scholarly journals Dihydrofolate Reductase Enzyme Inhibition Assay for Plasma Methotrexate Determination Using a 96-Well Microplate Reader

1999 ◽  
Vol 45 (2) ◽  
pp. 223-228 ◽  
Author(s):  
Brigitte C Widemann ◽  
Frank M Balis ◽  
Peter C Adamson
Keyword(s):  
Enzyme Inhibition ◽  
Sensitivity And Specificity ◽  
Dihydrofolate Reductase ◽  
Cross Reactivity ◽  
Small Sample ◽  
Plasma Samples ◽  
Inhibition Assay ◽  
Microplate Reader ◽  
Enzyme Inhibition Assay ◽  
Multiple Samples

Abstract Microplate reader assays offer several advantages over conventional spectrophotometric assays. We adapted the dihydrofolate reductase (DHFR) enzyme inhibition assay for use in a 96-well microplate reader to measure plasma methotrexate (MTX) concentrations. The assay is linear from 0.01 to 0.1 μmol/L. The within-run CVs at 0.03 μmol/L and 0.08 μmol/L MTX were 4.0% and 2.7%, respectively, and the interday (total) CVs were 7.6% and 1.8%. Cross-reactivity with the inactive MTX metabolite 2,4-diamino-N10-methylpteroic acid (DAMPA) was 3.9%, significantly less than that described with commercial immunoassays; with 7-hydroxymethotrexate cross-reactivity was 1.7%. In addition to sensitivity and specificity, the advantages of this assay are small sample volumes, simultaneous analysis of multiple samples, and rapid turnaround. Because of its greater specificity, the DHFR enzyme inhibition assay may be useful when DAMPA is present in plasma samples and HPLC is not available.

Development and Evaluation of an Enzyme Inhibition Assay for Methotrexate

1988 ◽  
Vol 25 (5) ◽  
pp. 516-521
Author(s):  
Michael G Kelly
Keyword(s):  
Reaction Time ◽  
Enzyme Inhibition ◽  
Sensitivity And Specificity ◽  
Dihydrofolate Reductase ◽  
Relative Concentration ◽  
Methotrexate Therapy ◽  
Inhibition Assay ◽  
Standard Curve ◽  
Brief Assessment ◽  
Enzyme Inhibition Assay

A method for the measurement of methotrexate based on its inhibition of dihydrofolate reductase (EC 1.5.1.3) is described. The performance of the method is evaluated and a brief assessment of its clinical usefulness made. Reaction time, pH, and the relative concentration of enzyme and inhibitor all have a significant effect on the shape of the standard curve. Pre-incubation of enzyme with methotrexate resulted in increased inhibition, but did not improve the sensitivity or linearity of the assay. Data are presented on the imprecision, specificity and accuracy of the method. The method is simple to perform, rapid and inexpensive. The sensitivity and specificity of the method are such as to allow for effective monitoring of patients on methotrexate therapy.

Plasma methotrexate as determined by liquid chromatography, enzyme-inhibition assay, and radioimmunoassay after high-dose infusion.

1980 ◽  
Vol 26 (6) ◽  
pp. 734-737 ◽  
Author(s):  
S K Howell ◽  
Y M Wang ◽  
R Hosoya ◽  
W W Sutow
Keyword(s):  
Liquid Chromatography ◽  
Enzyme Inhibition ◽  
Plasma Concentrations ◽  
Cross Reactivity ◽  
High Dose ◽  
Maximum Plasma ◽  
Inhibition Assay ◽  
Dose Infusion ◽  
Enzyme Inhibition Assay ◽  
Related Compounds

Abstract Three techniques for measuring methotrexate show various cross reactivities with methotrexate-related compounds: “high-pressure” liquid chromatography, by principle, is virtually specific for methotrexate; the enzyme-inhibition assay quantitates methotrexate, methotrexate diglutamate, and methotrexate triglutamate equally well, but has a 10% cross reactivity with 4-amino-4-deoxy-N10-methylpteroic acid and 1% with 7-hydroxymethotrexate; radioimmunoassay shows an equal cross reactivity with methotrexate, 4-amino-4-deoxy-N10-methylpteroic acid, methotrexate diglutamate and triglutamate, and a 5 to 10% cross reactivity with 7-hydroxymethotrexate. Radioimmunoassay almost always yielded the highest values for methotrexate, followed by enzyme-inhibition assay then liquid chromatography. The presence of two methotrexate-related compounds, 7-hydroxymethotrexate and 4-amino-4-deoxy-N10-methylpteroic acid, was confirmed in human urine samples and quantitated in patients’ plasma by liquid chromatography, the respective maximum plasma concentrations being 250 and 16 mumol/L. Materials cross reacting with methotrexate in radioimmunoassay of chromatographic fractions from plasma were also noted in fractions corresponding to methotrexate diglutamate and triglutamate peaks, in quantities estimated to be 47 and 30 nmol/L methotrexate equivalents, respectively. 7-Hydroxymethotrexate is eliminated more slowly than methotrexate and its production increases with dosages of methotrexate.

Plasma methotrexate as determined by liquid chromatography, enzyme-inhibition assay, and radioimmunoassay after high-dose infusion.

1980 ◽  
Vol 26 (6) ◽  
pp. 734-737 ◽  
Author(s):  
S K Howell ◽  
Y M Wang ◽  
R Hosoya ◽  
W W Sutow
Keyword(s):  
Liquid Chromatography ◽  
Enzyme Inhibition ◽  
Plasma Concentrations ◽  
Cross Reactivity ◽  
High Dose ◽  
Maximum Plasma ◽  
Inhibition Assay ◽  
Dose Infusion ◽  
Enzyme Inhibition Assay ◽  
Related Compounds

Abstract Three techniques for measuring methotrexate show various cross reactivities with methotrexate-related compounds: “high-pressure” liquid chromatography, by principle, is virtually specific for methotrexate; the enzyme-inhibition assay quantitates methotrexate, methotrexate diglutamate, and methotrexate triglutamate equally well, but has a 10% cross reactivity with 4-amino-4-deoxy-N10-methylpteroic acid and 1% with 7-hydroxymethotrexate; radioimmunoassay shows an equal cross reactivity with methotrexate, 4-amino-4-deoxy-N10-methylpteroic acid, methotrexate diglutamate and triglutamate, and a 5 to 10% cross reactivity with 7-hydroxymethotrexate. Radioimmunoassay almost always yielded the highest values for methotrexate, followed by enzyme-inhibition assay then liquid chromatography. The presence of two methotrexate-related compounds, 7-hydroxymethotrexate and 4-amino-4-deoxy-N10-methylpteroic acid, was confirmed in human urine samples and quantitated in patients’ plasma by liquid chromatography, the respective maximum plasma concentrations being 250 and 16 mumol/L. Materials cross reacting with methotrexate in radioimmunoassay of chromatographic fractions from plasma were also noted in fractions corresponding to methotrexate diglutamate and triglutamate peaks, in quantities estimated to be 47 and 30 nmol/L methotrexate equivalents, respectively. 7-Hydroxymethotrexate is eliminated more slowly than methotrexate and its production increases with dosages of methotrexate.

scholarly journals Pneumococcal Haemophilus influenzae Protein D Conjugate Vaccine Induces Antibodies That Inhibit Glycerophosphodiester Phosphodiesterase Activity of Protein D

2008 ◽  
Vol 76 (10) ◽  
pp. 4546-4553 ◽  
Author(s):  
Maija Toropainen ◽  
Anna Raitolehto ◽  
Isabelle Henckaerts ◽  
Dominique Wauters ◽  
Jan Poolman ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Enzymatic Activity ◽  
Enzyme Inhibition ◽  
Acute Otitis Media ◽  
Vaccine Antigen ◽  
Inhibition Assay ◽  
Igg Antibodies ◽  
Enzyme Inhibition Assay ◽  
Glycerophosphodiester Phosphodiesterase ◽  
Inhibition Index

ABSTRACT Haemophilus influenzae outer membrane protein D (PD) is a glycerophosphodiester phosphodiesterase (GlpQ) activity-possessing virulence factor and a promising vaccine antigen, providing 35.3% efficacy against acute otitis media caused by nontypeable H. influenzae (NTHI) when it was used as a carrier protein in a novel pneumococcal PD conjugate (Pnc-PD) vaccine. To study if PD-induced protection against NTHI could be due to antibodies that inhibit or neutralize its enzymatic activity, a GlpQ enzyme inhibition assay was developed, and serum samples collected from Finnish infants before and after Pnc-PD vaccination were analyzed for enzyme inhibition and anti-PD immunoglobulin G (IgG) antibody concentration. Before vaccination at age 2 months, the majority (84%) of infants (n = 69) had no detectable anti-PD IgG antibodies, and all were enzyme inhibition assay negative (inhibition index, <20). At age 13 to 16 months, all infants receiving three or four doses of Pnc-PD had detectable anti-PD IgG antibodies and 36% (8/22 infants) of the infants receiving three doses and 26% (6/23 infants) of the infants receiving four doses of Pnc-PD were inhibition assay positive (inhibition index, ≥20). No significant rise in anti-PD IgG antibodies or enzyme inhibition among control vaccinees (n = 24) receiving three doses of hepatitis B vaccine was detected. A modest correlation (r s , ∼0.66) between anti-PD IgG concentration and enzyme inhibition was detected; however, their kinetics were clearly different. These data suggest that measurement of antibody responses that inhibit PD's enzymatic activity could be a useful tool for assessing Pnc-PD vaccine-induced protective immunity against NTHI.

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