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.

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 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.

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.

Enzyme immunoassay of gentamicin with use of a centrifugal analyzer.

1981 ◽  
Vol 27 (8) ◽  
pp. 1460-1462 ◽  
Author(s):  
M A Pesce ◽  
S H Bodourian
Keyword(s):  
Enzyme Immunoassay ◽  
Significant Variation ◽  
Small Difference ◽  
Correlation Coefficients ◽  
Tris Buffer ◽  
Fluorescence Immunoassay ◽  
Standard Curve ◽  
Gentamicin Concentration ◽  
Sample Range ◽  
Homogeneous Enzyme Immunoassay

Abstract Gentamicin was determined in serum by the homogeneous enzyme immunoassay system (EMIT) adapted to the Multistat centrifugal analyzer. The standard curve can be extended to 16 mg/L; however, poor precision is obtained at concentrations greater than 10 mg/L because a small difference in change in absorbance there will produce a significant variation in gentamicin concentration. For example, the within-run precision of the method (CV) was 3.4% for a 5.0 mg/L sample (range 4.6--5.4 mg/L) and 11.1% for a 12.2 mg/L sample (range 10.4--15.4 mg/L). We recommend that all samples containing gentamicin at concentrations exceeding 10 mg/L be diluted with Tris buffer. Recovery of gentamicin added to four serum specimens averaged 102%. There is no interference from bilirubin at concentrations up to 200 mg/L or from moderately lipemic samples. Hemoglobin in serum in excess of 625 mg/L results in lower gentamicin values. Gentamicin is stable for six days in serum stored at 23, 4, or -20 degrees C. Comparison of gentamicin results by the proposed method with those by a manual EMIT procedure, and with those by a homogeneous fluorescence immunoassay method gave correlation coefficients of 0.986 and 0.947.

Avidin-Biotin Enzyme Immunoassay of Osteocalcin in Serum or Plasma

1992 ◽  
Vol 38 (10) ◽  
pp. 1968-1974 ◽  
Author(s):  
J Jaouhari ◽  
F Schiele ◽  
S Dragacci ◽  
P Tarallo ◽  
J P Siest ◽  
...  
Keyword(s):  
Human Serum ◽  
Enzyme Immunoassay ◽  
Healthy Subjects ◽  
Limit Of Detection ◽  
Serum Concentrations ◽  
Calcium Dependent ◽  
Standard Curve ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
High Concentrations

Abstract We describe a competitive enzyme immunoassay, the ExtrAvidin-biotin system, for determining osteocalcin in human serum or plasma. Antibodies were raised against bovine osteocalcin. Binding of the antibodies to osteocalcin was calcium-dependent. Limit of detection is 0.07 nmol/L (0.4 microgram/L). The standard curve for method is linear between 0.3 and 17.6 nmol/L (1.9 and 100 micrograms/L). Interassay CV over the range 0.9 to 14.8 nmol/L (5.3 to 84 micrograms/L) is 7.5% to 11.7%. Analytical recovery is 105% +/- 5% (mean +/- SD). The measurement, which is adapted to microtiter plates, requires only 20 microL of serum and 5 h. The coefficient of correlation between the concentrations measured by this method and by a commercially available radioimmunoassay kit (CIS Biointernational) is 0.91. Osteocalcin can be measured in serum or heparinized plasma. Hemolysis (174 mumol/L hemoglobin) reduces osteocalcin concentration by 54%. High concentrations of triglycerides (7 mmol/L) give an overestimation of 63%. Serum concentrations of osteocalcin measured in 130 healthy subjects (ages 15-64 years) and 86 children (ages 4-14 years) were 1.4 +/- 0.8 and 4.0 +/- 1.5 nmol/L (8.1 +/- 4.6 and 22.5 +/- 8.6 micrograms/L), respectively (mean +/- SD).

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).

scholarly journals Automated Trichloroacetic Acid Precipitation Method for Urine Total Protein

1987 ◽  
Vol 24 (2) ◽  
pp. 140-144 ◽  
Author(s):  
C K Cheung ◽  
Y T Mak ◽  
R Swaminathan
Keyword(s):  
Hydrochloric Acid ◽  
Total Protein ◽  
Trichloroacetic Acid ◽  
Acid Precipitation ◽  
Precipitation Method ◽  
Protein Determination ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Assay Precision ◽  
Method R

Urine total protein determination by a trichloroacetic acid (TCA) precipitation method was automated on a Cobas Bio centrifugal analyser. The assay measures the turbidity at 420 nm when a 50 μL sample is mixed with 150 μL of 30 g/L TCA at 25°C. Samples up to 5 g/L can be measured by this method and interference due to pigments and turbidity can be minimised by running a blank with 1.25% hydrochloric acid (HCl) instead of TCA. The standard curve is stable and can be stored in the microcomputer and used again. Within-assay precision (CV) varied from 2 to 4.6% and between-assay precision varied from 1.8 to 5.4%. Analytical recovery ranged from 95 to 106.5% and the results correlated well with those obtained by a manual TCA method ( r=0.98). The method is easy to perform and is considerably faster than the manual procedure, thus saving time and providing a faster turnround time.

Evaluation of a new valproic acid enzyme immunoassay and comparison with a capillary gas-chromatographic method.

1981 ◽  
Vol 27 (1) ◽  
pp. 169-172 ◽  
Author(s):  
S L Braun ◽  
A Tausch ◽  
W Vogt ◽  
K Jacob ◽  
M Knedel
Keyword(s):  
Valproic Acid ◽  
Enzyme Immunoassay ◽  
Chromatographic Method ◽  
Comparison Method ◽  
Manual Method ◽  
Analytical Recovery ◽  
Significant Difference ◽  
High Concentrations ◽  
Assay Precision ◽  
Gas Chromatographic Method

Abstract A new homogeneous immunoassay (EMIT) for valproic acid was evaluated. Besides testing the manual version of this enzyme immunoassay, we also developed two mechanized procedures for centrifugal analyzers (the CentrifiChem and the COBAS system), which take less time and are more precise than the manual method. Within-assay precision (CV) was 4.5% with the manual technique and 2% with the analyzers. Between-assay precision (CV) ranged from 4 to 13% for all three techniques. Accuracy of th manual method was checked by dilution and analytical recovery experiments. Our comparison of the EMIT results with those obtained by a comparison method (capillary gas chromatography) showed no significant difference. No interference from hemolysis, hyperbilirubinemia, or aliphatic amino acids was observed. At high concentrations of bile acids and with lipemic sera the analytical recovery rates decreased slightly, to 87% and 92%, respectively.

scholarly journals Protein C in human plasma determined by homogeneous enzyme immunoassay with use of a centrifugal analyzer.

1988 ◽  
Vol 34 (9) ◽  
pp. 1834-1838 ◽  
Author(s):  
M Shimamoto ◽  
T Yoshimura ◽  
N Kimura ◽  
M Kita ◽  
N Hoshino ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Human Plasma ◽  
Protein C ◽  
Standard Curve ◽  
Coagulation Protein ◽  
Adult Plasma ◽  
Antibody Conjugate ◽  
Antigen Protein ◽  
Antigen Antibody ◽  
Homogeneous Enzyme Immunoassay

Abstract We describe the simple and rapid enzyme immunoassay of protein C in human plasma with use of a Cobas Fara centrifugal analyzer. The antibody, labeled with horseradish peroxidase, is reacted with antigen (protein C) for 15 min. The peroxidase activity of the resulting antigen-antibody conjugate is measured at 500 nm for 5 min in the presence of excess H2O2, phenol, and 4-aminoantipyrine, as compared with that of free conjugates. Results are calculated from a stored standard curve and expressed as a percentage of the value determined for a pooled specimen of normal adult plasma. The standard curve is linear from 0% to 200%. The CV is generally less than 4% for different concentrations of protein C. In liver cirrhosis, hepatocellular carcinoma, therapy with warfarin, thrombosis, and disseminated intravascular coagulation, protein C concentrations are about 40-70% of normal. Results obtained with the present homogeneous enzyme immunoassay correlated well with those by enzyme-labeled immunosorbent assay (r = 0.97).

scholarly journals Comparison of Separation Methods in the 125I-Radioimmunoassay of Serum Cortisol

1983 ◽  
Vol 20 (5) ◽  
pp. 312-316 ◽  
Author(s):  
Sadie M Gray ◽  
J Seth ◽  
G J Beckett
Keyword(s):  
Solid Phase ◽  
Serum Cortisol ◽  
Steep Slope ◽  
Gas Chromatography Mass Spectrometry ◽  
Negative Bias ◽  
Separation System ◽  
Double Antibody ◽  
Target Values ◽  
Assay Precision ◽  
Better Than

Solid-phase first antibody (SP), liquid-phase double-antibody (DA), and preprecipitated double-antibody (PPT) separation methods have been compared in a radioimmunoassay (RIA) for cortisol in unextracted serum. Both double-antibody methods gave values for pools close to target values assigned by gas chromatography—mass spectrometry (GCMS) whereas the SP assay had a significant negative bias, p<0·001 (mean biases: SP —8%, DA —2%, PPT —3%). The SP and DA assays gave average values on patients' samples 12% and 4% lower than values by PPT. These differences could be attributed to different recoveries in the three methods. Between-assay precision of the DA and PPT systems was better than SP (mean CV: SP 11%, DA 7%, PPT 5%). This allowed sensitivity in the PPT assay comparable to that of SP and DA to be achieved, despite the less steep slope of the calibration curve. The DA and PPT assays have several practical advantages over SP. In addition, the PPT assay requires only a 1-hour incubation. It is concluded that the PPT separation system is the method of choice in terms of precision and practical convenience.

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