Competitive particle concentration fluorescence immunoassay for measuring 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (lometrexol) in serum

1991 ◽  
Vol 37 (2) ◽  
pp. 254-260 ◽  
Author(s):  
Larry D Taber ◽  
Peter O'Brien ◽  
Ronald R Bowsher ◽  
J Richard Sportsman
Keyword(s):  
Particle Concentration ◽  
Cross Reactivity ◽  
Minimum Detectable Concentration ◽  
Fluorescence Immunoassay ◽  
Standard Curve ◽  
Coefficients Of Variation ◽  
Analytical Recovery ◽  
Detectable Concentration ◽  
Tetrahydrofolic Acid ◽  
Cancer Agent

Abstract A competitive particle concentration fluorescence immunoassay (PCFIA) is described for measuring 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (lometrexol; Lilly) in human serum. b-Phycoerythrin-labeled lometrexol competes with free lometrexol for binding to a limiting concentration of lometrexol-specific antibodies immobilized by a second antibody to submicrometer-diameter polystyrene particles in specially designed 96-well plates. Reaction particles are washed and concentrated onto filter membranes in the wells of the plates and the fluorescence is measured at 575 nm. The method, including sample preparation and data reduction, is automated and can be completed in less than 2 h. The assay has a standard curve maximum measurable concentration of 1000 micrograms/L and a minimum detectable concentration of 0.1 microgram/L. Analytical recovery of lometrexol in serum is quantitative at concentrations greater than 1 micrograms/L. Intra- and interassay coefficients of variation at 50 micrograms/L in serum are 7.1% (n = 9) and 7.5% (n = 33), respectively. The cross-reactivity of naturally occurring folates, folic acid analogs, and the anti-cancer agent methotrexate is minimal. We report the use of the PCFIA during Phase I clinical studies designed to evaluate the pharmacokinetics of lomextrexol after intravenous administration to cancer patients.

Fluorescence polarization immunoassay of urinary vanillylmandelic acid

1990 ◽  
Vol 36 (1) ◽  
pp. 110-112 ◽  
Author(s):  
G W Mellor ◽  
G Gallacher
Keyword(s):  
Urine Sample ◽  
Fluorescence Polarization ◽  
Minimum Detectable Concentration ◽  
Vanillylmandelic Acid ◽  
Fluorescence Polarization Immunoassay ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Detectable Concentration ◽  
Hydroxyindoleacetic Acid ◽  
Neural Crest Tumors

Abstract This rapid fluorescence polarization immunoassay for urinary vanillylmandelic acid (VMA) involves use of our previously described antiserum and label and the program for 5-hydroxyindoleacetic acid in the Abbott TDx fluorimeter. Urine samples were measured directly, without pretreatment. The minimum detectable concentration was 0.3 mg/L, and the range of the standard curve was 0.3-200.0 mg/L. Precision, analytical recovery, and correlation of results with those by the Pisano method (Clin Chim Acta 1962;7:285-91) were all satisfactory. With this procedure one can determine the VMA concentration in 10 urine sample in 22 min. This is the first report of a clinical immunoassay for VMA and should greatly simplify screening for neural crest tumors.

Colorimetry of hemoglobin in plasma with 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS).

1984 ◽  
Vol 30 (3) ◽  
pp. 357-359 ◽  
Author(s):  
M Takayanagi ◽  
T Yashiro
Keyword(s):  
Hydrogen Peroxide ◽  
Calibration Curve ◽  
Plasma Proteins ◽  
Sulfonic Acid ◽  
Minimum Detectable Concentration ◽  
Colored Form ◽  
Analytical Recovery ◽  
Detectable Concentration ◽  
Precision And Accuracy

Abstract Hemoglobin in plasma can be determined by the color-developing action of 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid), which is oxidized to a colored form by a peroxidase-like effect of hemoglobin in the presence of hydrogen peroxide. Sensitivity, precision, and accuracy are discussed. The calibration curve is linear for hemoglobin concentrations up to 1 g/L; the minimum detectable concentration is 20 mg/L. The within-run precision (CV) was 2.39%, analytical recovery 101.8%. Interference from plasma proteins and lipids was eliminated by centrifuging the reaction mixture before measuring its absorbance at 410 nm.

scholarly journals Development of a Dual Monoclonal Antibody Immunoassay for Total Human Kallikrein 2

2001 ◽  
Vol 47 (7) ◽  
pp. 1218-1224 ◽  
Author(s):  
Judith A Finlay ◽  
John R Day ◽  
Cindy L Evans ◽  
Robert Carlson ◽  
Kristine Kuus-Reichel ◽  
...  
Keyword(s):  
Specific Antigen ◽  
High Specificity ◽  
Cross Reactivity ◽  
Minimum Detectable Concentration ◽  
Serum Samples ◽  
Bodily Fluids ◽  
Before And After ◽  
Detectable Concentration ◽  
Human Kallikrein 2 ◽  
Kallikrein 2

Abstract Background: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-α1-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease. Methods: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA. Using these mAbs, a sandwich assay was developed and its specificity for forms of hK2 was assessed. Serum samples (n = 1035) from healthy volunteers, patients with increased PSA, and men who had undergone radical prostatectomy were assayed for thK2. We also measured thK2 in samples before and after storage under common laboratory conditions. Results: The minimum detectable concentration in the thK2 assay was 0.008 μg/L, and PSA cross-reactivity was <0.001%. The assay detected prohK2 and three different hK2–serum protease complexes. The median serum concentration of thK2 in control samples (0.013 μg/L) was significantly lower than the median in samples from patients with increased PSA concentrations (0.085 μg/L). Immunoreactive hK2 changed little in samples stored for up to 1 month at −70 °C. Conclusions: The thK2 assay recognizes all forms of hK2 that have been found in bodily fluids to date.

scholarly journals Determination of salivary cortisol in donkey stallions

2018 ◽  
Author(s):  
MICAELA SGORBINI ◽  
Francesca Bonelli ◽  
Alessandra Rota ◽  
Christine Aurich ◽  
Natascha Ille ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Salivary Cortisol ◽  
Circadian Variation ◽  
Cross Reactivity ◽  
Commercial Enzyme ◽  
Standard Curve ◽  
Non Invasive ◽  
Detectable Concentration ◽  
Commercial Enzyme Immunoassay

Salivary cortisol provides information about free plasma cortisol concentration and salivary sampling is a non-invasive well-tolerate procedure. The aim of this study was to validate a commercial enzyme immunoassay for the determination of salivary cortisol in donkeys. Saliva samples were collected in 4 donkey stallions on thirteen non-consecutive days at 8:30 AM to avoid circadian variation. Animals were already accustomed to be handled. Saliva was collected by using a swab inserted at the angle of the lips, placed onto the tongue for 1 min and returned into a polypropylene tube. Tubes were centrifuged and at least 1 ml of saliva was aspirated from each sample and frozen at −20° C until analysis. A commercial enzyme immunoassay kit without extraction was used for determination of cortisol in saliva. Median cortisol concentrations with minimum and maximum value were calculated. Recovery of cortisol standard in donkey saliva was 107.9% and serial dilution of donkey saliva samples with assay buffer resulted in changes in optical density parallel to the standard curve. Cross-reactivity of the antiserum was 10.4% with 11-deoxycortisol, 5.2% with corticosterone, 0.4% with 11-deoxycorticosterone, 0.2% with cortisone and <0.1% with testosterone, progesterone and estradiol. The intra-assay coefficient of variation was 10.7%, the inter-assay variation was 8.0% and the minimal detectable concentration was 0.01 ng/ml. The results of the present study demonstrate the validity of a commercial kit to determine the concentration of cortisol in donkey saliva, as already reported in other species.

scholarly journals Analytical performance of the Tandem®-R free PSA immunoassay measuring free prostate-specific antigen

1997 ◽  
Vol 43 (7) ◽  
pp. 1203-1208 ◽  
Author(s):  
David L Woodrum ◽  
Chester M French ◽  
Timothy M Hill ◽  
Steven J Roman ◽  
Harold L Slatore ◽  
...  
Keyword(s):  
Prostate Specific Antigen ◽  
Accurate Determination ◽  
Specific Antigen ◽  
Cross Reactivity ◽  
Analytical Performance ◽  
Minimum Detectable Concentration ◽  
Free Psa ◽  
Detectable Concentration ◽  
Total Psa

Abstract The analytical performance of the Tandem®-R free PSA assay available from Hybritech Inc. was evaluated. Comparison of recoveries of purified free (unbound) prostate-specific antigen (PSA) diluted in female serum in the Tandem-R free PSA assay and the Tandem-R (total) PSA assay demonstrated a link in calibration between the assays and an accurate determination of percent free PSA. The cross-reactivity of the assay to purified PSA–α1-antichymotrypsin was determined to be &lt;1%. The minimum-detectable concentration was &lt;0.05 μg/L. The within-run and between-day CVs were ≤5% for samples with &gt;0.3 μg/L free PSA. Dilution and recovery showed no significant deviations from linearity across the assay range. The assay was insensitive to interference from blood components. The Tandem-R free PSA kit was shown to be an accurate, precise, and reliable assay for the measurement of free PSA.

Highly Sensitive Sandwich Enzyme Immunoassay for Alpha-Fetoprotein in Human Saliva

1992 ◽  
Vol 29 (5) ◽  
pp. 519-522 ◽  
Author(s):  
Xian Yang Yio ◽  
Jing Jiang ◽  
Fen Zhi Yin ◽  
Ke-He Ruan
Keyword(s):  
Enzyme Immunoassay ◽  
Human Saliva ◽  
Minimum Detectable Concentration ◽  
Igg Antibody ◽  
Coefficients Of Variation ◽  
Highly Sensitive ◽  
Detectable Concentration ◽  
Polystyrene Plate ◽  
Normal Human ◽  
Sandwich Enzyme Immunoassay

To determine α-fetoprotein (AFP) in human saliva, a highly sensitive sandwich enzyme immunoassay for saliva AFP was developed. AFP standards and saliva samples were added into the wells of a polystyrene plate coated with goat IgG antibody against human AFP. After incubation, the wells were washed and horseradish peroxidase-labelled antibody was added. The enzyme activity specifically bound to the well was assayed using 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide as substrate. The reaction was stopped by addition of 2 M sulphuric acid and the AFP concentration was determined from the absorbance at 450 nm. The minimum detectable concentration was 8 ng/L. The recovery of AFP mixed with human saliva was 91·1–102·4%. The within-assay and between-assay coefficients of variation were 6·5–8·9% and 7·6–10·8%, respectively. The assay correlated well with a radioimmunoassay for human AFP ( r = 0·985, n = 13, P < 0·001). The mean concentration of AFP in normal human saliva was 14·3 ng/L (SEM = 4·9 ng/L, n = 10) and significantly higher levels of saliva AFP were observed in hepatocellular carcinoma patients with positive serum AFP (mean 1367·8 ng/L, SEM 595·4 ng/L, n = 6; P < 0·001). Strong correlation was observed between saliva AFP and serum AFP ( r = 0·978, P < 0·01, n = 13).

A thyrotropin radioimmunoassay kit evaluated vs two reference assays.

1984 ◽  
Vol 30 (3) ◽  
pp. 437-439 ◽  
Author(s):  
S T Bigos ◽  
A E Pekary ◽  
J MacLean ◽  
C E Pierce ◽  
A W Reed ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Human Serum ◽  
Clinical Data ◽  
Clinical Status ◽  
Cross Reactivity ◽  
Minimum Detectable Concentration ◽  
Double Antibody ◽  
Human Choriogonadotropin ◽  
Detectable Concentration

Abstract We evaluated a double-antibody radioimmunoassay kit for thyrotropin that includes calibrators prepared in a matrix of human serum and involves overnight nonequilibrium. Results were compared with those from two reference assays for thyrotropin. The range of within-assay CVs for the kit for thyrotropin values between 0.9 and 2.4 milli-int. units/L was 2.2 to 5.3%, that for between-assay CVs was 8.3 to 30%. The estimated minimum detectable concentration of thyrotropin was 0.6 milli-int. unit/L. We saw no cross reactivity with human choriogonadotropin by any of 48 sera from pregnant women. The original lot of serum specified as thyrotropin-free contained small but measurable amounts of thyrotropin; a second lot did not. Clinical data generated with the kit and the reference assays correlated well and were consistent with the clinical status of various categories of patients.

Assay performance and clinical validation of a radioimmunoassay for serum thyroxine-binding globulin

2005 ◽  
Vol 24 (2) ◽  
pp. 115-119 ◽  
Author(s):  
Ivana Petrovic ◽  
Svetlana Savin ◽  
Dubravka Cvejic
Keyword(s):  
Specific Binding ◽  
Reference Ranges ◽  
Thyroxine Binding Globulin ◽  
Serum Thyroxine ◽  
Standard Curve ◽  
Coefficients Of Variation ◽  
Assay Performance ◽  
Analytical Recovery ◽  
Satisfactory Precision ◽  
Human Sera

A radioimmunoassay for the quantitation of serum thyroxine-binding globulin (TBG), RIA TBG (PEG) - INEP, was formed and characterised. Test sensitivity was found to be favorable (2.0 mg/L). The highly satisfactory precision of the assay was reflected in the low coefficients of variation (less than 5%) found for both intra- and interassay precision at three concentrations. Non-specific binding of the labeled antigen was less than 2% of the total binding. Linearity of the assay within the range of the standard curve (0-80.0 mg/L) was excellent (r =0.99) and the analytical recovery of TBG added to serum ranged from 91 to 115%. Reproducibility was checked by determining the concentration of TBG in samples of sera over a period of 32 days from the day of antigen iodination and F<Ftab 0.05, F0.05 7, 32 = 2.31. The assay was standardized for accuracy using Lyphochek Immunoassay Controls (Bio-Rad). A significant correlation was obtained between RIA TBG (PEG) and the Kodak Amerlite TBG Assay (r = 0.84; n = 25). The presented results show that RIA TBG (PEG) is a sensitive, precise and reliable method for determination of TBG in human sera. Assay reference ranges were established for sera from various subjects: euthyroid adults, hypothyroid and hyperthyroid patients and in healthy pregnant women.

Direct immunological determination of aspartate aminotransferase isoenzymes.

1981 ◽  
Vol 27 (9) ◽  
pp. 1597-1601 ◽  
Author(s):  
R Rej ◽  
C R Keese ◽  
I Giaever
Keyword(s):  
Aspartate Aminotransferase ◽  
Specific Binding ◽  
Cross Reactivity ◽  
Minimum Detectable Concentration ◽  
Tissue Extracts ◽  
Rabbit Igg ◽  
Catalytically Active ◽  
Detectable Concentration ◽  
Immunological Technique

Abstract We examined the suitability of a rapid immunological technique to determine the amount of aspartate aminotransferase (EC 2.6.1.1) isoenzymes in human serum or tissue extracts. Purified isoenzymes were absorbed as a monolayer to the surface of an indium metal film on glass. The enzyme retains immunological reactivity, allowing the specific binding of aspartate aminotransferase antibodies at the surface. The amount of isoenzyme in a specimen is estimated from the competition for the antibody between the free isoenzyme in the specimen and that at the surface. The surface is further incubated with goat antibodies to rabbit IgG, and the extent of antibody binding is determined by densitometry. There is no cross reactivity between the cytoplasmic and mitochondrial forms, so these two isoenzymes can be determined simultaneously. The minimum detectable concentration by this technique is about 50 micrograms of enzyme protein per liter. The within-day coefficient of variation for determination of either isoenzyme was about 20%. Our results suggest that normal and patients' sera contain considerably more immunologically active than catalytically active isoenzymes.

Direct solid-phase radioimmunoassay for screening 17 alpha-hydroxyprogesterone in whole-blood samples from newborns.

1985 ◽  
Vol 31 (7) ◽  
pp. 1127-1130 ◽  
Author(s):  
L F Hofman ◽  
J E Klaniecki ◽  
E K Smith
Keyword(s):  
Filter Paper ◽  
Whole Blood ◽  
Solid Phase ◽  
Cross Reactivity ◽  
Blood Samples ◽  
Standard Curve ◽  
Logit Transformation ◽  
Analytical Recovery ◽  
Solid Phase Radioimmunoassay ◽  
Direct Solid

Abstract We describe a direct, solid-phase RIA for 17 alpha-hydroxyprogesterone (17-OH-P) that we are using to screen neonates for congenital adrenal hyperplasia. Phosphate buffer containing danazol and anti-17-OH-P is placed in tubes coated with antibody to IgG. The tubes also contain standards, controls, or blood samples on filter paper discs 3 mm in diameter. 125I-labeled 17-OH-P is added to each tube. The mixture is vortex-mixed and incubated overnight. The fluid and disc are removed, the radioactivity remaining in the tubes is counted, and the amount of 17-OH-P per disc is calculated by using a log-logit transformation of the standard curve. Results compare favorably with those by two extraction assays. Inter- and intra-assay CVs were less than 11% and less than 9%, respectively. Sensitivity was 2 pg per assay tube. There is no significant cross reactivity with structurally related steroids at their physiological concentrations. Analytical recovery of added 17-OH-P averaged 104%. 17-OH-P in whole blood spotted on filter paper is stable for at least six months.

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