Immunoassay of 5-methyltetrahydrofolate: use of /sup 125/I-labeled protein A as the tracer molecule for specific antibody
A sensitive and specific solid-phase radioimmunoassay for 5-methyltetrahydrofolate (5-MTHFA) has been developed. /sup 125/I-Labeled staphylococcal Protein A (/sup 125/I-PA) was used as the tracer molecule for rabbit IgG antibodies bound to 5-MTHFA immobilized on polyacrylamide beads. The dose-dependent inhibition of antibody binding by fluid-phase drug was reflected in decreased binding of /sup 125/I-PA. This inhibition, determined in the presence of known amounts of 5-MTHFA, served as the basis for quantification of 5-MTHFA in test samples. An early bleeding was relatively specific; 4.5 ng 5-MTHFA inhibited immune binding by 50% compared to 7700 ng folinic acid or 1200 ng tetrahydrofolate. Other folic acid analogs, including methotrexate, failed to inhibit significantly. The assay using a later bleeding was more sensitive since 1.6 ng 5-MTHFA gave 50% inhibition (detection limit 0.2 ng), but folinic acid cross-reacted significantly. Absorption with immobilized folinic acid markedly enhanced the specificity of this antiserum and resulted in a 15 to 20% increase in maximum inhibition by 5-MTHFA. The assay could be carried out in the presence of 0.025 ml human serum or urine without affecting the standard curve, and was used to determine levels of 5-MTHFA in serum of drug-treated rabbits.
- Research Organization:
- National Cancer Inst., Bethesda, MA
- OSTI ID:
- 5180014
- Journal Information:
- Anal. Biochem.; (United States), Vol. 104:2
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
CITROVORUM FACTOR
RADIOIMMUNOASSAY
ANTIBODIES
ANTIGEN-ANTIBODY REACTIONS
BIOCHEMICAL REACTION KINETICS
CPB
FOLIC ACID
IMMUNE SERUMS
INHIBITION
IODINE 125
LABELLED COMPOUNDS
METABOLISM
METABOLITES
METHOTREXATE
AMINO ACIDS
ANTIMETABOLITES
ANTIMITOTIC DRUGS
BETA DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PTERIDINES
RADIOASSAY
RADIOISOTOPES
REACTION KINETICS
TRACER TECHNIQUES
VITAMIN B GROUP
VITAMINS
550601* - Medicine- Unsealed Radionuclides in Diagnostics
550201 - Biochemistry- Tracer Techniques