Claims for Patent: 4,517,288

Summary for Patent: 4,517,288

Title:	Solid phase system for ligand assay
Abstract:	A method for conducting a ligand assay in an inert porous medium wherein a binding material is immunologically immobilized within the medium, which includes the steps of immunologically immobilizing a binding material within a finite zone of the medium, applying an analyte to the zone containing the immobilized binding material, applying a labeled indicator to the zone which becomes immobilized within the zone in an amount which can be correlated to the amount of analyte in the zone, applying a solvent to substantially the center of the zone to chromatographically separate the unbound labeled indicator from the zone, and measuring the amount of labeled indicator remaining in the zone.
Inventor(s):	Giegel; Joseph L. (Miami, FL), Brotherton; Mary M. (Miami, FL)
Assignee:	American Hospital Supply Corp. (Evanston, IL)
Application Number:	06/227,664
Patent Claims:	1. A competitive method for conducting a solid phase enzyme immunoassay of a fluid sample within the interstices of a solid, inert porous medium, said fluid sample containing an unknown level of analyte, the method comprising a. immobilizing a binding material within a finite zone of the interstices of the solid, inert porous medium, said binding material being capable of immunological reaction with said analyte from among the constituents of the fluid sample; b. applying, under binding conditions, to substantially the center of said finite zone, containing said immobilized binding material, a fluid sample containing the analyte for which said binding material is specific, said analyte being applied as a solution so as to permit diffusion thereof within the interstices of a reaction zone of the porous medium; c. applying an enzyme-labeled indicator to substantially the center of said reaction zone, under conditions which allow said indicator and said analyte to compete for binding sites on said immobilized binding material, said indicator, which comprises an enzyme conjugated to a ligand, being immunochemically bound to said immobilized binding material in a amount which can be correlated to the amount of analyte in said reaction zone; d. applying, to substantially the center of said reaction zone a stream of eluting solvent containing a substrate for the enzyme of said enzyme-labeled indicator, the quantity of said eluting solvent being sufficient to effect radial chromatographic separation, within said porous medium, of unbound enzyme-labeled indicator from bound enzyme-labeled indicator within said reaction zone; and e. observing the extent to which the bound enzyme-labeled indicator is present within a delimited area of said reaction zone by measurement of the level of chromophore or fluorophore produced by the action of the bound enzyme-labeled indicator on said substrate, said delimited area of said reaction zone being essentially free of unbound indicator. 2. The enzyme immunoassay of claim 1, wherein the binding material is immunologically immobilized within a finite zone of the inert, porous medium. 3. The enzyme immunoassay of claim 1, wherein the binding material is an antibody specific for said analyte and is immunologically immobilized within said zone by applying a solution of said antibody to said zone and applying an antiserum to said antibody to said zone, thus effecting an immunological reaction in said zone. 4. The enzyme immunoassay of claim 1, wherein the assay is a competitive enzyme immunoassay for digoxin in a biological fluid; the binding material is digoxin antibody; the labeled indicator is digoxin labeled with alkaline phosphatase enzyme; the eluting solvent is a buffered solution of 4-methylumbelliferyl phosphate, which acts as a substrate for the alkaline phosphatase enzyme and releases 4-methylumbelliferone upon enzyme hydrolysis; and the amount of 4-methylumbelliferone so released is determined by front surface fluorometry. 5. The enzyme immunoassay of claim 1, wherein the analyte is a therapeutic drug and the binding material is an antibody specific for said therapeutic drug. 6. The enzyme immunoassay of claim 5, wherein said therapeutic drug is selected from the group consisting of dilatin, phenobarbital, theophylline, gentamycin, quinidine, and propranolol and said antibody is derived from an animal selected from the group consisting of rabbit, goat, horse, donkey, sheep, chicken and human. 7. The enzyme immunoassay of claim 1, wherein the analyte is a steroid and the binding material is an antibody specific for said steroid. 8. The enzyme immunoassay of claim 7, wherein said steroid is selected from the group consisting of cortisol, aldosterone, testosterone, progesterone and estriol. 9. The enzyme immunoassay of claim 1, wherein the analyte is a thyroid hormone and the binding material is an antibody specific for said thyroid hormone. 10. The enzyme immunoassay of claim 9, wherein said thyroid hormone is triiodothyronine or thyroxine. 11. The enzyme immunoassay of claim 1, wherein the analyte is a peptide hormone and the binding material is an antibody specific for said peptide hormone. 12. The enzyme immunoassay of claim 11, wherein said peptide hormone is selected from the group consisting of insulin, corticotropin, gastrin, angiotensin and proangiotensin. 13. The enzyme immunoassay of claim 1, wherein the analyte is a polypeptide hormone and the binding material is an antibody specific for said polypeptide hormone. 14. The enzyme immunoassay of claim 13, wherein said polypeptide hormone is selected from the group consisting of thyrotropin, luteotropin and somatotropin. 15. The enzyme immunoassay of claim 1, wherein the binding material is a specific binding protein selected from the group consisting of vitamin B-12 intrinsic factor, thyroxine binding globulin, folate binding protein and sex hormone binding protein. 16. In an analytical method for conducting a solid phase enzyme immunoassay of a fluid sample by immunological differentiation of complex molecules in such fluid by first contacting such fluid, containing an unknown level of analyte, and an enzyme-labeled indicator with an immobilized binding material within a reaction zone of a porous medium under conditions favoring immunochemical interaction of said binding material with the analyte and the enzyme-labeled indicator, whereby the analyte of interest of said fluid and the enzyme-labeled indicator compete for available sites on said binding material, said enzyme-labeled indicator comprising an enzyme conjugated to a ligand, applying a wash fluid to said porous medium to remove unreacted enzyme-labeled indicator from said medium, and measuring the amount of enzyme-labeled indicator immunochemically bound to the binding material within the porous medium, the improvement comprising: a. applying, to substantially the center of a reaction zone within an inert porous medium, a stream of eluting solvent containing a substrate for the enzyme of the enzyme-labeled indicator, the quantity of such eluting solvent applied to said reaction zone being sufficient to effect radial separation, within said inert porous medium, of unbound enzyme-labeled indicator from bound enzyme-labeled indicator within said reaction zone; and b. observing the extent to which the bound enzyme-labeled indicator is present within a delimited area of said reaction zone by measurement of the level of chromophore or fluorophore produced by the action of the bound enzyme-labeled indicator on said substrate, said delimited area of said reaction zone being essentially free of unbound indicator. 17. A sandwich method for conducting an enzyme immunoassay of a fluid sample within the interstices of a solid, inert porous medium, said fluid sample containing an unknown level of analyte, the method comprising: a. immobilizing a binding material within a finite zone of a solid, inert porous medium, said binding material being capable of immunological reaction with said analyte from among the constituents of the fluid sample; b. applying, under binding conditions, to substantially the center of said finite zone containing said immobilized binding materials, a fluid sample containing the analyte for which the said binding material is specific, said analyte being applied as a solution so as to permit diffusion thereof within a reaction zone of the solid, porous medium containing the immobilized binding material, whereby substantially all of said analyte is bound to the binding material; c. applying an enzyme-labeled indicator to substantially the center of said reaction zone, under conditions which allow said labeled indicator to become immunochemically bound to said analyte in a manner which can be correlated to the amount of analyte in the reaction zone, said indicator comprising an enzyme conjugated to a ligand; d. applying, to substantially the center of said reaction zone, a stream of eluting solvent containing a substrate for the enzyme of said enzyme labeled indicator, said solvent being applied in a quantity sufficient to effect radial separation, within said porous medium, of unbound enzyme-labeled indicator from the indicator which is bound within said reaction zone; and e. observing the extent to which the bound enzyme-labeled indicator is present within a delimited area of said reaction zone by measurement of the level of chromophore or fluorophore produced by the action of the bound enzyme-labeled indicator on said substrate, said delimited area of said reaction zone being essentially free of unbound indicator. 18. Th enzyme immunoassay of claim 17, wherein the binding material is immunologically immobilized within the porous medium. 19. The enzyme immunoassay of claim 17, wherein the analyte is immunoglobulin G, the binding material is an anti-human immunoglobulin G and the enzyme-labeled indicator comprises an enzyme conjugated to anti-human immunoglobulin G. 20. The enzyme immunoassay of claim 19, wherein the enzyme-labeled indicator is anti-human immunoglobin G and the label is an enzyme selected from the group consisting of E. coli alkaline phosphatase, betagalactosidase and horse radish peroxidase. 21. The enzyme immunoassay of claim 19, wherein the enzyme-labeled indicator is anti-human immunoglobulin G and the label is a fluorophore selected from the group consisting of fluorescein, rhodamine and fluorescamine. 22. The enzyme immunoassay of claim 19, wherein the analyte is hepatitis suface antigen (HB.sub.s Ag), the binding material is anti-HB.sub.s Ag, and the enzyme-labeled indicator is enzyme-labeled anti-HB.sub.s Ag. 23. In an analytical method for a solid phase enzyme immunoassay of a fluid sample by immunological differentiation of complex molecules in such fluid by first contacting such fluid, containing an unknown level of analyte, with an immobilized binding material within a reaction zone of a porous medium under conditions favoring immunochemical interaction of said binding material with the analyte, whereby substantially all of the analyte of interest of said fluid is immunochemically bound to the binding material, contacting the analyte with an enzyme-labeled indicator under conditions favoring immunochemical interaction of said indicator with the analyte, said indicator comprising an enzyme conjugated to a ligand, applying a wash fluid to said porous medium to remove unreacted enzyme-labeled indicator from said medium and measuring the amount of enzyme-labeled indicator which is immunochemically bound to the analyte, the improvement comprising: a. applying, to substantially the center of a reaction zone within an inert, porous medium, a stream of eluting solvent containing a substrate for the enzyme of said enzyme-labeled indicator, the quantity of solvent applied to said reaction zone being sufficient to effect radial separation within said porous medium, of unbound enzyme-labeled indicator from bound enzyme-labeled indicator within said reaction zone; and b. observing the extent to which the bound enzyme-labeled indicator is present within a delimited area of said reaction zone by measurement of the level of chromophore or fluorophore produced by the action of the bound enzyme-labeled indicator on said substrate, said delimited area of said reaction zone being essentially free of unbound labeled indicator.

Details for Patent 4,517,288

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Csl Behring Ag	CARIMUNE, CARIMUNE NF, PANGLOBULIN, SANDOGLOBULIN	immune globulin intravenous (human)	For Injection	102367	07/27/2000		2040-01-28
Csl Behring Ag	PRIVIGEN	immune globulin intravenous (human), 10% liquid	Injection	125201	07/26/2007		2040-01-28
Csl Behring Ag	PRIVIGEN	immune globulin intravenous (human), 10% liquid	Injection	125201	10/02/2009		2040-01-28
Csl Behring Ag	PRIVIGEN	immune globulin intravenous (human), 10% liquid	Injection	125201	02/07/2013		2040-01-28
Bio Products Laboratory	GAMMAPLEX	immune globulin intravenous (human)	Injection	125329	09/17/2009		2040-01-28