Determination of Fetal Hemoglobin by Time-Resolved Immunofluorometric Assay

1992 ◽  
Vol 38 (10) ◽  
pp. 2013-2018 ◽  
Author(s):  
U Turpeinen ◽  
U H Stenman
Keyword(s):  
High Performance ◽  
Fetal Hemoglobin ◽  
Gamma Chain ◽  
Time Resolved ◽  
Assay Procedure ◽  
Analytical Recovery ◽  
Sandwich Type ◽  
Capture Antibody ◽  
Europium Chelate

Abstract We have developed a "sandwich"-type time-resolved immunofluorometric assay (IFMA) for fetal hemoglobin (HbF) in hemolysates from adults and newborns, amniotic fluid, and plasma, based on a polyclonal and a monoclonal antibody against human fetal hemoglobin. Microtiter wells are coated with polyclonal capture antibody, and the gamma-chain-specific monoclonal tracer antibody is labeled with a europium chelate. In a simple and fast assay procedure, prediluted hemolysates are incubated in the microtiter wells first with capture antibody for 1 h and, after washing, for 1 h with tracer antibody. The wells are washed and the fluorescence of europium is measured. The mean analytical recovery is 102% and results by IFMA agreed well with values obtained by high-performance liquid chromatography. The analytical range of IFMA is large and well suited for clinical purposes. The detection limit of the assay is 0.2 microgram/L and the measuring range extends to 500 micrograms/L.

Double monoclonal time-resolved immunofluorometric assay of beta 2-microglobulin in serum

1990 ◽  
Vol 36 (11) ◽  
pp. 1961-1964 ◽  
Author(s):  
A Tienhaara ◽  
J U Eskola ◽  
V Näntö
Keyword(s):  
Serum Samples ◽  
Time Resolved ◽  
Assay Procedure ◽  
Analytical Recovery ◽  
Sandwich Type ◽  
Capture Antibody ◽  
Polyclonal Antisera ◽  
Europium Chelate ◽  
Beta 2 ◽  
Beta 2 Microglobulin

Abstract Previously reported immunochemical assays of beta 2-microglobulin (beta 2m) have usually been based on polyclonal antisera. We have developed a "sandwich"-type time-resolved immunofluorometric assay (TR-IFMA) for beta 2m in serum, based on two monoclonal antibodies against human beta 2m. Microtiter wells are coated with the capture antibody, and the tracer antibody is labeled with a europium chelate. In a simple and fast assay procedure, prediluted serum samples are incubated with the tracer for 1 h in the microtiter wells, after which the wells are washed and the fluorescence of Eu is measured. The mean analytical recovery was 101.8% and results by TR-IFMA showed a good linear correlation with those by an established radioimmunoassay. The analytical range of TR-IFMA is large and well suited for clinical purposes.

Specific determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine by liquid chromatography with post-column reaction.

1988 ◽  
Vol 34 (1) ◽  
pp. 87-90 ◽  
Author(s):  
K Abe ◽  
R Konaka
Keyword(s):  
High Performance ◽  
Signal To Noise Ratio ◽  
High Performance Liquid Chromatographic ◽  
Reference Intervals ◽  
Reversed Phase ◽  
Sodium Metaperiodate ◽  
Liquid Chromatographic Method ◽  
Analytical Recovery ◽  
Liquid Chromatographic

Abstract We describe a "high-performance" liquid-chromatographic method for determining 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) in human urine. MHPG is separated on a reversed-phase column with isocratic elution, oxidized with sodium metaperiodate, and its absorbance measured at 365 nm. This method shows higher specificity, less interference for MHPG than methods involving electrochemical or fluorescence detection. Post-column derivatization of MHPG with periodate yields vanillin. The detection limit (twice the signal-to-noise ratio) in urine samples was 0.08 mg/L. Mean analytical recovery was 72%. Within-assay and day-to-day CVs were 2.9% and 6.5%, respectively. Reference intervals for MHPG in 24-h urine from apparently healthy subjects were 0.85-3.24 mg/day for men and 0.63-2.20 mg/day for women. In terms of creatinine excretion, the respective reference intervals were 0.55-1.99 and 0.70-1.96 mg per gram of creatinine.

Sensitive, rapid procedure for time-resolved immunofluorometry of lutropin.

1988 ◽  
Vol 34 (8) ◽  
pp. 1640-1644 ◽  
Author(s):  
M J Khosravi ◽  
R C Morton ◽  
E P Diamandis
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Detection System ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Practical Procedure ◽  
Daily Monitoring ◽  
Fluorescence Measurements ◽  
Capture Antibody ◽  
Europium Chelate

Abstract In this new immunofluorometric method for quantification of lutropin in serum, the "sandwich" principle is combined with time-resolved fluorescence measurements, with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) used as label. A monoclonal antibody to the alpha-subunit of lutropin is adsorbed onto the walls of white-opaque microtiter wells to form the solid-phase capture antibody, and a biotin-labeled soluble monoclonal antibody is used for antigen quantification. The detection system is completed with streptavidin, which has been linked to a protein bulking agent labeled with multiple BCPDA residues. In the presence of excess europium, the fluorescence of the final complex attached to captured lutropin molecules is measured on the dried solid phasse with an automated time-resolved fluorometer. The assay can be performed as a rapid (less than 60 min incubation) or regular (150 min incubation) procedure. The rapid assay is well-suited for routine daily monitoring of increasing or ovulatory lutropin concentrations; the regular assay, with its greater sensitivity (0.5 int. unit/L), is a practical procedure for lutropin measurements in hyposecretory states. The assay measures up to 240 int. units/L, and results compare well with those by a commercially available radioimmunoassay, an immunoradiometric assay, and another time-resolved immunofluorometric procedure.

Homogeneous time-resolved fluoroimmunoassay of thyroxin in serum.

1988 ◽  
Vol 34 (11) ◽  
pp. 2320-2322 ◽  
Author(s):  
I Hemmilä ◽  
O Malminen ◽  
H Mikola ◽  
T Lövgren
Keyword(s):  
Time Resolved ◽  
Fast Measurement ◽  
Europium Chelate

Abstract We describe a rapid, simple nonseparation fluoroimmunoassay for determination of thyroxin in serum. The assay is based on the labeling of thyroxin directly with a fluorescent europium chelate, the fluorescence of which is quenched on binding to an antithyroxin antibody. With the assay buffer we used, maximum quenching is 90%. The rapid achievement of equilibrium in the assay solution, regardless of the sequence of reagent additions, allows fast measurement of thyroxin. Precision was good (CV less than 5%) within the clinical range for total thyroxin (50-300 nmol/L), and results correlated well with those by a commercial radioimmunoassay.

Use of alumina, sephadex G10, and ion-exchange columns to purify samples for determination of epinephrine, norepinephrine, dopamine, homovanillic acid, and 5-hydroxyindoleacetic acid in urine.

1982 ◽  
Vol 28 (8) ◽  
pp. 1745-1748 ◽  
Author(s):  
B H Westerink ◽  
F J Bosker ◽  
J F O'Hanlon
Keyword(s):  
Ion Exchange ◽  
High Performance ◽  
Homovanillic Acid ◽  
Clinical Chemistry ◽  
Simultaneous Quantification ◽  
Analytical Recovery ◽  
Hydroxyindoleacetic Acid ◽  
One Year ◽  
Catecholamines In Urine

Abstract We investigated the usefulness of small Sephadex columns, prepared in Pasteur pipettes, in purifying samples to be analyzed for catecholamines, homovanillic acid, and 5-hydroxyindoleacetic acid. After free catecholamines in urine samples were purified on alumina followed by Sephadex G10, a reliable and simultaneous quantification of epinephrine, norepinephrine, and dopamine was achieved by using high-performance liquid-chromatography with electrochemical detection. Purification of urine on Bio-Rad prepacked ion-exchange columns followed by Sephadex G10 resulted in a reliable, fast (200 samples processed per week) method for determination of free catecholamines in urine. Analytical recoveries of both methods were between 80 and 95%, with a CV of about 3%. Single purification on Sephadex G10 sufficed to allow simultaneous determination of homovanillic acid and 5-hydroxyindoleacetic acid in urine. Analytical recovery for this method was about 90%, with a CV of about 5%. Sephadex G10 columns, which can be re-used without regeneration for at least one year, appear to have a great potential in clinical chemistry.

Rapid enzymatic determination of urinary oxalate.

1989 ◽  
Vol 35 (12) ◽  
pp. 2330-2333 ◽  
Author(s):  
M G Li ◽  
M M Madappally
Keyword(s):  
Enzyme Assay ◽  
Divalent Cations ◽  
Urinary Oxalate ◽  
Assay Sensitivity ◽  
Color Development ◽  
Assay Procedure ◽  
Enzymatic Determination ◽  
Analytical Recovery ◽  
Urine Specimens

Abstract This new reagent kit for the quantitative measurement of oxalate in urine is a modification of an earlier Sigma oxalate assay procedure (procedure no. 590), a coupled enzyme assay involving oxalate oxidase and horseradish peroxidase. The new analytical procedure includes methods for processing urine specimens to eliminate interference with oxalate color development at 590 nm by ascorbic acid, divalent cations, and other urinary constituents. The reaction is complete in less than 5 min, and results are linearly related to oxalate concentration up to at least 1 mmol/L. Assay sensitivity and within-run and between-run precision were within the limits acceptable for other urinary oxalate procedures. Analytical recovery of added oxalate was close to 100%. This specific, simple, rapid procedure is suitable for routine clinical use.

Reverse Phase High Performance Liquid Chromatographic Determination of Phenprocoumon in Tablets

1982 ◽  
Vol 65 (3) ◽  
pp. 753-756
Author(s):  
Walter F Schmidt
Keyword(s):  
High Performance ◽  
Internal Standard ◽  
Chromatographic Determination ◽  
High Performance Liquid Chromatographic ◽  
Reverse Phase ◽  
Assay Procedure ◽  
Chromatographic Procedure ◽  
Liquid Chromatographic Determination ◽  
Liquid Chromatographic

Abstract A high performance liquid chromatographic procedure has been developed for the assay of phenprocoumon in tablets. In comparison to the present official USP assay procedure, it is equivalent in precision and accuracy and is faster and more specific. A mobile phase consisting of a 1% solution of acetic acid in acetonitrile-water (4 + 3) separates phenprocoumon from warfarin internal standard on a 6 μm octadecylsilane (ODS) column with UV detection at 311 nm. The method enables the concurrent determination of phenprocoumon and possible contaminants such as salicylic acid.

Comparison of an Isoelectric Focusing Technique and High-Performance Liquid Chromatography for Determination of Fetal Hemoglobin Levels

1991 ◽  
Vol 96 (1) ◽  
pp. 109-110 ◽  
Author(s):  
Enid F. Gilbert-Barness ◽  
Katherine S. Kenison ◽  
Earl Shrago ◽  
Terry L. Spennetta ◽  
Gary G. Giulian
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Isoelectric Focusing ◽  
High Performance ◽  
Fetal Hemoglobin
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