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

Definitions

• the invention relates to a preparation for immunoas- say consisting of a solid support, to which two li- gands are linked, and to a method for producing the preparation.
• Immunoassays are among other things used for quanti ⁇ tatively and qualitatively measuring a single compo ⁇ nent, such as antigens or antibodies in a complex li ⁇ quid, such as blood fractions, secretions, tissue extracts, plant sap, cultures of bacteria, food ex- tracts and similar liquids.
• Antibodies linked to a solid surface are used in many of these assays.
• the primary binding or coating to the surface forms an essential part of the method, in that all the basic properties of the assay depend on particularly the purity of the active component in the coating mixture and of the strength of the bond between the components linked to the surface and the specific complementary components in soluble form in the sub ⁇ sequent immunoassay. Concerning the purity or the relative amount of foreign components in the coating mixture which at the same time are capable of attaching themselves to the solid support, these components will compete with the specific component for the available binding sites on the surface.
• polyclonal and mono- clonal antibodies are decisive since there is a distinction between polyclonal and mono- clonal antibodies.
• the affinity distribution or avi ⁇ dity of polyclonal antibodies may vary from one pre ⁇ paration to the other from the same as well as from different animals.
• Monoclonal antibodies always have the same affinity which will often be insufficient, i.e. of average or low affinity.
• Monoclonal antibodies are furthermore very expensive to develop and produce in large quan- tities.
• immunocom ⁇ plexes are a poorely defined unit. Their production in pure form is carried out by means of e.g. precipi ⁇ tation with low concentrations of polyethylene glycol or by binding on a solid support to Clq or protein A.
• the purpose of the present invention is not to ana ⁇ lyze the composition of immunocomplexes formed in vi ⁇ vo, but merely to provide a fully quantitative immu- nochemical measuring tool.
• the immunoprecipitates of the present invention are thus well-defined at a num ⁇ ber of levels in that they are produced in vitro from monospecific antibody with well-defined titer, and besides measures have been taken to prevent attach- ment of contaminating serum complement factors, etc.
• EP published specification no. 80,108 describes the detection of antigen by means of components of immu ⁇ nocomplexes linked to solid supports, so that antigen and antibody no longer have the form of immunocom ⁇ plexes but are linked separately to the solid sup ⁇ port.
• the present invention aims at an in ⁇ complete dissociation of the immunoprecipitates so that apart from the binding to the solid support, a well-defined population of small immunoaggregates with high antibody activity is" provided.
• This method has turned out td increase the binding capacity of the surface considerably for the complementary anti ⁇ gen.
• Graduated dissociation tests have shown that the antibody activity which can be released at extreme pH-values, i.e.
• the present invention de ⁇ scribes a process which selects immunoaggregates both with regard to size and average antibody binding force.
• the pH-conditions mentioned in the present example used during the dissociation do not form a general recipe in that individual adjustments may be necessary from one measuring system to the other. Species of animal ' and molecular size are important parameters and certain immunization methods create antibodies which possess very low avidity and have little tendency to form optimum immunopre ⁇ ipitations for the present purpose.
• antibody prepa ⁇ rations are often used which are purified by physi ⁇ cal/chemical methods, such as salt precipitation and ion exchange chromotography. Such preparations are free from the main component of globulins but still contain between 95% and 99% of inactive and irrele ⁇ vant antibodymolecules which may take up a lot of room on the surface.
• a preparation for im- munoassay consisting of a solid support, to which two ligands are linked, as referred to in the" pre ⁇ amble of claim 1, and by a method for producing said preparation, as referred to in the preamble of claim 2, said preparation being characterized in that the two ligands are an antigen and the corresponding mo ⁇ nospecific antibody, and that the two ligands origi ⁇ nate from a well-defined immunoprecipitate produced in vitro from an antigen solution and an animal-de ⁇ rived antibody, and a method being characterized in that antigen and antibody from a well-defined immu ⁇ noprecipitate are used as antigen and antibody, and that the immunoprecipitate is in a partially dis ⁇ sociated form during the binding to the solid support in the form of small and highly active immunoaggre- gates.
• sedimentable immunoprecipltates which is a molecular complex between antibodies and their corresponding antigens from soluble antigen and antibody takes place in accordance with the generally known precipitin reaction.
• Antigen is taken to mean protein, nucleic acid, polysaccharide, hapten and cell, and by antibody is meant immunocompound or im- munoglobulins from any species of animal.
• the concept of antibody includes polyclonally produced antibodies as well as monoclonally produced antibodies, monoclo ⁇ nal antibodies, however, often requiring two or more clone products in order to achieve a precipitation.
• the optimum reaction is found at or close to the "point of equivalence", where antigen as well as an ⁇ tibody is quantitatively used in the formation of the immunoprecipitate.
• the optimum ratio between antigen or antibody can be established for instance by weigh- ing or by nitrogen determination on the washed preci ⁇ pitate. Simpler methods comprise turbidimetry, nephe- lometry or visual inspection of milky precipitations.
• Isolation of insoluble immunoprecipitates formed in the liquid phase can take place by simple centrifu- ging and several washes.
• the ability of the complexes to adsorb complement factors will cause a contamina ⁇ tion which can be reduced by adding complement- ⁇ onsu- ming reagents prior to the actual precipitation.
• a partial blocking of the binding of complement factors can be achieved by adding EDTA.
• Dissociation of the immunoprecipitate meaning any i- nfluence which is capable of partially dissociating the bond between antibodies and their corresponding antigens, such as high temperature, high ion strength, high pH, low pH, impact of particular ions (e.g. chaotropi ⁇ ions) and competitive molecules.
• This dissociation thus takes place at high tempera ⁇ ture, high or low pH, influence of particular ions such as chaotropic ions and competitive molecules. Analyses have shown that dissociation at a high pH is best suited for the present purpose.
• This step comprises binding of the components, i.e. antigens or antibodies, to a solid support, which may take place by both covalent as well as non-covalent forces.
• the components i.e. antigens or antibodies
• Binding of molecules to a solid support can take- place by , means of both covalent as well as non- ⁇ qva- lent forces.
• the surface may be in the nature of a plastic surface, e.g. polystyrene, or be composed of
• the support can also consist of other materi ⁇ als possessing the quality that a molecule or a com ⁇ ponent can be adsorbed or linked thereto, either on the basis of the surface structure or on the basis of
• This assay is based on the coupling of immunoprecipi ⁇ tates formed between human placental lactogen and an- ti-HPL-antibody from sheep. After the coupling, HPL as well as anti-HPL is reactive to the complementary soluble component. An enzyme is used as an indicator principle.
• the patient sample containing the unknown amount of HPL is mixed with a defined amount of enzyme conjugated HPL of high purity. After mixing, a volume thereof is transferred to a hole in a polystyrene microtitre tray (NUNC immunoplate), which prior to this has been coated with partially dissociated HPL-anti-HPL immunoprecipitates.
• NUNC immunoplate polystyrene microtitre tray
• Enzyme conjugated HPL and Patient-HPL will then compete for the accessible binding sites on the anti-HP -antibo ⁇ dies on the plastic surface and bind themselves to this corresponding to their relative concentration.
• concentration of HPL in the patient sample will be inversely related to the insolubilized enzyme ac ⁇ tivity.
• the antigen mixture consists of a crude HPL-contain- ing extract of placenta which was produced as fol- lows: 1 placenta was homogenized for 5 minutes in the presence of 500 ml 0.1 M phosphatebuffered saline, pH 7.4. Solid pieces of tissue were removed by ⁇ entri- fuging at 1000 G for 5 minutes. Cell organelles and the like were removed by further centrifuging at 50000 G for 15 minutes. EDTA was added to a final concentration of 10 mM. In order to find the optimum ratio between antigen and antibody, a simple floccu- lation test was instituted, in which variable volumes of the antigen preparation was mixed with variable volumes of the antibody preparation, e.g.:
• Crude placenta extract 50 100 200 400 1000 ( ⁇ l) Monospec. antibody 1000 400 200 100 50 ( ⁇ l)
• polystyrene mi- crotitre trays As solid support for coating was used polystyrene mi- crotitre trays (NUNC F 16 modules, high binding). 200 microliters of coating mixture were added to each microtitre well and incubated 4-15 ' hours at room tem ⁇ perature in a moisture-proqf box. The wells .were washed twice with 0.01 M citric acid buffer, pH 2.3, with 0.02% Tween 20 added, followed by 2 x washing with 0.01 M tris-buffered saline added (0.14 M NaCl) with 0.1% NaN 3 and 0.02% Tween 20 added. The coated microtitre trays were then ready for use.
• the above example describes a competitive measuring construction.
• the advantages of the coupling method can also be used for the sandwich method (double an ⁇ tibody method).
• account must be taken of the presence of immunoreactive an ⁇ tigen on the surface, which is coupled side by side with the corresponding antibody, it is assumed that the molecule X should be quantitized in serum.
• This molecule can be cleaved enzymatically in two or seve- ral well-defined fragments XI, X2 and X3. Presumably, these fragments can be separated from each other by known separation methods, and thus monospecific anti ⁇ bodies against them can also be produced.
• a sandwich method can then be initiated by forming immunopreci ⁇ pitates between XI and anti-Xl, followed by coupling of these to a support.
• Monospecific antibodies against either X2 or X3 can then be used as a secon ⁇ dary indicator-reagent, provided these are conjugated with an indicator principle. Antibodies against X2 or X3 will not react with the insolubilized Xl-antigens.
• both competitive and sandwich immunoassays can be constructed so that the antigen component rather than the antibody can be used as the primary ligand, however with the li ⁇ mitation that epitopes can be lost, while new ones will emerge when the dissociation agent influences the antigen.

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Citations (5)

• 1986
  • 1986-06-19 DK DK289686A patent/DK289686A/da not_active Application Discontinuation
• 1987
  • 1987-06-11 AU AU75887/87A patent/AU7588787A/en not_active Abandoned
  • 1987-06-11 JP JP50389787A patent/JPS63503569A/ja active Pending
  • 1987-06-11 WO PCT/DK1987/000074 patent/WO1987007956A1/en not_active Application Discontinuation
  • 1987-06-11 EP EP19870904240 patent/EP0271554A1/en not_active Withdrawn

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