (Trade Mark) X-ray films. Plate-binding Immunoassay To the wells of the prepared plates, 50 microlitres antibody preparations (dilutions 1:1000 for anti-I(Ma)seru and, 1:100 for anti-SSEA-1 ascites or 125 I-labell R. communis (60 ng containing 1 x 10 cpm in 1% BSA in PB were added and the plates incubated at 4°C for 16 hours. The plates were then washed five times with PBS. 125 For the antibody assays, I-labelled rabbit antibodies to human [for the anti-I (Ma) antibody] and mouse (for anti-SSEA-1) immunoglobulins were added (50 microlitres containing 10 cpm and 30 ng of protein per well). For the radioassay using 125I labelled lectin, the plates were dried and the wells counted using a Nuclear Enterprises 1600 counter. Inhibition assay procedures were adapted from Wood, E. , et_.___.- (1979) Mol. Immunol. 16, 813-819; and Gooi, H. C. , e_t.a ; (1985) Mol. Immunol. 22, 689-693. using serial, dilutions of the neoglycolipids incorporated into liposomes as inhibitors of the binding of the anti-lCMa) (1:10 dilution) or R. communis lectin (30 ng containing 5 x 10 cpm) to sheep gastric mucins or meconium glycoproteins of the non-secretor type (500 ng per well). The composition of the liposomes was neoglycolipid, lecithin (dimiristoyl phosphatidyl choline) and cholesterol. Further details are discussed in Tang P.W. e_t al_ Biochem Biophys Res Comm Vol. 132, pages 474-480 (1985) Results and Discussion (i) The anti-I(Ma) antibody bound to oligosaccharides 0 , 0 and N- which, all contain the saccharide sequence Gal-beta-l-4GlcNAc-beta-1-6- . Reference is made to Fig.2A which shows the reactivity of anti-I(Ma) to the neoglycolipids on PVC plates. Positive binding was observed with as little as 10 to 50 ng (10 to 100 picomoles) of the oligosaccharides but no binding was detected with normal human serum or with a negative control of conjugate containing PPEADP linked to lactose. - 31- (ii) It has previously been reported that the carbohydrate chains of sheep gastric mucins express I(Ma) as well as other antigenic determinants and that there is a 10,000-fol reduction of the antigenicity of the oligosaccharides after release from the bound peptide moiety [Wood, E. , et.al., (1981) Carbohydr. Res., 90, 269-282]. Fig. 2B shows the results for inhibition-of-binding assay of anti-I(Ma) to sheep gastric mucins on PVC plates. The data indicate tha when oligosaccharides derived from sheep gastric mucins are incorporated into a conjugate of this invention, using PPEADP and incorporated into liposomes, the antigenic activity of the oligosaccharide in inhibition assays is virtually indistinguishable from that of the original untreated ucin glycoprotein . (iii) The oligosaccharide 0., containing the I(Ma) antigen sequence produced a radioactive band on HP-TLC with immunostaining as described above, with as little as three micrograms of oligosaccharide. No immunostaining was detectable for lactose. Fig. 2C shows the results of binding of anti-I(Ma) to neoglycolipids on a TLC plate. (iv) In PVC wells coated with the neoglycolipid obtained b reductive amination of LNFP III alditol which contains the carbohydrate sequence Gal-beta-1-4(Fuc-alpha-1-3)GlcNAc of the stage specific embryonic antigen of mouse, SSEA-1, the oligosaccharide specifically bound the hybridoma antibody, anti-SSEA-1, (Fig.2D). The chemically synthesised trisaccharide analogue 3F, after reduction to the 3F aldito and 32- conjugation to PPEADP, gave no binding above the control mouse serum even at the highest level (500 ng) tested. The difference may be due to the loss of one carbon unit from the N-acetylglucosamine residue of the 3F alditol during periodate oxidation, since the conjugate containing 3F without prior reduction gave substantial binding, or to difference in orientation of the oligosaccharide moiety. (v) Conjugates containing reduced oligosaccharide 0. and N-acetyllactosamine were coated on to PVC plates, and show to bind the 125I- labelled lectin R. co munis which is known to react strongly with the sequence Gal-beta-l-4GlcN (Fig.2E). A conjugate containing maltose and PPEADP gave no binding, nor did PPEADP itself. (vi) The conjugates containing the oligosaccharide 0. and N-acetyllactosamine were incorporated into liposomes and were shown to inhibit specifically binding of the lectin t human meconium glycoproteins (Fig 2F). EXAMPLE 3 Reduced 0-linked and reducing N-linked oligosacchari chains were obtained from galactosyltransferase, an enzyme from human milk, by alkaline borohydride degradation and hydrazinolysis and purified by ion-exchange chromatography. Three preparations of the enzyme were used: (1) combined galactosyltrans erase from donors of unknown blood group; (2) combined milk from blood group A donors; and, (3) milk from a single donor of blood group 0 (non-secretor). For deglycosylation an aliquot of galactosyltransferase - 33- preparation 3 was treated with anhydrous hydrogen fluoride for one hour at 4 C. The mixture was extensively dialysed against water then lyophilised and the residue redissolved in isotonic saline. Galactosyltransferase (600 micrograms of preparations 1 and 3 or 300 micrograms of preparation 2)' containing 5% o hexose as determined by phenol sulphuric acid assay using preparation 3, was treated with 0.05M NaOH/M NaBH 4 (200 microlitres) at 50 C for 24 hours to release the 0-linked oligosaccharides. After acidification with acetic acid to pH 5.0 the mixture (250 microlitres) was applied to a colum (0.4 x 1cm) of Dowex AG50W-X8 (Trade Mark) (H + ) resin (200-400 mesh). Elution with water (1.2 ml) gave the 0-linked oligosaccharide fraction. The fraction was evaporated to dryness at 60 C in vacuo and methanol (200 microlitres) was thrice evaporated from the residue to remove boric acid. Elution with 3M ammonium hydroxide (1.2 ml) yielde the peptides and glycopeptides with the N-linked oligosaccharides. This fraction was evaporated to dryness at 60 C in vacuo and water (500 microlitres) was evaporated from the residue which was then subjected to hydazinolysis and N-acetylation. The products (250 microlitres) were chromatographed on the same resin as described above. The unretained fraction contained the N-linked oligosaccharides. Fractions corresponding to the 0- and N-linked oligosaccharides were also obtained from preparation 3 which had been deglycosylated as described above. The oligosaccharides were resolved by HP-TLC. on silica gel 60 (Merck 5641, Trade Mark) using butan-1-ol: acetone:water, 6:5:4, and visualised with 0.2% orcinol in 2 ethanolic sulphuric acid. For antibody binding assays on PVC plates and inhibition-of-binding assays, reduced 0-linked fractions released from galactosyltransferase (100-600 micrograms) were dried at 60 C in vacuo and oxidised with sodium periodate (10-60 micrograms as appropriate) in 40mM imidazole hydrochloride (pH 6.5, 100 microlitres) at 0°C for 45 minutes. To the resulting mixtures, chloroform/ methanol (1:1) was added (240-480 microlitres as appropriate) containing PPEADP (240-480 micrograms) and NaCNBH, (100-200 micrograms) and the mixtures incubated at 50°C for 16 hours. The reducing oligosaccaharides released by hydrazinolysis of galactosyltransferase (100 to 600 micrograms) were coupled to PPEADP without periodate oxidation. The entire reaction mixtures were used for antibody binding on PVC plates and inhibition-of-binding assays. For chromatogram binding assays, 0-linked oligosaccharides from 15 micrograms of galactosyltransferas (preparation 1) were subjected to HP-TLC, oxidised with periodate ji situ and coupled to PPEADP as described in Example 2. N-linked oligosaccharides from 15 micrograms o galactosytransferase were chromatographed and coupled to PPEADP without periodate oxidation. EXAMPLE 4 Immunochemical and chromatographic studies were carried out, using the procedures described in Example 2, on keratan sulphate proteoglycans which express antigenic determinants recognised by several monoclonal antibodies. Ten sulphated oligosaccharides were purified from bovine corneal keratan sulphate after end-beta-galactosidase digestion according to the procedure described in Scudder e_t a^, Eur. J. Biochem. 157, 365-373 (1986). The oligosaccharides were converted to neoglycolipids with PPEADP. For chroraatograhic studies, the conversion was carried out i j i situ. When bound to PVC plates or to the chromatograhic support, it was possible to effect a variety of immunoiogical tests on the oligosaccharides which in this immobilised form were presented in spatial configuration which permitted antibody binding. The results of this work are reported in Eur.J. Biochem. (1986) 157, 385-391

Claims: CLAIMS 1. A biochemical reagent comprising a conjugate of an oligosaccharide and an immobilising carrier, said oligosaccharide being bound in spaced relationship to the carrier by means of an interposed spacer molecule whereby the oligosaccharide is presented in antigenically active steric configuration for binding on encountering an antibody. 2. A reagent as claimed in claim 1, in which the spacer molecule is a lipid and the combination thereof with the oligosaccharide is a neoglycolipid. 3. A reagent as claimed in claim 2, in which the lipid has at least two hydrophobic chains. 4. A reagent as claimed in claim 3, in which the neoglycolipid has the general formula: R 2 \ R J NH X — R ' / R 4 1 2 where R represents a carbohydrate group, R 3 represents an alkyl, alkoxy or alkoyl group, R 4 represents an alkyl, alkoxy or alkoyl group, R represents an alkyl, alkoxy or alkoyl group, or 1 1 A hydrogen, R , R and R being the same or different groups, z is zero or 1,2, or 3 and X represents a linking group selected from: O II - M 2 ) —O -P— O — C ^ , I ό O I O " I I I 5. A reagent as claimed in claim 2 or 3 or 4, in which the group X is a residue of a compound selected from phosphatidyl ethanolamine dipalmitoyl (PPEADP) , L-alpha- phosphatidyl-L-serine, L-alpha-phosphatidyl- ethanolamine (PPEA) dilauroyl, PPEA dimyristoyl, PPEA distearoyl, PPEA beta-oleoyl-gamma-palmitoyl. 6. A reagent as claimed in any preceding claim, in which the oligosaccharide is derived from an glycoprotein or proteoglycan. i 7. A reagent as claimed in any preceding claim, in which the carrier is selected from a chromatographic support material, a plate of plastics material and a liposome. 8. A reagent as claimed in claim 7, in which the plastics material is polyvinyl chloride. 9. A reagent as. claimed in claim 7, in which the chromatographic support is silica gel. 10. A method of preparing a biochemical reagent, comprising reacting a reducing oligosaccharide and an amine derivative of a spacer compound to produce a neoglycolipid and bonding the reaction product to an immobilising carrier. 11. A method of preparing a biochemical reagent as claimed in claim 10, in which the oligosaccharide is liberated from an immunogenic glycoprotein or proteoglycan. - 39- 12. A method of preparing a biochemical reagent as claime in claim 11, in which the oligosaccharide is liberate by enzyme action. 13. A method of preparing a biochemical reagent as claime in claim 11, in which the oligosaccharide is liberate by the action of alkaline borohydride, followed by oxidation with periodate to form an aldehyde group. 14. A method of preparing a biochemical reagent as claime in claim 11, in which the oligosaccharide is liberate by the action of hydrazine, followed by acylation and oxidation with periodate to form an aldehyde group. 15. A method of preparing a biochemical reagent as claime in any of claims 10 to 14, in which the amine and the oligosaccharide are reacted in the presence of cyanoborohydride to form a neoglycolipid. 16. A method of conducting a biochemical assay, comprisin providing a biochemical reagent comprising a conjugat of an oligosaccharide and an immobilising carrier, said oligosaccharide being bound in spaced relationship to the carrier by means of an interposed spacer molecule whereby the oligosaccharide is presented in antigenically active steric configuratio for binding on encountering an antibody, contacting said conjugate with an antibody to the oligosaccharide whereby binding of the antibody and the oligosaccharide moiety of the conjugate occurs and detecting that with an analytically detectable reagent. 17. A method of conducting an immunoassay as claimed in claim 16, including the step of treating the conjugate-bound antibody with a second conjugate of an oligosaccharide with a liposome carrier. 18. A method of conducting an immunoassay as claimed in claim 16, in which the analytically detectable reagent is a radiolabelled antibody to the bound antibody and detection thereof is effected by autoradiography. 19. A biochemical assay kit comprising a test plate having thereon an immobilised oligosaccharide, as claimed in any of claims 1 to 9 , a reagent comprising a solution of a neoglycolipid of which the glyco group is the said oligosaccharide and a further reagent comprising a solution of liposome-forming components.

Inventors: FEIZI TEN, TANG PING WOON, FEIZI, TEN, TANG, PING, WOON
Applicants: RESEARCH CORP, RESEARCH CORPORATION LIMITED
Application year: 1986
Main classification: G01N33
IPC codes: G01N33