Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

• PROTEINACEOITS SPECIFIC BINDING AGENTS AND MATERIALS BINDABIE THERETO
• This invention concerns improvements in or relating to processes and materials for detecting and determining proteinaceous specific binding agents and materials bindable thereto.
• the invention has particular application for example to the detection and determination of antibodies and their corresponding antigens or haptens, among other materials which take part in avid specific binding reactions.
• haptens and antibodies for which analytical methods have been devised are for example insulin and anti-insulin; human chorionic gonadotrophin and antibodies thereto; antibodies against disease-producing organisms such as Toxoplasma, Entamoeba and Treponema, and antigenic components of such organisms themselves; virus components such as hepatitis antigen and antibodies thereto; normal and abnormal tissue components, derivatives thereof and antibodies thereto, such as proteins including immunoglobulins themselves such as IgG and IgM, and degradation products thereof, such as Fc fragments of IgG, low molecular weight hormones, such as oestradiol; nutritional factors such as folate; and drugs such as opium alkaloids and cardiac steroids such as digoxin.
• materials for the detection or determination of an analyte which is a component of the reaction between proteinaceous binding agents and materials bindable thereto, in which one of the reaction components, i.e. a binding agent or a material bindable thereto is fixed or coupled to the surface of vesicles which contain a quantity of enzyme-containing aqueous material which is occluded from contact with the bulk of the suspending medium in the absence of lytic conditions, and in which one of the reaction components is immobilised on a solid carrier in a form which is able to accept the binding of a complementary reaction component when fixed or coupled to the vesicles.
• This latter immobilised component is referred to herein as an immunoadsorbent.
• the materials for the detection or determination of an analyte which is one of the reaction components can conveniently be in the form of a test kit having dosed quantities of the components in a form suitable for their application to assay. They can also include a lytic agent for releasing or rendering accessible the contents of vesicles, for reacting with vesicles which have taken part in a detection or determination reaction, and a substrate yielding detectable product upon which the enzyme can react once released or rendered accessible by the lytic agent.
• binding specificities of the reaction components fixed or coupled to the surface of the vesicles and immobilised on the solid carrier can take any of a very wide variety of forms and combinations which in themselves can be analogous to the specificities and configurations of known binding assays and immunoassays and in themselves do not constitute this invention. They will be determined by the identity of the material which it is desired to assay and by the desired assay configuration. A number of examples of these are given below to facilitate understanding of the present invention and illustrate particular applications of it.
• the materials provided according to the invention are used according to the invention in a binding assay method for one of the above-mentioned reaction components in which both the vesicle-fixed or -coupled reaction component mentioned above, and the immunoadsorbent capable of accepting the binding of vesicles which have a complementary reaction component bound or coupled thereto, are used, and in which the detection or determination of a portion of the vesicles which has reacted in an amount which is dependent upon the amount of any analyte present is carried out by lysing any such reacted vesicles with the lytic agent so as to release or render accessible the enzyme contained therein, and determining the amount or presence of any such released enzyme by reaction with a substrate that gives rise to a detectable product.
• an assay method according to the invention comprises:
• an antibody B to an antigen A (to be detected or determined), which can for example be an amphipathic antigen, onto an activated solid surface to form immunoadsorbent;
• an assay method according to the invention can comprise:
• the sample contains analyte (antigen A)
• this will react with B fixed on the vesicles; then there will be less free B available to react with the immobilised A, and the level measured in step 5 will be correspondingly reduced as compared with the level found in the absence of analyte in the sample.
• analyte antigen A to be determined is at least bivalent
• a preferred process according to the invention can involve the following steps:
• the amphipathic antigen is bound to the bifunctional complex B' which consists of an antibody C specific to the amphipathic antigen and an antibody D to antigen A.
• Antibody D can be a preparation similar to antibody B.
• Antibody D can alternatively be capable of binding to antigen A through different antigenic determinants of antigen A, as compared with the specificity of antibody B.
• any adsorbed enzyme has suffered either chemical coupling or avid binding in a way which generally affects both its activity, (whether by denaturation or the kinetic effect of adsorption or steric hindrance, or by more than one of these causes), and its ease of handling. Furthermore, it is easy to arrange for the occluded aqueous environment of the enzyme within the vesicles to be relatively conducive to its satisfactory storage and preservation, and after such occlusion it remains physically relatively unaffected by subsequent stages of the preparation and assay.
• materials and methods of this invention can provide a "double amplification" in the number of detectable product molecules relative to the number of analyte molecules which is greater than either the amplification available with single enzyme molecules chemically coupled to immunological reagents or that given by previous attempts to "amplify" enzyme-linked immunoassay in that both the ratio of the molecules of enzyme associated with a given number of molecules of analyte or its complementary reaction component can be higher, and also the state of preservation of activity in those enzyme molecules can be better, i.e. more active, because of their occlusion during many of the reaction stages, as mentioned above. This can lead to highly satisfactory sensitivity relative to background in a suitable assay configuration.
• the vesicles referred to in this specification are in themselves known, as sac-like structures, e.g. liposomes, which are artificial spherules composed of phospholipids arranged to form a bi-lamellar membrane in which polar groups are arrayed externally and and hydrocarbon chains form a hydrophobic core.
• liposomes can be prepared according to the method of Gregoriadis et al, PEBS letters, 14, 95 (1971) or according to the method of Batzri et al Biochem. Biophys. Acta, 298, 1015. Because of the hydrophobic core it is possible to introduce a variety of amphipathic antigens into the membrane, to give an immunological valency to the liposomes.
• One very convenient such material is bacterial lipopolysaccharide, which can conveniently be that obtained from E.coli 0149 strains (well known and commercially available).
• liposomes are referred in for example US Patent No. 3,850,578, which concerns free-radical-loaded liposomes used in agglutination-type and complement-mediated immunological reactions.
• One method of preparing liposomes useful for example connection with the present invention is to suspend a membrane-forming lipid mixture, comprising phosphatidyl ⁇ choline, phosphatidylethanolamine and sterol, into an aqueous medium containing bile salt or equivalent detergent, e.g. deoxycholate, any amphiphilic antigen to be incorporated into the sacs, e.g. lipopolysaccharide 0149, and any enzyme to be occluded in the liposomes.
• bile salt or equivalent detergent e.g. deoxycholate
• any amphiphilic antigen to be incorporated into the sacs, e.g. lipopolysaccharide 0149, and any enzyme to be occluded in the liposomes.
• the detergent and non-occluded materials can then be removed by gel-filtration.
• suitable vesicles e.g. liposomes
• the vesicles can for example have a volume per particle corresponding to diameters of equivalent spheres (or can be roughly spherical with diameters) of about 0.01-10 microns.
• the methods described herein can result in the occlusion of for example 3 to 30 microliter, e.g. 10 microliter, of enzyme-containing liquid per micromole of lipids used to form the liposomes.
• the vesicles have fixed or coupled thereto an immunologically reactive material of specificity appropriate to the determination of interest.
• an immunologically reactive material of specificity appropriate to the determination of interest.
• This can be achieved for example by fixation of an antigen into the structure of the vesicle, e.g. lipopolysaccharide 0149 of E.coli, or by coupling of a desired immunologically reactive material to the vesicle by covalent bonding using a chemical coupling agent or by avid binding, or both.
• a material desired to be coupled to the vesicle can be coupled to a component of the vesicle, e.g.
• a coupling reagent that reacts with said component to give an activated product that subsequently couples covalently with the material to be coupled.
• Suitable coupling reagents include for example dialdehydes such as glutaraldehyde, and adipimidate (see for example GB Specification 1,470,955). It is generally desirable to use coupling agents that give rise to linkage groups between vesicle and coupled material that are as long as possible, to avoid steric hindrance, e.g.
• the material to be coupled in this way can be for example an immunoglobulin, a protein or polypeptide antigen, or a non-protein antigen capable of reacting with the coupling agent, e.g. having a free aminogroup.
• An alternative or additional and desirable coupling method consists of binding to an immunologically reactive group which itself is fixed or coupled to a vesicle, e.g. by one of the methods just described above, a compound bearing immunological valency of the complementary specificity, optionally Itself chemically or physically coupled to a material having a desired further immunological specificity.
• a compound bearing immunological valency of the complementary specificity optionally Itself chemically or physically coupled to a material having a desired further immunological specificity.
• liposomes bearing bacterial lipopolysaccharide can be treated with antibody thereto, so that they bear an immunological valency corresponding to the antigenic type of the immunoglobulin constituting the antibody.
• the liposomes can be reacted with a bifunctional coupled complex, consisting of such an anti-lipop ⁇ lysaccharide antibody which is chemically coupled to a further antigen or antibody of interest, e.g. anti-(human alpha-fetoprotein), e.g. by reaction with glutaraldehyde, or ethyl N-(carbomoyl-cyano-methyl)-1-iminoacetate followed by diazotisation and further coupling (see GB Specification No. 1,316,990).
• the resulting liposome preparation then has an immunological valency with the specificity of the anti-(human alpha-fetoprotein) or other material coupled to the anti-lipopolysaccharide.
• a protein fixed or coupled to liposomes can have its complementary binding partner coupled thereto by their avid binding reaction, and the resulting complex covalently linked by treatment with dimethyl suberi ⁇ iidate (see Gersten and Marchalonis, J. Immunol.
• an alternative covalent bridging reagent for example, protein-A from Staphylococcus aureus can be coupled to vesicles using glutaraldehyde, and bound by Its characteristic affinity to the Fc group of an immunoglobulin having a desired antibody specificity. The linkage between protein A and the bound antibody can then be made covalent by reaction with the dimethyl suberimidate, to give a vesicle preparation with the specificity of the antibody.
• an immunologically reactive material of desired specificity is coupled to the vesicles, e.g.
• liposomes via a linkage comprising a linkage group which is equivalent in length at least to a carbon chain of 5 atoms, e.g. up to 20 atomsthe linkage may comprise a plurality of such linkage groups and/or one or a plurality of protein molecules.
• the particular coupling methods chosen aim to provide the desired immunological valency sufficiently free of steric hindrance to react with an immunosorbent of suitably complementary valency, as described below.
• the methods described herein can be used to give attachment of 0.3-10 micrograms, e.g. 1-5 micrograms, of antibody to liposomes for each micromole of lipids used to form the liposomes.
• Each liposome may have a number of effective binding sites for example in the range 10-100 per liposome.
• vesicle preparations to those noted above can incorporate (instead of bacterial lipopolysaccharide) other amphiphilic antigens, such as erythrocyte membrane antigens and myelin antigens.
• the result and desired effect is to produce according to the invention a composition of lipid vesicles having an aqueous preparation of an enzyme releasably occluded therein, said vesicles having fixed to their surfaces via a spacer group or spacer molecule a homologue or specific binding partner of an analyte to be detected or determined.
• the spacer molecule or group can be for example the residue of a dialdehyde group of at least 5 carbon atoms (or the residue of a coupling reagent of at least equivalent molecular size or length thereto the residue of dimethyl adipimidate, protein A of S.aureus, or antibody directed against amphipathic lipid such as lipopolysaccharide included in the vesicles.
• compositions form the test kits described herein.
• the methods given above can be used to produce vesicles loaded with enzyme and sensitised by fixation of an Immuno logically reactive material via a. spacer molecule or linkage group sufficiently large to leave the reactive material adequately unhindered sterically for binding to sensitised immunoadsorbent.
• the enzyme which is chosen for its occlusion in the vesicles can be any enzyme of choice. The most desirable those available, in quantity with high activity and stability and which possess safe convenient substrates leading to easily measurable products.
• Hydrolases, oxidases and reductases provide suitable examples.
• the most preferred convenient enzyme is alkaline phosphatase for which pnitrophenyl phosphate is a convenient substrate leading to a convenient easily measurable yellow product, p-nitrophenol.
• Another convenient enzyme is horseradish peroxidase, usable with substrates hydrogen peroxide and chromogen (see, e.g., GB Specification No. 1,348,935/8).
• the nature of the immunoadsorbent is an important feature of the invention, in combination with the remaining material characteristics described herein.
• the solid support must be in a form which when immunologically sensitised, can accept the binding of immunologically sensitised vesicles having suitable valency
• the solid support has a solid (i.e. nonporous) surface, e.g. that of a solid polymer such as polystyrene or nylon, to which immunologically reactive material can be coupled.
• a solid polymer such as polystyrene or nylon
• inorganic solids to which such material can be coupled.
• antigen or antibody can be coupled to nylon using adipimidate reagent after partial hydrolysis of nylon to yield amino groups, e.g. as the use of this coupling agent is described in GB Specification No. 1,470,955.
• antibody can be adsorbed to polystyrene.
• a suitable preparation and precautions are for example exposure of the polystyrene surfaces to the antibody (e.g. about 5 ⁇ g/ml) and incubation, e.g. overnight at 37°C, followed by conducting any further reactions with materials which do not include detergent (until it or another lytic agent is finally required to lyse any adherent vesicles) and conducting any further reactions in the presence of ovalbumin (e.g.
• the form of the solid surfaces carrying the immunoadsorbent reactive groups is generally immaterial in the practice of the invention: convenient forms of solid support are for example tubes, wells, granules, grains, flecks, plates and slides of the material forming the solid support surface.
• a tube carries a binding reagent or its specific binding partner, it can be made to fit automated continuous flow equipment so that certain embodiments of the tests and assays can be integrated into automatic analyser systems with exchangeable tube-boundbinding reagents.
• the lytic agents used according to this invention can be any suitable means for releasing enzyme from Its occluded state in the vesicles.
• Suitable lytic agents include for example detergents, such as Triton X-100, but may consist of hypotonic solutions when the tonicity of the occluded phase is sufficiently high to lead to lysis of the vesicles in more dilute solutions.
• the final essential component of the test materials is a substrate for reacting with the occluded enzyme after its release from the vesicles.
• Any convenient substrate giving a conveniently measurable product is suitable. Examples include p-nitrophenyl phosphate for use with alkaline phosphatase, giving a yellow product p-nitrophenol; 4-methyl-umbelliferyl phosphate for use with alkaline phosphatase giving a highly fluorescent product 4-methyl-umbelliferone; and hydrogen peroxide and any of several chromogens for use with horseradish peroxidase, leading to easily measurable coloured products. Many other examples have been describe in connection with enzyme-linked immunoassay systems and are applicable here too.
• materials can be provided for a competitive binding assay or test in which enzyme-loaded vesicles bearing a specific binding valency homologous to that of the analyte are provided together with immunoadsorbent having a limiting quantity of the complementary specific binding valency.
• the assay can then be carried out by mixing the analyte and assay or test materials, separating bound and free vesicles, lysing one of the separated vesicle portions and allowing the liberated enzyme to act on its substrate, yielding a measurable product.
• Other competitive binding configurations are Illustrated for example by the enzyme-coupled assay schemes of US Patent No. 3,654,090, 3,839,153 and 3,850,752. In modifications of these configurations the reactants can be reacted sequentially instead of competing simultaneously.
• a preferred assay or test configuration involves test materials in which the enzyme-loaded vesicles and the immunoadsorbent can both bind to the analyte but not to each other.
• the "sandwich” configuration is generally applicable only to antibodies and their binding partners which themselves are of high molecular weight so that they are bivalent, i.e. have two or more specific binding sites. The other configurations are used when low molecular weight binding partners are the analytes, e.g. digoxin and haptens, which generally are monovalent.
• the present invention is applicable to the detection and determination of a very large variety of materials that take part in avid specific binding reactions: wherever immunological reagents or reaction components are referred to herein, analogous non-immunologlcal reagents or reaction components are also understood as included.
• alpha-fetoprotein examples are alpha-fetoprotein, carcinoembryonic antigen, thyroxin binding globulin, pregnancy-associated alpha(2)glycoprotein, antibodies to Toxoplasma organisms, hepatitis antigen, rubella antigens, allergens, immunoglobulins E (reagins), Treponema antigens, cardiollpln, haemoglobins, immunoglobulins M, digoxin, insulin, human chorionic gonadotrophin, streptococcal antigens, bacterial lipopolysaccharides, and antibodies corresponding to those of the above-mentioned materials that are antigens. Further materials which can be detected and determined by the use of assay materials described herein and having corresponding specificity are listed for example in US Patent No. 3,996,345 ⁇
• assays are carried out in buffered aqueous media, e.g. pH 5-5 to 10, often 6.5 to 9, the buffers being inert and for present purposes free of detergent at least until the stage of the assay when the lytic agent is called for.
• Citrate and phosphate are suitable buffer examples.
• present can be non-detergent inhibitors of non-specific binding phenomena, e.g. inert proteins such as purified ovalbnmin. from hen's egg, e.g. at 0.1-1% w/v.
• the buffer solution can be salinated, e.g. with NaCl to be isotonic with about 0.9% NaCl.
• concentrations of analytes which can be of interest in the assays can be for example in the wide range of about micromolar to fractions of picomolar, e.g. 10 -6 M to 10 -13 M although with, certain assays concentrations outside these ranges may be of interest and detectable.
• kits of assay materials the quantities of assay materials are chosen to set their quantities in relation to the analyte concentrations of interest.
• a vesicle preparation or immunosorbent which iscomplementary (and can bind) to the analyte can be present from about 0.1 times the minimum concentration to be detected up to about 100 times the maximum to be detected, based on equivalence of binding capacity; usually up to about 10 times the maximum to be detected.
• Another component of the reaction can be present either in excess or in comparable quantity, depending on the chosen assay configuration.
• the components are present in amounts in which the quantity of sensitised vesicles reacting with the immunoadsorbent is dependent on the quantity of analyte present in the concentration range of interest.
• the assays and tests can be carried out so that the binding reactions reach equilibrium, or alternatively so that the amount of product depends on the rate of a critical reaction carried on for a standard time.
• the reaction times for each assay step where more than one can range from 0.1 minute to about 8 hours, preferably about 1-30 minutes, e.g. 5 to 15 minutes.
• the required reaction times where a ratedependent assay is used can be adjusted within limits by choice of reagent concentration: standardised reaction times of 15 seconds to 30 minutes can be used, at standard temperatures, e.g. 15°C-40°C.
• the order of reagent additions will usually be dictated by the assay configuration chosen.
• assays In carrying out the assays and tests and preparing the test materials, it is desirable to avoid non-specific adsorption of materials important in the assay both to vesicles and to immunoadsorbents, and for this purpose assays can generally be carried out in the presence of a non-detergent inhibitor of non-specific adsorption, e.g. an inert protein (e.g. ovalbumin where this particular material plays no part in the assay in question), and in the absence of detergent until this or another lytic agent is called for to release enzyme from the vesicles at the appropriate time.
• a non-detergent inhibitor of non-specific adsorption e.g. an inert protein (e.g. ovalbumin where this particular material plays no part in the assay in question)
• an inert protein e.g. ovalbumin where this particular material plays no part in the assay in question
• the quantity of product from enzyme liberated from vesicles measured at the end of a given test will show either a direct or inverse relation to the quantity of analyte over a region of dependence on analyte concentration, usually accompanied by a background value.
• low background levels can normally be arranged in assay configurations using bivalent analytes, such as large molecular weight antigens and antibodies, in a "sandwich" format in which immunoadsorbent and sensitised vesicles each bind specifically to the analyte but not to each other. Larger background levels are naturally encountered where there is an inverse relation between analyte concentration and final measured enzyme product.
• the invention also provides a test complex for the detection or determination of an analyte selected from proteinaceous binding reagents and their specific binding partners, said complex comprising said analyte in combination with (a) a reaction component selected from specific binding partners and homologues of the analyte in coupled form to a surface of a solid support material, and (b) a reaction component selected from specific binding partners and homologues of said analyte coupled to the surface of lipid vesicles having releasably occluded therein an aqueous preparation of an enzyme.
• the complex will be a "sandwich” type complex with sensitised vesicles bound to the immunoadsorbent via the analyte, and in others the immunoadsorbent will have some of its binding sites occupied by analyte and some occupied by sensitised vesicles of complementary binding specificity.
• the invention also embraces the complementary alternative embodiments in which the place of antigen is taken by antibody and vice versa.
• the following procedure can be used to produce a suspension in aqueous medium of liposomes having entrapped enzyme (in this instance it is alkaline phosphatase) in a separate aqueous phase occluded therein, and incorporating antigenic lipopolysaccharide from E.coli 0149 in the liposome structure, (a) E.coli 0149 E91 (116) "Abbotstown” strain (commercially available) or an equivalent strain of the well-known E.coli 0149 is grown on 100 ml of BAB culture medium (4%) + 0.1% glucose (w/v) in Roux flasks for 48 hours at 37°C.
• the resultant bacterial lawn is scraped off the surface in about 2 ml saline per flask and the flasks washed in saline before autoclaving at 121° and 15 psi.
• the autoclaved preparations are centrifuged and a clear supernatant rich in lipopoly saccharide is separated, and concentrated to one-tenthvolume by dialysis against polyethylene glycol (20M grade).
• the pellet from the centrifugation is extracted into an equal volume ofphenoltwater (90:10 v/v) and is dialysed successively against water (to remove phenol) and polyethylene glycol (to concentrate).
• the two lipopolysaccharide concentrates are pooled, heated at 56°C in 0-5M NaOH for 1 hour, returned to pH 7 with HCl and dialysed 18 hours against water, before freeze-drying in the presence of 0.1% glucose as cryopreservative.
• this lyophilisate is taken up at the rate of 5 mg/ml in citrate or other suitable buffer and sonically disrupted to give a slightly opalescent solution.
• Equivalent preparations of lipopolysaccharide can be used if desired.
• Hpids and aqueous material containing lipopolysaccharide e.g.
• alkaline phosphatase e.g. Sigma Type 711 (Trade Mark)
• alkaline phosphatase e.g. Sigma Type 711 (Trade Mark)
• the lipids used are egg yolk lecithin, cholesterol and L-alpha-phosphatidylethanolamine (molar ratio 7:2:1) dissolved in chloroform at respective concentrations 11.2 mg/ml, 1.6 mg/ml, 0.15 mg/ml.
• the mixed chloroform solution is evaporated under reduced pressure at 50°C to form a film of solid in the containing vessel, which is then suspended in a volume of 0.1M bicarbonate buffer pH 7.2 equal to that of the chloroform removed, also containing 0.25 mg/ml alkaline phosphatase, 0.24 mg/ml lipopolysaccharide 0149 and 4.2 mg/ml sodium deoxycholate.
• the milky suspension first obtained is ultrasonically disrupted at 0-4°C until it becomes translucent. At this time the suspension contains mixed micelles of lipid and deoxycholate.
• the deoxycholate is removed by gel-filtration on cross-linked agarose (Sepharose 4B (Trade Mark)): the liposomes appear in the eluate close to the void volume.
• Inaccessibility to substrate of the occluded enzyme in the product liposomes can be verified by testing aliquots of the liposome preparation (0.1 ml) with 1 ml of pH 9.8 carbonate buffer containing chromogenic substrate (pnitrophenyl phosphate) 1 mg/ml, in respective tests in the absence and in the presence of a lytic agent and liberating agent for the occluded phase, detergent Triton X-100 (0.1% w/v).
• the eluate fractions showing; highest activity in the presence of detergent are pooled. In the absence of lytic agent only low background levels of enzyme activity are seen.
• liposomes having enzyme occluded therein and bearing antigen on their surfaces can be used for assays according to the invention.
• the liposome preparation described here is found on electron-microscopic examination under negative staining to comprise particles of average size about 1500 angstroms, composed of one or a few bilayers with large internal spaces.
• Preparation 2 liposomes having material of another immunological specificity than that of lipopolysaccharide of E.coli (i.e. of any desired specificity), bound to their surfaces can be made by the following procedure.
• Rabbit or sheep antibody specific for E.coli 0149 lipopolysaccharide is obtained as the IgG fraction of pooled immune rabbit or sheep sera showing high-titre antibody activity against the lipopolysaccharide after immunisation in the normal way.
• Sheep antibody specific for human, alpha-fetoprotein is obtained in conventional manner (also commercially available, from Seward Laboratory, UAC House, Blackfrlars Road, London SEl, England, under code BN516 (nephelometrlc grade)).
• the anti-(E.coli 0149) is coupled with the anti-(human alpha-fetoprotein)inthe following way.
• Anti-(E.coli 0149) globulin fraction (6 mg/ml) is reacted for several hours at room temperature with 20 mg/ml ethyl N-(carbamyl-cyanomethyl) l-iminoacetate and dialysed against saline buffer pH 7-2.
• the resulting antibody solution (derivatisedwit a substituted 2-amino-1-imidazolyl group) is diazotlsed at 4oC by the successive addition with mixing of 1.5 volumes 0.4% aqueous NaNO 2 , 1.5 volumes of N aqueous HCl and 1.5 volumes of 2% aqueous ammonium sulphamate. After 20 seconds subsequent reaction at 4°C the diazonium product is added dropwise to an excess of sheep anti-(human alpha-fetoprotein) in pH 4.7 buffered saline (10 mg/ml) at 4°C, controlling the (falling) pH to its original value with N aqueous NaOH, and after 10 minutes raising the pH to 7.2. After several hours storage at 4°C and centrifugation, the supernatant containing coupled protein product is taken and stored at -20oC till further use.
• One volume of the coupled protein product (about 5 mg/ml), and one volume of the liposome preparation of Preparation are mixed at room temperature 30 minutes, and treated by gelfitration over cross-linked agarose (Sepharose 4B). Close to the void volume there are eluted fractions containing the product, liposomes as of Preparation 1 having bound thereto coupled complexes of anti-(E.coli 0149)-anti-(human alphafetoprotein). (Uncoupled proteins are separated by the gel filtration and emerge in later eluate.) These are suitable for the assay of human alpha-fetoprotein.
• anti-(human alpha-fetoprotein) there can be used in the preparation other antibodies or antigens, such as for example sheep anti-(human IgM/Fc) or sheep anti-(human pregnancy-associated alpha (2) glycoprotein) in which case the resulting product is useful in assays with the corresponding specificity, or the antibody can be substituted with antigen such as, for example, purified human alphafetoprotein itself, in which case the product is suitable use in alternative assay configurations as described above corresponding to this specificity.
• Preparation 3 Preparation 3
• Preparation 1 is reacted with an equal volume of 0.25% w/v glutaraldehyde in 0.1M bicarbonate buffer pH 7.2. for 10 minutes at room temperature and dialysed against 0.1M bicarbonate pH 7.2 for several hours to remove unreacted glutaraldehyde completely.
• the liposomes bear free aldehyde groups on their surfaces, at the distal end of substituents derived by condensation of one aldehyde group of glutaraldehyde with the ethanolamine amino group of each reacted phosphatidylethanolamine molecule of the liposomes.
• the activated liposomes are incubated at room temperature (24 hours) with 0.1 mg/ml of the protein of desired immunological specificity to be coupled thereto, e.g. sheep anti-(human alpha-fetoprotein), human alpha-fetoprotein itself, or sheep anti-(human lgE) or other desired antigen or antibody.
• the protein of desired immunological specificity e.g. sheep anti-(human alpha-fetoprotein), human alpha-fetoprotein itself, or sheep anti-(human lgE) or other desired antigen or antibody.
• the protein-A from Staphylococcus aureus (which has a binding avidity for immunoglobulin G) is obtained in known manner and coupled to liposomes loaded with alkaline phosphatase in the manner described in Preparations 1 and 3 above.
• the protein-A is used as the protein to be coupled to the aldehyde-activated liposomes.
• the so-treated liposomes (in pH 8.5 buffer, 0.1M bicarbonate) are mixed with an excess (on a protein:coupled protein molar basis, e.g. 0.1 mg protein per ml of liposome preparation) with immunoglobulin G and incubated for several hours up to about a day at room temperature.
• a tenfold weight excess (based on the immunoglobulin) of dimethyl suberimi date is gradually added to the mixture and if necessary the pH is readjusted to pH 8.5. After 30 minutes at room temperature the mixture is dialysed overnight against carbonate buffer containing 0.1M ethanolamine and separated from low molecular weight reagents and unlinked protein by elution from Sepharose 4B as described in Preparations 1 to 3.
• the product has immunoglobulin G of the chosen specificity covalently bound to the liposome by a protein-A bridging group linked covalently both to the immunoglobulin and to the phosphatldylethanolamine molecule of the liposome by glutaraldehyde-derived and suberimidate-derived links respectively, and is useful for assay according to this invention for an analyte corresponding to the specificity of the immunoglobulin used.
• Dipalmitoylphosphatidylcholine (83 micromole, 65 mg) is dissolved in each 7.5 ml of isopropyl ether and each 2ml methanol at 45°C.
• phosphate buffered solution containing 0.5 mg lipopolysaccharide e.g. prepared as described above and 2.5 mg alkaline phosphatase was added. The mixture is thoroughly disrupted ultrasonically (aoubt 15 minutes) at 45°C.
• Antibodies, or antigens can be formed into immunosorbents by linkage to nylon surfaces via glutaraldehyde to give products which can accept the specific binding of suitably activated liposomes, by the following method.
• Nylon cups 7 mm by 9 mm diameter e.g. shaped as standard microtiter trays, or as individual cups, are filled with 3.5M HCl and incubated at 50°C for 1 hour to expose free amino groups by hydrolysis. After thorough washing in phosphate buffered saline (pH 7.2) the cups are filled with glutaraldehyde solution in distilled water (12.5% v/v), chilled to 4° to 6°C. After 30 minutes at 4° to 7°C and thorough washing in phosphate buffered saline, an antibody or antigen to be coupled and itself having free amino groups is added to the cups as a solution at 0.02 to 3 mg/ml concentration and incubated there 16 to 18 hours at 37°C.
• phosphate buffered saline pH 7.2
• the buffer for this stage e.g. pH 7.2 phosphate saline
• the buffer for this stage e.g. pH 7.2 phosphate saline
• the immunosorbent is prepared for use by washing in plain buffer, and blocking of any residual activated coupling groups by exposure to ethanolamine (1.5M) as the chloride (pH 8.5 with HCl) for 1 hour at 37°0, followed by further washing in plain buffer.
• Materials according to an example of this invention can be prepared as follows.
• a preparation of liposomes having alkaline phosphatase occluded therein, and bearing immunological valency with the specificity anti-(human alpha-fetoprotein) coupled to their surface is prepared according to Preparations 1, 3 and 4 above (using anti-(human alpha-fetoprotein) (obtainable from Seward Laboratory) as the immunoglobulin G used in Preparation 4).
• the preparation is kept free of detergent and made 1% w/v with purified ovalbumin.
• An immunoadsorbent is prepared according to Preparation 6, again using anti(human alpha-fetoprotein) as the antibody to be coupled.
• the remaining materials required for the assay are a lytic agent, in this case 0.1% w/v Triton X-100 detergent, and phosphate substrate obtainable commercially as watersoluble pellets, which are dissolved in water before use.
• Assay is carried out by filling each of a set of similarly prepared Immunoadsorbent cups with dilutions of analyte samples suspected of containing alpha-fetoprotein, dilutions of standard solutions of alpha-fetoprotein (including saturation and null controls), all free of detergent and made 1% w/v in ovalbumin (purified).
• the assay for alpha-fetoprotein given above can be adapted for use as an assay for human chorionic gonadotrophin as follows.
• Assay materials are provided as a liposome preparation made as described above in Preparations 1, 3 and 4 above, using anti-(human chorionic gonadotrophin) as the immunoglobulin G in Preparation 4. The preparation was kept free of detergent and made 1% with purified ovalbumin.
• An immunoadsorbent is prepared according to Preparation 6, using, human chorionic gonadotrophin as the antigen to be coupled to the solid support. The remaining assay materials used are as in Part (1) above.
• a limited standardised quantity of the vesicle preparation is used after quantitation of its binding capacity for human chorionic gonadotrophin by standard method.
• the limited quantity chosen is in excess of the highest concentration (at lowest dilution) of interest in the assay.
• the materials are used for assay by exposing the standardised allquots of vesicles bearing anti-(human chorionic gonadotrophin) to dilutions of the analyte possibly containing the hormone of interest, to dilutions of standard solutions of it, and to saturation and null controls. After 2 hours at 37°C the reaction mixtures are transferred to the immunoadsorbent cups and incubated for a further 2 hours at 37°C. Then the liquid phase is removed from each cup. Either the removed liquid in this case or the remaining well surface can be treated with detergent to release enzyme from the liposomes, and treated to establish corresponding calibration curves and assay results in the usual way.
• the coloured product quantity is directly related to the amount of analyte present, and where the enzyme released from the immunoadsorbed liposomes is used, the quantity of coloured product is inversely related to the amount of analyte present.
• This Example illustrates materials for the immunoadsorbent immunoassay using enzyme-loaded liposomes in the most simple form.
• liposomes filled with an occluded aqueous preparation of alkaline phosphatase and bearing on their surface alkali-treated lipopolysaccharide extracted from E.coli, serotype 0149 were suspended in a solution of ovalbumin in phosphate-buffered saline (PBS) and placed in 300 yul polystyrene wells. The inside walls of the wells had been coated with rabbit-derived antibodies to E.coli 0149 organisms.
• PBS phosphate-buffered saline
• the lipopolysaccharide was prepared as follows: Batches of E.coli, serotype 0149, K.91 were cultured in 100 ml lots of blood agar No. 2 (Oxoid) containing 0.2% glucose for 48 hours at 37°C. The cells were removed from the surface by the addition of 2 ml saline (0.85%) and sweeping of the surface with a glass rod. The cells were recovered by centrifugation, washed in saline and then suspended in 0.5% formalin for 16 hours. The formaldehyde was removed by dialysis against distilled water. Lipopolysaccharide was extracted from these killed bacteria by treatment with 90% phenol in water.
• the IPS extract was then treated with alkali, to increase its critical micelle concentration (CMC), and so increase its ability to insert into liposome membranes.
• CMC critical micelle concentration
• the suspension was mixed with an equal volume of N sodium hydroxide solution and heated at 56°C for 1 hour. Finally, the extract was neutralised and then dialysed against 0.1% glucose in distilled water in preparation for freeze-drying.
• the antibodies were obtained as follows:
• the antibodies used were in the form of IgG fractions from the sera of rabbits immunised with E.coli 0149.
• the IgG fractions were prepared from whole sera by (l) double precipitation with sodium sulphate (to 16%) and (2) subsequent ion exchange chromatography on Whatman DE.52 cellulose.
• the immunosorbent was produced as follows:
• Immunosorbent surfaces were prepared by attaching rabbit anti-0149 antibodies onto the inside walls of
• Dy ⁇ atech polystyrene "Removawells”. This was achieved by simply filling the wells with antibody solution in pH 9-8 carbonate buffer at a total protein concentration of 5 ⁇ g per ml, and incubating them in a sealed container at 37oC overnight.
• the enzyme-filled, antigen-sensitised liposomes were prepared as follows: liposomes were prepared from a lipid mixture containing lecithin (16.42 mg), chlesterol (5-80 mg) and dicetyl phosphate (1.20 mg) giving a molar ratio of 2:1.5:0.22. The molar quantity of lecithin used for this preparation of liposomes was 20 ⁇ moles. Alkali-treated IPS (120 ⁇ g) and alkaline phosphatase (1,500 ⁇ g) were included in the aqueous suspending fluid to provide sensitising antigen and marker enzyme.
• the lipid components were dissolved in 25 ml ⁇ f chloroform and were deposited on the inside surface of a 100 ml round bottom flask, by rotary evaporation under reduced pressure at 40°C. Last traces of solvent were removed by placing the flask under high vacuum for 1 hour.
• the dried lipid film was suspended in a 2 ml solution of IPS (120 ⁇ g) and alkaline phosphatase (Sigma Type VII, 1,500 ⁇ g) in PBS. To facilitate dispersion of the lipids, the mixture was agitated by means of "Whirlimix" Vortex Mixer and the milky-white suspension was left to stand overnight at 4oC.
• IPS aqueous suspending medium
• a standard, stock solution by taking up 5 mg of the freeze-dried material in 1 ml of distilled water and subjecting this to sonication until it was only slightly opalescent.
• 24 ⁇ ml were added to the aqueous suspending medium to give required quantity of 120 ⁇ g.
• Free (unentrapped) enzyme was removed from the suspension by chromatography on Sepharose 4B.
• the liposomes eluted from the column in the void volume but free enzyme was retained and came out in later fractions.
• substrate solution nitro phenyl phosphate, 1 mg/ml in carbonate buffer, pH 9-8
• the amount of product formation after one hour was measured in terms of the optical density (OD) at 400 nm.
• OD optical density
• liposomes can be bound by immobilised antibodies, through surface membrane antigens. This binding is specific and is firm enough to withstand a relatively vigorous rinsing process. The specificity is confirmed by the absence of binding, even when a non-specific protein (i.e. ovalbumin) is included in the sample, thus precluding the possibility of non-specific adsorption of liposomes onto a layer of adsorbed protein at the polystyrene/water interface. Finally, the enzyme activity detected in those wells bearing antibodies to 0149 IPS can only be attributed to the binding of liposomes through their surface IPS. The release of this activity upon incubation with substrate and detergent confirms that such bound liposomes can be made to yield their contents quantitatively, as needed.
• a non-specific protein i.e. ovalbumin
• test materials described above are applicable in immunoassay according to the procedures described above, in any of several forms, for example as follows.
• Antibody against E.coli 0149 or its lipopolysaccharide can be assayed by exposing aliquots of the liposomes bearing the lipopolysaccharide to contact with the antibody under assay before placement in the wells. Any antibody present in the material under assay depresses the extent of adherence of liposomes to the well walls. Detection of the vesicles and estimation of their amount is carried out by detergent lysis, exposure to substrate, and optical measurement, as described above. Control and calibration are performed in the usual way.
• E.coli 0149 or its native lipopolysaccharide can be assayed by contacting the material under assay with the immunoadsorbent (well walls coated with antibody) before or during exposure of the immunosorbent to the liposomes. Any antigen present in the material under assay depresses the extent of adherence of liposomes to the well walls. Detection and estimation are carried out as before. Control and calibration are performed in the usual way.
• Antibody against E.coli 0149 or its lipopolysaccharide can also be assayed using a modified Immunosorbent in the system to give a "sandwich" technique.
• a heterologous anti-immunoglobulin G or (even better) anti-Fc globulin directed against the species from which the material under assay comes can be used to coat the walls.
• the material under assay is then exposed first either to the liposomes, or to the well walls, or it is placed in the wells in admixture with the liposomes: then the extent of adherence of liposomes to the well walls is directly related to the amount of anti-E.coli 0149 present in the test material.
• test materials described above can be applied to the immunoassay of for example the human alpha-fetoprotein mentioned above.
• the immunosorbent can be made by using an antibody to human alpha-fetoprotein (instead of the anti-E.coli 0149) to coat the wells in the above-described immunosorbent preparation. Then the ex ⁇ ent of adherence of liposomes to the walls of the well is directly related to the amount of human alpha-fetoprotein in the material under assay. Detection and estimation are carried out as described above. Control and calibration are performed in the usual way.
• substitutions can be severally made in each and every one of the embodiments of the test methods and materials described hereinabove with the achievement of satisfactor results. Where such substitutions are made, the substrate for the enzyme is of course omitted, and the released marker measured after exposure of the vesicles to the lytic agent if need be. Any marker substance, i.e. a substance capable of being occluded in the vesicles in place of the enzymes first described herein, can be used instead. Such occlusion of marker substances in the vesicles enables achievement of a high ratio of marker to binding reagent (or its binding partner) in conjunction with releasabllity and the ease of handling conferred by the use of solid-phase reagents.

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• 1980
  • 1980-01-18 JP JP50022780A patent/JPS56500030A/ja active Pending
  • 1980-01-18 ES ES487849A patent/ES487849A0/es active Granted
  • 1980-01-18 GB GB8029219A patent/GB2051360B/en not_active Expired
  • 1980-01-18 AT AT80300172T patent/ATE6698T1/de active
  • 1980-01-18 EP EP80300172A patent/EP0014530B1/en not_active Expired
  • 1980-01-18 AU AU54717/80A patent/AU5471780A/en not_active Abandoned
  • 1980-01-18 DE DE8080300172T patent/DE3066899D1/de not_active Expired
  • 1980-01-18 WO PCT/GB1980/000010 patent/WO1980001515A1/en unknown
  • 1980-01-18 IE IE100/80A patent/IE50457B1/en unknown
• 1984
  • 1984-10-24 AU AU34660/84A patent/AU3466084A/en not_active Abandoned

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