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Case Western Reserve University Patents
Show moreThe present invention relates, as indicated, to a competitive radioisotopic enzyme or biochemical cofactor assay for measuring the concentration of folate in serum. The term "folate" is well understood by those skilled in biochemistry. It is generic and includes not only the derivatives of pteroylmonoglutamic acid but also the salts and other neutralization products thereof. Whether a material is in the acidic or basic form will depend upon the pH of the medium in which it is found. Thus, the term folate includes substituted derivatives of pteroylmonoglutamic acid and the polyglutamate forms as well as esters, salts and other such derivatives involving not only the carboxyl group but also other points of substitution on the molecule. It is often helpful to know the serum folate concentration in order to confirm the etiology of a patient's anemia. Presently known microbiological assays for measuring the serum folate concentration, however, are time-consuming and, like other microbiological assays, are affected by antimicrobial and antineoplastic agents which may be present in the serum sample. Reference may be had to the work by Herbert entitled, "Asceptic Addition Method for Lactobacillus Casei of Folate Activity in Human Serum," J. Clin. Path. 19: pp 12-16, 1966; and Grossowicz et al, "Microbiologic Determination of Folic Acid Derivatives in Blood," appearing in Blood 20, pp 609-616, 1962. The present process is distinguished from these microbiological assays in utlizing a radio assay procedure characterized by a direct competitive binding between a labeled folate and a second folate, in one case the second folate being known and in the second case the second folate being a serum folate, whereby the concentration of folate in the serum may be determined. This assay may be completed in a morning's work and the results obtained by the end of the day. This assay employs .sup.3 H-pteroylmonoglutamate (hereinafter identified as .sup.3 H-PGA) as the labeled folate, d,1,5-methyltetrahydrofolic acid (hereinafter called 5CH.sub.3 FH.sub.4) as a known folate, a partially purified folate binder extracted from hog kidney or other suitable animal organ. This assay is sensitive to 25 picograms of 5CH.sub.3 FH.sub.4, the main serum folate (see Herbert et al, "Studies on the Identification of a Folate Compound of Human Serum," J. Clin. Invest. 41: pp 1134-1138, 1962). Unlike microbiological assays, the present assay is unaffected by antimicrobials or antineoplastic agents such as Methotrexate (amethopterin), 4-(amino-N.sup.10 -methylpteroylglutamic acid). Reference may also be had to Rothenberg et al, "A Radio Assay for Serum Folate: Use of a Two-Phase Sequential-Incubation, Ligand-Binding System," New England Journal of Medicine 286: 1335-1339, 1972.
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