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
• • 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
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
• • 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
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• • 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
  • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/815—Test for named compound or class of compounds

Definitions

• This invention relates to methods of immuno-Analysis using monoclonal antibodies.
• Specific antisera are widely applied to tite detection and quantitation of a target antigen when it is present in a complex mixture of molecules, and many quantitative methods depend upon precipitation resulting from the formation of insoluble antigen/antibody complexes.
• the insoluble complexes are of relatively small size and are held in suspension.
• Several techniques of specific protein determination depend upon the light scattering .or absorption properties of such complexes.
• One such, automated, method is based on difference turbidi metric measurements resulting from the increased (apparent) absorption properties of such complexes, held in suspension when a polyclonal antibody interacts with a multivalent antigen.
• a method of determining the amount of a particular protein in a sample comprises reacting the sample with a combination of two monoclonal antibody preparations which are respectively specific to two distinct antigenic sites (determinants) on the macromolecule of the protein under investigation, and determining from the resulting antigen/antibo-dy complexes formed a quantitative measure of the original protein coneentration.
• the aforesaid two monoclonal antibodies are selected so as to be specifically directed against relatively widely spaced antigenic determinants present on a given macromolecule, whereby the bindin g of one monoclonal antibody to its specific antigenic determinant does not interfere, spatially, with the binding of the second monoclonal antibody to the antigenic determinant for which said second antibody has specificity.
• the macromolecule comprises an immunoglobulin and the monoclonal antibodies are selected so as to be specific to antigenic determinants expressed by the light and heavy chains respectively of the immunoglobulin.
• the method comprises reacting a sample including first and second immunoglobulin populations with a combination of first and second monoclonal antibody preparations, both of which axe specific to antigenic determinants expressed on the heavy chains of both immunoglobulins, determining from the resulting complex the total immunoglobulin level in the sampe reacting the sample with a combination of said first monoclonal antibody and a third monoclonal antibody which is specific for an antigenic determinant expressed by only one of said immunoglobulin population, and determining from the resulting complex the quantity of said one immunoglobulin population.
• Figure 1 is a representation of an IgG molecule, indicating the regions against which test antibodies were directed;
• Figure 2 is a graph of turbidity against time, for several antigen/antibody reactions.
• Figure 3 is a graph of change in turbidity obtained from antigen/antibody reactions with varying antibody concentrations.
• Figure 1 represents a macromolecule of immunoglobulin G (IgG) having two "heavy" polypeptide chains H and two "light” polypeptide chains L, the chains H and L being linked by disulphide bonds.
• the molecule is symmetrical and has two antigen binding fragments Fab, each of which includes a light chain L and a portion of the heavy chain H.
• a crystalisable fragment Fc of both heavy chains H comprises C ⁇ 2 and C ⁇ 3 regions of equal lengths.
• the fragment Fc may be separated from the remainder of the molecule by enzyme action, and this fragment Fc may be subjected to further enzyme action to leave a residual fragment pFc' whose extent is effectively .that of the CY3 region.
• Each chain in the Fab fragment has a variable region V and a constant region C .
• Normal human IgG includes sub-populations of IgG kappa and IgG lambda, the kappa and lambda characteristics being defined by the structures of the constant regions C of the molecules.
• antibodies were elicited using polyclonal human IgG as an immunogen, and the antibody products were selected on the basis of their ability to agglutinate sheep red blood cells sensitised with human IgG.
• the specificity of the cloned antibody products was further defined by their ability to agglutinate sheep red blood cells sensitised with kappa light chain, lambda light chain, Fc fragments, or pFc' fragments.
• Sample II was also found ro agglutinare cells sensitised with pFc' fragments, and this sample was thus directed against: an antigenic determinant in the C ⁇ 3 region of the IgG molecule.
• samples III and IV In the absence of agglutination of pFc' sensitised cells by samples III and IV, these samples were presumed to be specific to antigenic determinants in the C ⁇ 2 region of the IgG molecule, or to be conformational determinants dependent on the structural integrity of the Fc fragment of the molecule for expression
• the antigen with which the foregoing antibodies were reacted was an IgG kappa paraprotein.
• Preparations of the antibody samples I to IV were reacted with the antigen, separately and in combinations of antibody.
• the reactions were carried out at 25°C in phosphate buffered solutions containing 4% polyethylene glycol (Mw. 3000).
• the difference in turbidity of the fluid in which the reactions were performed was determined using light of wavelength 260 nm, (Jacobsen and Steensgaard Immunology, 36 (1979) at 293-298).
• Figure 2 shows the turbidity differences, D, obtained for a number of values of antigen concentration, C, expressed in mg/ml.
• the curve shown was obtained by titration of the IgG kappa paraprotein, at differenting concentrations, against a standard concentration of an antibody preparation.
• the antibodies used in this case were those from samples I and II, that is those directed against the spatially distinct kappa light chain and C 3 region of the antigen.
• the IgG content of human serum is made up of sub-populations of IgG kappa and IgG lambda.
• the ratio of IgG kappa to IgG lambda lies between 3:1 and 1:1, a typical ratio being approximately 1:8:1.
• a significant departure from this range of ratios may indicate an early stage of myeloma, which otherwise may develop for up to twenty years before showing symptoms Since detection and treatment at an early stage may significantly prolong life expectancy, it is clearly advantageous that the quantities of the above sub-populations should be capable of being determined.
• Monoclonal antibody preparations have been used to determine the relative proportions of sub-populations of immunoglobulins present in such polyclonal IgG.
• a test sample including IgG kappa and IgG lambda which is reacted with antibodies from sample II and sample III, both of which are directed against the Fc portion of the IgG molecule, allows quantiration of the total IgG conrent of the test sample.
• An identical IgG test sample reacted with a combination of antibodies from samples I and II will allow quantitation of the IgG kappa content.

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• Health & Medical Sciences (AREA)
• Immunology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Analytical Chemistry (AREA)
• Cell Biology (AREA)
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• Food Science & Technology (AREA)
• Biotechnology (AREA)
• Physics & Mathematics (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)

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• 1981
  • 1981-07-13 EP EP81303203A patent/EP0044219A1/en not_active Ceased
  • 1981-07-13 WO PCT/GB1981/000136 patent/WO1982000364A1/en unknown
  • 1981-07-13 JP JP56502348A patent/JPS57501147A/ja active Pending
• 1982
  • 1982-03-08 DK DK99682A patent/DK99682A/da not_active Application Discontinuation
  • 1982-03-15 NO NO820836A patent/NO820836L/no unknown
• 1985
  • 1985-06-19 US US06/745,837 patent/US4618589A/en not_active Expired - Fee Related

Non-Patent Citations (5)

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