# US 4,399,228 A · Polate competitive protein binding assay > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 4,399,228 A - Document ID: US4399228A - Status: Granted - Kind: A - Application number: 06/288,477 - Filed: 1981-07-30 - Granted: 1983-08-16 - Earliest claimed: 1981-07-30 - Assignees: Corning Glass Works - Inventors: Louis J. Riceberg - Web page: https://patentgrep.com/patent/US4399228A ## CPC - G01N 33/82: involving vitamins {or their receptors} - Y10S 436/804: Radioisotope, e.g. radioimmunoassay - Y10S 436/808: Automated or kit - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract Improvement in a competitive protein binding assay for N-5-methyltetrahydrofolic acid wherein any folate standard used is N-5-methyltetrahydrofolic acid complexed with folate binder protein. The protein of the complex is destroyed prior to the addition of immobilized folate binder protein. The immobilized folate binder protein participates in competitive binding with unknown folate and labeled folate reagents. ## Claims ### Claim 1 A process for determining the concentration of N-5-methyltetrahydrofolic acid in a biological sample of unknown N-5-methyltetrahydrofolic acid concentration which comprises separately admixing the biological sample and at least one standard reagent with known quantities of labeled folic acid or labeled folic acid derivative to yield at least two separate test samples, said at least one standard reagent comprising a reconstituted lyophilized complex of N-5-methyltetrahydrofolic acid-folate binding protein, destroying the folate binding protein of said at least one standard reagent and destroying any binding protein present in the biological sample, adding a known quantity of folate binding protein to each test sample, measuring the amount of labeled folic acid or labeled folic acid derivative which does or does not bind to the added binding protein in each test sample and from said measurements determining the concentration of N-5-methyltetrahydrofolic acid in the biological sample. ### Claim 2 (depends on 1) The process of claim 1 wherein the known quantity of folate binding protein added to each test sample is immobilized on an inert support. ### Claim 3 A process comprising: (A) complexing N-5-methyltetrahydrofolic acid with folate binding protein in an aqueous based medium; (B) lyophilizing said aqueous based medium containing said complex; and (C) carrying out a competitive protein binding assay for N-5-methyltetrahydrofolic acid using the lyophilized complex in reconstituted form as a N-5-methyltetrahydrofolic acid reagent. ### Claim 4 (depends on 2, 3) The process of any of claims 2 or 3 wherein the assay being carried out is a simultaneous vitamin B.sub.12 /folate assay. ### Claim 5 (depends on 2, 3) The process of any of claims 2 or 3 wherein a plurality of reconstituted lyophilized N-5-methyltetrahydrofolic acid reagents, each having a different N-5-methyltetrahydrofolic acid concentration, wherein the N-5-methyltetrahydrofolic acid is complexed with folate binding protein, are run side-by-side along with the unknown sample and then the concentration of the folate in the unknown is determined by generating a standard curve from said known concentration reagents and comparing said unknown with said standard curve. ### Claim 6 (depends on 4) The process of claim 4 wherein a plurality of reconstituted lyophilized N-5-methyltetrahydrofolic acid reagents, each having a different N-5-methyltetrahydrofolic acid concentration, wherein the N-5-methyltetrahydrofolic acid is complexed with folate binding protein, are run side-by-side along with the unknown sample and then the concentration of the folate in the unknown is determined by generating a standard curve from said known concentration reagents and comparing said unknown with said standard curve. ### Claim 7 (depends on 2, 3) The process of any of claims 2 or 3 wherein the label is radioactive. ### Claim 8 (depends on 4) The process of claim 4 wherein the label is radioactive and wherein a radioactive vitamin B.sub.12 standard reagent is used in a competitive binding assay for vitamin B.sub.12 and the radioactive label of said vitamin B.sub.12 reagent is different than the radioactive label of said labeled folic acid or labeled folic acid derivative. ### Claim 9 A competitive protein binding assay kit comprising: (A) N-5-methyltetrahydrofolic acid complexed with folate binding protein and (B) folate binding protein immobilized on an inert support. ### Claim 10 (depends on 9) The assay kit of claim 9 as a radioassay kit, said kit containing a plurality of standard lyophilized N-5-methyltetrahydrofolic acid reagents, each having upon reconstitution a different N-5-methyltetrahydrofolic acid concentration, wherein the N-5-methyltetrahydrofolic acid is complexed with folate binding protein, so that a standard folate binding curve can be generated from said standard reagents. ### Claim 11 (depends on 10) The radioassay kit of claim 10 wherein the inert support is silane-glutaraldehyde derivatized glass particles and the folate binding protein is covalently bound thereto.