Specific binding assays utilizing analyte-cytolysin conjugates
AssigneeE. I. Du Pont de Nemours and Company
InventorsJ. William Freytag, William J. Litchfield
Life of this patent
Estimating the term…
Abstract
A novel analyte-cytolysin conjugate and its use in a lipid vesicle mediated measurement process is described for a wide variety of analytes present at very low concentration. The method involves forming a reaction system consisting of analyte, analyte specific binding agent, analyte-cytolysin conjugate, and vesicles containing detectable marker material in such proportions that uncombined conjugate alters the permeability of the vesicles resulting in the release and quantitative detection of marker material which can be correlated with the amount of analyte initially present.
Claims40
A conjugate for the detection of an analyte in a test sample, comprising in combination:
- (1) cytolysin means for altering the permeability of a membrane of a vesicle, said means being conjugated to
- (2) at least one analyte molecule.
An analyte-cytolysin conjugate comprising a substituted melittin molecule of the formula:
- ##STR1## wherein at least one of R.sub.1 through R.sub.8 is an analyte derivative molecule selected from the group consisting of drug, vitamin, hormone, metabolite, steroid, pesticide, environmental pollutant, food toxin, protein, microbial surface marker, cancer cell marker, fungus, protozoan, cell or tissue antigen, provided that when at least one of R.sub.2, R.sub.6, or R.sub.7 is said analyte derivative, attachment is through an .epsilon.-amino group of the amino acid side chain;
- when at least one of R.sub.3, R.sub.4, or R.sub.5 is said analyte derivative, attachment is through a hydroxyl group of said amino acid side chain;
- when R.sub.1 is said analyte derivative, attachment is through the N-terminal amino group;
- and when R.sub.8 is said analyte derivative, attachment is through the C-terminal carboxyl group.
A method for determining the amount of an analyte in liquid medium comprising the steps of:
- (A) forming a reaction system by contacting said liquid medium with
- (1) analyte specific binding agent;
- (2) analyte-cytolysin conjugate;
- and
- (3) vesicles containing marker material sequestered within the vesicle
- in proportions such that the interaction of unbound analyte-cytolysin conjugate with said vesicles results in the release of said marker material;
- and
- (B) measuring said marker material thereby released which is related to the amount of said analyte initially present in said liquid medium.
A method for determining the amount of digoxin in liquid medium comprising the steps of:
- (A) forming a reaction system by contacting said liquid medium with
- (1) digoxin specific antibody;
- (2) ouabain-melittin conjugate;
- (3) lipid vesicles containing sequestered alkaline phosphatase;
- and
- (4) p-nitrophenyl phosphate
- in proportions such that the interaction of unbound ouabain-melittin conjugate and said lipid vesicles results in the conversion of said p-nitrophenyl phosphate by said alkaline phosphatase;
- and
- (B) measuring the concentration of p-nitrophenlate anion produced which is related to the amount of said digoxin initially present in said liquid medium.
A conjugate for the detection of an analyte in a test sample, comprising in combination:
- (1) cytolysin means for altering the permeability of a membrane of a vesicle, said means being conjugaed to
- (2) an antibody molecule capable of binding to the analyte.
A heterogeneous immunoassay for detecting an analyte in a liquid sample, comprising:
- (1) forming a reaction mixture by contacting the sample with a cytolysin/anti-analyte antibody conjugate in immunochemical excess over the analyte, whereby a fraction of said conjugate binds to analyte to form a complex and a fraction of said conjugate remains free,
- (2) contacting the reaction mixture with a solid phase having analyte or analyte-analog bound thereto to separate the complex from the free conjugate, and
- (3) contacting either the complex or the free conjugate with vesicles having marker material sequestered therein, whereby the permeability of the vesicle increases, thereby releasing marker material,
- (4) measuring the amount of released marker material, and
- (5) relating the amount of released marker material to the amount of analyte initially present in the test sample.
Dependencies are inferred from the claim text.