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
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody

Definitions

• AN IMPROVED METHOD OF NON HOMOGENOUS ENZYME IMMUNOASSAY This invention relates to an improved method of the technique known as non homogenous enzyme immunoassa , described, for example, in “Quantitative Enzyme Immunoassay", Ed. E. Engvall and A.J. Pesce, Blackwood Scientific Publications (Scand. J. Immunol. 8, Suppl. 7, 1978) .
• non homogenous enzyme immunoassay is based on the competition for the active site of an anti ⁇ body between a ligand and a covalently coupled derivative of that same ligand to an enzyme. With separation of the bound and free enzyme ligand phases, enzyme assay of either fraction can then be made. To aid in the separation of the phases, it is often convenient to bind the antibody to an insoluble particle such as the wall of the reaction vessel itself, or a polysaccharide particle. Plotting of the percent bound against the ligand concentration, results in a typical immunoassay curve.
• the enzyme immunoassay system is also capable of simple modification to provide for the measurement of serum antibody levels by making enzyme-anti- body derivatives and using a solid phase ligand to assist in the separation step.
• the antibody characteristics required for the development of a successful non homogenous enzyme immunoassay for a particular ligand are those of high specificity for the ligand, as well as a high apparent affinity constant (as 1/mol) for the ligand.
• an antibody demonstrating a high specificity and a high affinity constant towards a particular ligand will also bind substances which are closely similar in chemical structure to the ligand itself. Such substances are herein referred to as "analogues of the ligand”.
• the affinity constant of the antibody observed with respect to the analogue may differ markedly from that observed for the antibody with the ligand itself. It is the object of
• the present invention provides a method of non homogenous enzyme immunoassay including the steps of: (i) binding an analogue of a ligand to an enzyme to form an enzyme-ligand analogue complex; (ii) contacting the enzyme-ligand analogue complex with antibody for the ligand so as to bind the complex to the antibody;
• a suitable analogue of the ligand is selected such that it is bound specifically by the antibody, though to a much lower affinity than is the case for the original ligand itself.
• Such an analogue is then covalently joined to an appropriat enzyme by known methods of chemistry.
• preparations of the antib are linked by appropriate means to an insoluble particle which may be the surface of the inner wall of the small tube or may be a small insoluble particle, such as a polysaccharide or a glass bead.
• an insoluble particle which may be the surface of the inner wall of the small tube or may be a small insoluble particle, such as a polysaccharide or a glass bead.
• Such a "formed solid phase” antibody is required to retain its function as an antibody.
• the enzyme-labelled analogue is then brought into contact with the solid phase antibody, such that all the binding sites of the solid phase antibody are occupied by the enzyme-labelled analogue. Such an.
• enzyme-labelled analogue may then be displaced from the binding site of the solid phase antibody by addition under appropriate conditions of the original ligand to which the antibody has been derived, or a different analogue having a higher affinity for the antibody than the enzyme-labelled analogue.
• liberated enzyme analogue By separation of the liberated enzyme analogue from the enzyme analogue remaining bound to the solid phases , it is found that the quantity of liberated enzyme analogue, as measured by whatever means, bears an analytical relation ⁇ ship to the quantity of antigen or other ligand which causes the displacement.
• the amount of enzyme analogue liberated by known concentration of ligand in appropriate standards, the measurement of an unknown concen ⁇ tration of the ligand in a sample can be effected.
• known amounts of the enzyme-labelled analogue bound to the antibody can be dispensed into vials and subjected to the process known as freeze-drying.
• freeze-drying By adding a required volume of water to the vial to reconstitute the active enzyme-analogue-antibody-complex, the assay can then be performed directly.
• appropriate precautions to maintain stability of the reagent it would be possible to utilise a previously defined calibration curve from which the amount of ligand in a sample can be calculated without the necessity of so performing a calibration curve at that time. This method thus has application in times of emergency when speed
• an assay for the anti- epilepsy drug dilantin, in the patients taking this drug is described.
• Antibodies were raised to dilantin in goats by appropriate means and the antibodies demonstrated an affinity for dilantin of 10 1/mole, while having an affini for meth l-dilantin of 10 1/mole.
• Methyl dilantin was the covalently joined to bacterial beta-galactosidase using water soluble carbo-dii ide, and the antibody covalently bound to sepharose beads. Aliquots of the sepharose antibo methyl dilantin-beta-galactosidase complexes were formed an all available antibody binding sites saturated.
• Tube No. Dilantin Cont b-Galactosidase activity (Mg/L) (A405/min/ml)
• ligand or ligand analogue such as the systems phenobarbitone (meta-nitro phenobarbitone) theophylline (l-methyl-3-carboxy butyl xanthine) thyroxine (3,3' 5 - triiodo-5 '-bromo thyronine) and triiodothyronine (3,5 - diiodo-3'-bromo thyronine).
• phenobarbitone metal-nitro phenobarbitone
• theophylline l-methyl-3-carboxy butyl xanthine
• thyroxine l-methyl-3-carboxy butyl xanthine
• thyroxine l-methyl-3-carboxy butyl xanthine
• thyroxine l-methyl-3-carboxy butyl xanthine
• thyroxine l-methyl-3-carboxy butyl x

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• Health & Medical Sciences (AREA)
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• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 1980
  • 1980-11-19 WO PCT/AU1980/000090 patent/WO1981001414A1/en not_active Application Discontinuation
  • 1980-11-19 JP JP50264580A patent/JPS56501583A/ja active Pending
  • 1980-11-19 EP EP19800902222 patent/EP0040224A4/en not_active Withdrawn

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