Enzyme immunoassay of thyroxin with a centrifugal analyzer.

1982 ◽  
Vol 28 (1) ◽  
pp. 123-125 ◽  
Author(s):  
J M Izquierdo ◽  
P Sotorrío ◽  
A Quirós
Keyword(s):  
Fatty Acids ◽  
Enzyme Immunoassay ◽  
Protein Complex ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Target Values ◽  
Assay Precision ◽  
Homogeneous Enzyme Immunoassay ◽  
Control Samples

Abstract We have applied a homogeneous enzyme immunoassay for determination of thyroxin in serum to the "Cobas Bio" centrifugal analyzer. To unbind thyroxin from its protein complex, serum is treated for 20 min with a solution of NaOH containing "Lipex," an agent for sequestering free fatty acids. The immunoenzymic reaction is then automatically performed by the analyzer at 37 degrees C. To 20 microL of sample mixture is added 125 microL of reagent (thyroxin antibodies and NAD+) and this mixture is incubated for 10 s. Then 25 microL of start reagent (enzyme-thyroxin conjugate and malate substrate) is added and the change in absorbance is monitored at 340 nm. The standard curve is linear up to at least 200 micrograms of thyroxin per liter. Within-assay precision (CV) varied from 1.1 to 2.9%, between-assay precision from 3.1 to 7.8%. Analytical recovery of thyroxin was complete. The deviation of control samples from target values ranged from -2.1% to 7.0%. Interference by hemoglobin or bilirubin is negligible. Results compare favorably with those by radioimmunoassay.

Homogeneous enzyme immunoassay for cortisol with a centrifugal analyzer.

1984 ◽  
Vol 30 (11) ◽  
pp. 1824-1826 ◽  
Author(s):  
J M Izquierdo ◽  
A Quirós ◽  
J Alvarez-Uría ◽  
P Sotorrío
Keyword(s):  
Enzyme Immunoassay ◽  
Protein Complex ◽  
Acidic Solution ◽  
Serum Cortisol ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Target Values ◽  
Assay Precision ◽  
Homogeneous Enzyme Immunoassay ◽  
Control Samples

Abstract We determine serum cortisol by a homogeneous enzyme immunoassay in the Cobas Bio centrifugal analyzer. To unbind cortisol from its protein complex, serum is treated for 15 min with an acidic solution. The reaction then proceeds automatically in the analyzer at 37 degrees C. To 50 microL of sample mixture is added 125 microL of reagent (cortisol antibodies, glucose 6-phosphate, and NAD+). This mixture is incubated for 60 s, after which 25 microL of a cortisol derivative labeled with glucose 6-phosphate is added; the increase in absorbance is monitored at 340 nm. The standard curve was linear from 10 to 500 micrograms of cortisol per liter. Within-assay precision (CV) varied from 0.2 to 0.6%, between-assay precision from 6.2 to 10.6%. Analytical recovery ranged from 100 to 103%. Results for control samples deviated from target values by 1.4 to 7.8%. Results compared well with those by radioimmunoassay. The method is reliable and practicable and will usefully replace previous routine methods for serum cortisol.

Homogeneous enzyme immunoassay for tobramycin evaluated and compared with a radioimmunoassay.

1982 ◽  
Vol 28 (6) ◽  
pp. 1370-1374 ◽  
Author(s):  
J Woo ◽  
M A Longley ◽  
D C Cannon
Keyword(s):  
Enzyme Immunoassay ◽  
Room Temperature ◽  
Correlation Study ◽  
Stability Study ◽  
Data Handling ◽  
Handling System ◽  
Analytical Recovery ◽  
Assay Precision ◽  
Absorbance Data ◽  
Homogeneous Enzyme Immunoassay

Abstract We evaluated a commercially available homogeneous enzyme immunoassay (EMIT, Syva Co.) for tobramycin against a reference radioimmunoassay (RIA) method. Between-assay precision (CV) was 2.9% at 6.2 mg/L and 3.0% for values in the range of 1.0-7.6 mg/L. Accuracy based on a recovery experiment (1.0-13.0 mg/L) yielded an analytical recovery of 88-112%. A correlation study with 75 sera from patients on tobramycin therapy showed that EMIT = 0.984 RIA - 0.0808, r = 0.993. Neither the EMIT nor the RIA procedure was affected by the presence of gentamicin, amikacin, and vancomycin. Absorbance data from the EMIT system calculated with the conventional RIA logit-log algorithm correlate well with results generated by the Syva data-handling system (logit-log = 1.077 Syva - 0.318, r = 0.998). A reagent stability study indicated that the EMIT reagents, once reconstituted, remain stable for at least 17 days when stored at refrigerated temperatures, or 11 days if stored at room temperature, thus enabling frequent "stat" assays without the need to prepare a calibration curve each time.

Improved determination of aluminum in serum and urine with use of a stabilized temperature platform furnace.

1982 ◽  
Vol 28 (10) ◽  
pp. 2139-2143 ◽  
Author(s):  
F Y Leung ◽  
A R Henderson
Keyword(s):  
Reference Interval ◽  
Matrix Interference ◽  
Linear Calibration ◽  
Standard Curve ◽  
Steady State Temperature ◽  
Analytical Recovery ◽  
Standard Additions ◽  
Assay Precision ◽  
Serum And Urine

Abstract This method for determining aluminum in serum and urine is essentially free from matrix interference and gives a linear response with concentration to at least 500 micrograms/l. Use of a stabilized temperature platform (L'vov platform, Perkin-Elmer Corp.) to approach a "steady-state" temperature, addition of matrix modifiers [especially Mg(NO3)2], and the use of peak area integration all helped substantially diminish spectral interference. With the platform furnace, serum protein concentrations as great as 260 g/L did not interfere with the determination of Al. The within- and between-assay precision (CV) was less than or equal to 3.5% and less than or equal to 7.4%, respectively. Analytical recovery of Al added to serum ranged between 95 and 101% throughout the linear calibration range (to 500 micrograms/L), either when measured directly from the standard curve or by the method of standard additions. The reference interval for Al in 28 healthy subjects was 2-14 micrograms/L (mean 6.5, SD 4.1 micrograms/L), and for 130 patients on hemodialysis, 20-550 micrograms/L (mean 87.5, SD 62.5 micrograms/L).

Determination of creatinine in serum and urine by a rapid liquid-chromatographic method

1990 ◽  
Vol 36 (6) ◽  
pp. 830-836 ◽  
Author(s):  
R Paroni ◽  
C Arcelloni ◽  
I Fermo ◽  
P A Bonini
Keyword(s):  
Signal To Noise Ratio ◽  
Liquid Chromatographic Method ◽  
Analytical Recovery ◽  
Internal Standardization ◽  
Assay Precision ◽  
Fuller’S Earth ◽  
Liquid Chromatographic ◽  
Specific Evaluation ◽  
Serum And Urine

Abstract We describe an HPLC ion-pair procedure for rapid and specific evaluation of creatinine in serum and urine. We used a 15 cm X 4.6 mm ODS column with a 50/50 (by vol) mixture of sodium decanesulfonic acid (10 mmol/L, pH 3.2) and methanol and measured absorbance at 236 nm. Serum (100 microL) or 30-fold-diluted urine (100 microL) was added to 400 microL of acetone. After centrifugation, the supernates (300 microL) were dried, reconstituted with the mobile phase, and injected into the HPLC. Assay precision was tested for concentrations of 10, 29, and 130 mg/L and yielded, respectively, 3.1%, 2.1%, and 1.1% for within-day CV and 2.8%, 2.1%, and 2.2% for total CV. Analytical recovery was 102 (+/- 6.7%). Linearity was demonstrated in the 0-200 mg/L range for serum and 0-3.5 g/L range for urine (r greater than or equal to 0.999). The detection limit for creatinine (signal-to-noise ratio = 3) was 0.5 mg/L. We used cimetidine for internal standardization. Correlation was good between this procedure and the Jaffé kinetic, the enzymatic (creatinine amidohydrolase), and the Fuller's earth alkaline picrate methods.

Kinetic Enzymatic Method for Determining Serum Creatinine

1975 ◽  
Vol 21 (10) ◽  
pp. 1422-1426 ◽  
Author(s):  
Gerald A Moss ◽  
Richard J L Bondar ◽  
Diane M Buzzelli
Keyword(s):  
Serum Creatinine ◽  
Accurate Determination ◽  
Test Subject ◽  
Rate Of Change ◽  
Enzymatic Method ◽  
Inhibitory Effects ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Enzymatic Procedure

Abstract Creatinine amidohydrolase is used to measure serum creatinine in a totally enzymatic procedure. Creatine, produced by hydrolysis, is acted upon by creatine kinase, and then by pyruvate kinase and lactate dehydrogenase, to result in a change in absorbance at 340 nm. The amount of creatinine present is related to the rate of change in A340 and is determined from a standard curve. Absorbance and concentration are linearly related to 100 mg/liter and only 250 µl of serum is required. At 1.0 g/liter, heparin, oxalate, citrate, ethylenediaminetetraacetate, ascorbate, or glucose had no significant effect on the accurate determination of creatinine; higher concentrations (30 g/liter) had inhibitory effects on the test. Analytical recovery of creatinine added to either normal or abnormal sera averaged 102%. When results of this procedure and of the standard direct Jaffé test were compared, the latter were significantly higher. Unlike the Jaffé method, the present method of determining creatinine is rapid (about 10 min per test), subject to few or no interfering substances, and requires no serum deproteinization.

Evaluation of a modified alcohol dehydrogenase assay for the determination of ethanol in blood.

1982 ◽  
Vol 28 (10) ◽  
pp. 2125-2127 ◽  
Author(s):  
A Poklis ◽  
M A Mackell
Keyword(s):  
Gas Chromatography ◽  
Reaction Time ◽  
Alcohol Dehydrogenase ◽  
Correlation Coefficients ◽  
Least Square ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Sigma Chemical ◽  
Enzymic Assay

Abstract We evaluated a new alcohol dehydrogenase (EC 1.1.1.1) enzymic assay (ADH-glycine, Sigma Chemical Co.) for the determination of ethanol in blood. This assay differs from the manufacturer's previous assay (ADH-pyrophosphate) in that glycine replaces pyrophosphate as the buffer and hydrazine replaces semicarbazide as the trapping agent. The standard curve for the assay was linear over blood ethanol concentrations of 0.50-5.00 g/L. The reaction time of the assay was 10 min. At 1.00 g/L within-run and between-run CVs were 3.96% (n = 20) and 4.01% (n = 20), respectively. Mean analytical recovery of ethanol added to whole blood at 0.50-5.00 g/L was 99.7% (SD 2.6%). We performed 100 consecutive clinical and forensic determinations by the ADH-glycine assay, the ADH-pyrophosphate assay, and gas chromatography. Correlation coefficients of the results by least-square linear regression were 0.995 for ADH-pyrophosphate vs ADH-glycine, and 0.990 for gas chromatography vs ADH-glycine. The major advantage of the ADH-glycine assay over the ADH-pyrophosphate assay is the shorter reaction time, 10 min vs 30 min.

Semiautomated homogeneous enzyme immunoassay of total serum thyroxine with a kinetic analyzer.

1980 ◽  
Vol 26 (3) ◽  
pp. 501-503
Author(s):  
H Bhatia
Keyword(s):  
Enzyme Immunoassay ◽  
Total Serum ◽  
Serum Thyroxine ◽  
Assay Procedure ◽  
Homogeneous Enzyme Immunoassay

Abstract A new semiautomatic procedure for determination of total serum thyroxine was adapted to a kinetic analyzer, LKB 2086 Mark II. The ABA thyroxine assay kit was used, and although the assay procedure was modified, the advantage of small reagent volumes, short measuring times, and automation were retained. The results were analyzed off-line with a programmable desk-top calculator. The method has a precision of 7% (CV) and a sensitivity of 8 nmol/L. Values in sera from 64 patients analyzed by enzyme immunoassay and by radioimmunoassay correlated well (r = 0.975). One kit contains enough reagents for 500 assays, and an operator could do 25 samples (i.e., 64 assays) in about 4 h.

Liquid-chromatographic determination of acetaminophen in serum.

1979 ◽  
Vol 25 (3) ◽  
pp. 409-412 ◽  
Author(s):  
C G Fletterick ◽  
T H Grove ◽  
D C Hohnadel
Keyword(s):  
Chromatographic Determination ◽  
Pharmacokinetic Studies ◽  
Structural Isomers ◽  
Internal Standards ◽  
Standard Curve ◽  
Liquid Chromatographic Method ◽  
Analytical Recovery ◽  
Liquid Chromatographic Determination ◽  
Liquid Chromatographic

Abstract We describe a sensitive and precise "high-pressure" liquid-chromatographic method for determining acetaminophen in serum. The 2-acetaminophenol and 3-acetaminophenol structural isomers of acetaminophen are used as internal standards. The method, which involves solvent extraction and adsorption chromatography on silica, provides excellent sensitivity, accuracy, and selectivity. The standard curve is linear over the range of acetaminophen concentrations of 0.5 to 300 mg/L, which makes the method useful for both pharmacokinetic studies and overdose monitoring. Analytical recovery is 97% for acetaminophen concentrations ranging from 5 to 300 mg/L. Many commonly used drugs were tested and found not to interfere. The procedure has been successfully adapted as a microscale method requiring only 50 microL of sample. The microscale method is particularly useful for pediatric and neonatal patients for whom sample size is a major concern.

Evaluation of four assay methods for determination of tobramycin in human serum.

1982 ◽  
Vol 28 (1) ◽  
pp. 177-180 ◽  
Author(s):  
W J Acton ◽  
O M Van Duyn ◽  
L V Allen ◽  
D J Flournoy
Keyword(s):  
Human Serum ◽  
Enzyme Immunoassay ◽  
Correlation Coefficient ◽  
Clinical Laboratory ◽  
The Other ◽  
Serum Enzyme ◽  
Analytical Recovery ◽  
Assay Methods

Abstract Four assay procedures for tobramycin in serum--enzyme immunoassay (I), substrate-labeled fluorescent immunoassay (II), radioimmunoassay (III), and bioassay (IV)--were compared and evaluated by replicate and analytical recovery studies. I and II were about 50% more precise than III and IV. II was substantially more nearly accurate than the other methods and also gave the best reproducibility (correlation coefficient 0.992 between-day). The least expensive method was IV. Ease of handling favored I and II. Overall, we find II to be the most acceptable procedure for use in the clinical laboratory.

Enzyme immunoassay and enzyme inhibition assay of methotrexate, with use of the centrifugal analyzer.

1981 ◽  
Vol 27 (3) ◽  
pp. 380-384 ◽  
Author(s):  
M A Pesce ◽  
S H Bodourian
Keyword(s):  
Enzyme Immunoassay ◽  
Enzyme Inhibition ◽  
Correlation Coefficients ◽  
High Dose ◽  
Inhibition Assay ◽  
High Dose Therapy ◽  
Standard Curve ◽  
Competitive Protein Binding ◽  
Enzyme Inhibition Assay ◽  
Homogeneous Enzyme Immunoassay

Abstract Methotrexate was determined by the homogeneous enzyme immunoassay (EMIT) with the Multistat and CentrifiChem centrifugal analyzers and by the enzyme inhibition assay with use of the Multistat centrifugal analyzer. With both methods, the standard curve extends from 0.2 to 2.0 mumol/L and there is no interference from bilirubin in concentrations up to 100 mg/L. Moderately lipemic samples do not interfere with the EMIT method, but lower the values obtained with the enzyme inhibition assay. Hemoglobin concentrations as great as 1 g/L do not affect results for methotrexate obtained by the enzyme inhibition assay. With the EMIT assay, methotrexate values are lowered in samples containing hemoglobin in concentrations exceeding 750 mg/L. With the EMIT assay, the following compounds in concentrations of 1 mmol/L do not interfere: leucovorin, 5-methyl-tetrahydrofolate, 5-fluorouracil, and 6-mercaptopurine. When folic acid (100 mumol/L) was added to a serum that did not contain methotrexate, a response equivalent to 0.15 mumol/L was obtained. Methotrexate is stable for five days in serum stored at 23, 4, or -20 degrees C. Within-run precision (CV) for the enzyme inhibition method ranged from 4.7 to 8.1% and for the EMIT assay from 2.5 to 6.2%. Methotrexate concentrations in the serum of children receiving high-dose therapy were compared by three methods: competitive protein binding, EMIT, and enzyme inhibition assays. The correlation coefficients averaged 0.95.

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