scholarly journals Determination of Haptoglobin in Bovine Serum using Polyclonal and Monoclonal Anti-human Haptoglobin Antibodies

2010 ◽  
Vol 79 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Paulina Jawor ◽  
Tadeusz Stefaniak ◽  
Iwona Kątnik-Prastowska
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Bovine Serum ◽  
Solid Phase ◽  
Low Cost ◽  
Reference Method ◽  
Serum Samples ◽  
Incubation Periods ◽  
Short Time

Two ELISA procedures to determine haptoglobin (Hp) in bovine serum were developed. Equine haemoglobin was used as the solid phase. Self-developed goat polyclonal antibody (variant I) and monoclonal antibody (variant II) raised against human Hp were used. The results were compared with the guaiacol method. High correlation was found (r = 0.96 and r = 0.90, respectively) based on the results of 548 bovine serum samples, of which 357 were from clinically healthy cows and 191 from cows and calves monitored during treatment for the most common diseases. The Hp detection limit of ELISA using polyclonal Ab was 0.1 mg/l and using MoAb 0.21 mg/l. The addition of 2% PEG 6000 at the antibody-binding steps enabled major shortening of the incubation periods. The relatively short time, low cost of reagents, and high correlation with the reference method support the use of these ELISA variants in bovine diagnostics.

scholarly journals Direct Assay for Cobalamin Bound to Transcobalamin (Holo-Transcobalamin) in Serum

2002 ◽  
Vol 48 (3) ◽  
pp. 526-532 ◽  
Author(s):  
Marius Ulleland ◽  
Ingar Eilertsen ◽  
Edward V Quadros ◽  
Sheldon P Rothenberg ◽  
Sergey N Fedosov ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Quantitative Determination ◽  
Reliable Method ◽  
Solid Phase ◽  
Reference Interval ◽  
Magnetic Microspheres ◽  
Affinity Constant ◽  
Capture Assay

Abstract Background: Only cobalamin carried by transcobalamin (holo-transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-transcobalamin are available. We report a novel holo-transcobalamin assay based on solid-phase capture of transcobalamin. Methods: A monoclonal antibody specific for human transcobalamin with an affinity constant >1010 L/mol was immobilized on magnetic microspheres to capture and concentrate transcobalamin. The cobalamin bound to transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-transcobalamin was accomplished using calibrators composed of recombinant, human holo-transcobalamin. Results: The assay was specific for holo-transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV <20%) was 5–370 pmol/L. Dilutions of serum were linear in the assay range. The recovery of recombinant, human holo-transcobalamin added to serum was 93–108%. A 95% reference interval of 24–157 pmol/L was established for holo-transcobalamin in 105 healthy volunteers 20–80 years of age. For 72 of these sera, holo-haptocorrin and total cobalamin were also determined. Whereas holo-haptocorrin correlated well (r2 = 0.87) with total cobalamin, holo-transcobalamin correlated poorly (r2 = 0.23) with total cobalamin or holo-haptocorrin. Conclusions: The solid-phase capture assay provides a simple, reliable method for quantitative determination of holo-transcobalamin in serum.

scholarly journals Dual-Label Immunoassay for Simultaneous Measurement of Prostate-specific Antigen (PSA)-α1-Antichymotrypsin Complex Together with Free or Total PSA

2003 ◽  
Vol 49 (1) ◽  
pp. 97-103 ◽  
Author(s):  
Lei Zhu ◽  
Jari Leinonen ◽  
Wan-Ming Zhang ◽  
Patrik Finne ◽  
Ulf-Håkan Stenman
Keyword(s):  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Simultaneous Measurement ◽  
Solid Phase ◽  
Specific Antigen ◽  
Cathepsin G ◽  
Serum Samples ◽  
Total Psa ◽  
Dual Label

Abstract Background: A major portion of prostate-specific antigen exists in circulation as a complex with α1-antichymotrypsin (PSA-ACT), whereas a minor part is free (fPSA). The proportion of PSA-ACT is increased in prostate cancer (PCa), but immunologic determination of PSA-ACT is hampered by a background produced by nonspecific adsorption of ACT to the solid phase. To reduce the nonspecific interference, we produced an antibody specific for complexed ACT and developed immunofluorometric assays (IFMAs) for simultaneous measurement of fPSA + PSA-ACT (fPSA/PSA-ACT) and PSA-ACT + total PSA (tPSA, PSA-ACT/tPSA). Methods: Monoclonal antibodies (MAbs) were produced by immunization with PSA-ACT. The dual-label time-resolved IFMAs for fPSA/PSA-ACT and PSA-ACT/tPSA used a capture MAb to tPSA, an Eu3+-labeled MAb to fPSA or complexed ACT, and an Sm3+-labeled MAb to complexed ACT or to tPSA as tracer antibodies. The clinical utility was evaluated using serum samples from individuals with or without PCa with PSA concentrations of 2.0–20.0 μg/L. Results: One MAb (1D10) showed low cross-reactivity with free ACT and cathepsin G-ACT. A sandwich assay for PSA-ACT with 1D10 as tracer had a detection limit of 0.05 μg/L, and with this assay, PSA-ACT was undetectable in female sera. The detection limit for fPSA was 0.004 μg/L. Determinations of the ratio of fPSA to PSA-ACT and the proportions of fPSA/tPSA and PSA-ACT/tPSA provided the same clinical specificity for PCa and provided significantly better clinical specificity than did tPSA. Conclusions: Background problems observed in earlier PSA-ACT assays are eliminated by the use of a MAb specific for complexed ACT as a tracer. The same clinical validity can be obtained by determination of fPSA or PSA-ACT together or in combination with tPSA.

scholarly journals Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2

2004 ◽  
Vol 50 (9) ◽  
pp. 1607-1617 ◽  
Author(s):  
Ville Väisänen ◽  
Susann Eriksson ◽  
Kaisa K Ivaska ◽  
Hans Lilja ◽  
Martti Nurmi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Solid Phase ◽  
Specific Antigen ◽  
Control Group ◽  
Serum Samples ◽  
Human Kallikrein 2 ◽  
Kallikrein 2 ◽  
Capture Antibody ◽  
Total Psa

Abstract Background: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. Methods: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. Results: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 μg/L. The median total hK2 concentration was 0.022 μg/L (range, 0.0015–0.37 μg/L). hK2 concentrations were 0.1–58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41–50 and 51–60 years of age. The ratio of hK2 to PSA steadily decreased from 5–30% at PSA <1 μg/L to 1–2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 μg/L (range, 0.0015–16.2 μg/L). The median free hK2 concentration was 0.070 (range, 0.005–12.2) μg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%). Conclusions: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.

scholarly journals Quantitative Determination of Azithromycin in Human Plasma by Liquid Chromatography–Mass Spectrometry and its Application in Pharmackokinetic Study

2012 ◽  
Vol 11 (1) ◽  
pp. 55-63 ◽  
Author(s):  
Maizbha Uddin Ahmed ◽  
Mohammad Safiqul Islam ◽  
Tasmin Ara Sultana ◽  
AGM Mostofa ◽  
Muhammad Shahdaat Bin Sayeed ◽  
...  
Keyword(s):  
Mobile Phase ◽  
Solid Phase ◽  
Internal Standard ◽  
Extraction Process ◽  
Serum Samples ◽  
Limit Of Quantification ◽  
Relative Standard ◽  
Linear Concentration Range ◽  
Linear Concentration

Azithromycin is an effective and well-known antimicrobial agent. In the present study, a simple, sensitive and specific LC/MS/MS method has been developed and validated for the quantification of Azithromycin in  human serum samples using Clarithromycin as internal standard. Azithromycin was extracted from biological matrix  by using solid phase extraction process. The chromatographic separation was performed on Luna C18 (3 ?, 2x150   mm) column with a mobile phase consisting of 35 mM ammonium acetate buffer (mobile phase-A) and acetonitrile  and methanol in ratio of 90:10 ( as mobile phase-B) at a flow rate of 0.25 mL/min. The method was validated over a  linear concentration range of 0.5?50.0 ng/mL and limit of quantification (LOQ) was 0.5 ng/mL with a coefficient of  correlation (r2) = 0.9998. The intra-day and inter-day precision expressed as relative standard deviation were 1.64% – 8.43% and 2.32% – 9.92%, respectively. The average recovery of azithromycin from serum was 98.11%. The method  was successfully applied to a pharmacokinetic study after oral administration of Azithromycin 200 mg/5 ml suspension in healthy Bangladeshi volunteers. DOI: http://dx.doi.org/10.3329/dujps.v11i1.12488 Dhaka Univ. J. Pharm. Sci. 11(1): 55-63, 2012 (June)

Resonance Rayleigh Scattering Determination of Trace Hemin Basing on Aptamer-Modified Nanogold Catalysis of HAuCl4-Vitamine C

2013 ◽  
Vol 787 ◽  
pp. 400-403
Author(s):  
Jin Chao Dong ◽  
Ai Hui Liang ◽  
Zhi Liang Jiang
Keyword(s):  
Detection Limit ◽  
Rayleigh Scattering ◽  
Resonance Rayleigh Scattering ◽  
Serum Samples ◽  
Buffer Solution ◽  
Strong Resonance ◽  
Scattering Spectra ◽  
Vitamine C ◽  
Nanogold Catalysis

Hemin aptamer was used to modify gold nanoparticles (AuNPs) to obtain a stable aptamer-nanogold probe (AussDNA). In the condition of pH 8.0 Tris-HCl buffer solution containing 50mmol/L NaCl, the substrate chain of AussDNA was cracked by hemin to produce a short single-stranded DNA(ssDNA) and then further combined with hemin to form a stable hemin-ssDNA conjugate. The AuNPs released from AussDNA would be aggregated in the condition of 50mmol/L NaCl and exhibited a strong resonance Rayleigh scattering (RRS) peak at 368nm. Under the selected conditions, the increased RRS intensity (ΔI368nm) was linear to hemin concentration in the range of 5-750nmol/L, with a detection limit of 66 pmol/L. This RRS method was applied to determination of residual hemin in serum samples, with satisfactory results. The remnant AussDNA in the solution exhibited a strong catalytic activity on the gold particle reaction of HAuCl4-vitamine C (VC) that can be monitored by RRS technique at 368 nm. When the hemin concentration increased, the AussDNA decreased, the catalysis decreased, and the RRS intensity at 368nm decreased. The decreased RRS intensity ΔI368nmwas linear to the hemin concentration in the range of 1-200nmol/L, with a detection limit of 54 pmol/L. Accordingly, a sensitivity, selectivity, and simplicity new method of resonance Rayleigh scattering spectra to detect hemin using aptamer-modified nanogold as catalyst was established.

scholarly journals Synchronous fluorescence as a green and selective tool for simultaneous determination of bambuterol and its main degradation product, terbutaline

2018 ◽  
Vol 5 (10) ◽  
pp. 181359 ◽  
Author(s):  
Samah Abo El Abass ◽  
Heba Elmansi
Keyword(s):  
Degradation Product ◽  
Low Cost ◽  
Reference Method ◽  
Cost Effective ◽  
Zero Crossing ◽  
Cost Effective Method ◽  
Validation Parameters ◽  
Main Degradation Product ◽  
20 Nm

A green, sensitive and cost-effective method is introduced in this research for the determination of bambuterol and its main degradation product, terbutaline, simultaneously, relying on the synchronous spectrofluorimetric technique. First derivative synchronous spectrofluorimetric amplitude is measured at Δ λ = 20 nm, so bambuterol can be quantitated at 260 nm, and terbutaline can be measured at 290 nm, each at the zero crossing point of the other. The amplitude–concentration plots were linear over the concentration ranges of 0.2–6.0 µg ml −1 and 0.2–4.0 µg ml −1 for both bambuterol and terbutaline, respectively. Official guidelines were followed to calculate the validation parameters of the proposed method. The low values of limits of detection of 0.023, 0.056 µg ml −1 and limits of quantitation of 0.071, 0.169 µg ml −1 for bambuterol and terbutaline, respectively, point to the sensitivity of the method. Bambuterol is a prodrug for terbutaline, and the latter is considered its degradation product so the established method could be regarded as a stability-indicating one. Moreover, the proposed method was used for the analysis of bambuterol and terbutaline in their single ingredient preparations and the results revealed statistical agreement with the reference method. The suggested method, being a simple and low-cost procedure, is superior to the previously published methods which need more sophisticated techniques, longer analysis time and highly toxic solvents and reagents. It could be considered as an eco-friendly analytical procedure.

Direct solid-phase enzymoimmunoassay of testosterone in saliva.

1989 ◽  
Vol 35 (10) ◽  
pp. 2044-2047 ◽  
Author(s):  
K Howard ◽  
M Kane ◽  
A Madden ◽  
J P Gosling ◽  
P F Fottrell
Keyword(s):  
Detection Limit ◽  
Analytical Procedure ◽  
Solid Phase ◽  
Medical Applications ◽  
Microtiter Plates ◽  
Large Numbers ◽  
Coefficients Of Variation ◽  
Serial Sampling ◽  
Direct Solid

Abstract This competitive, solid-phase enzymoimmunoassay for testosterone in saliva is carried out on microtiter plates and involves no chromatographic or extraction steps. With an overnight incubation the detection limit of the assay is 230 fg per well (16.1 pmol/L). There was a good correlation (correlation coefficient 0.95) between testosterone concentrations measured with and without prior extraction of the saliva samples. Repeated assay of three control saliva samples containing a range of testosterone concentrations (200-1000 pmol/L) gave within- and between-assay coefficients of variation of 5.5-13.2%. The analytical procedure is simple and closely resembles already published procedures for the determination of progesterone and estrone (with extraction) in saliva. One person can assay 200 samples in 24 h and the assay is suitable for reproductive and sports medical applications, particularly for projects involving serial sampling and yielding large numbers of samples.

scholarly journals Preparation of Cerium Oxide-MWCNTs Nanocomposite Bulk Modified Carbon Ceramic Electrode: A Sensitive Sensor for Tamoxifen Determination in Human Serum Samples

2021 ◽  
Author(s):  
Sepideh Shafaei ◽  
Elyas Hosseinzadeh ◽  
Gulsah Saydan Kanberoglu ◽  
Balal Khalilzadeh ◽  
Rahim Mohammad-Rezaei
Keyword(s):  
Cerium Oxide ◽  
Limit Of Detection ◽  
Low Cost ◽  
High Electron ◽  
Range Limit ◽  
Serum Samples ◽  
Important Species ◽  
Carbon Ceramic Electrode ◽  
Carbon Ceramic

Abstract In this study, cerium oxide and multi-walled carbon nanotubes nanocomposite was incorporated into the carbon ceramic electrode (CeO2-MWCNTs/CCE) as a renewable electrode for the electrocatalytic purposes. To demonstrate capability of the fabricated electrode, determination of Tamoxifen as an important anticancer drug with differential pulse voltammetry technique was evaluated. Linear range, limit of detection and sensitivity of the developed sensor were found to be 0.2-40 nM, 0.132 nM and 1.478 µA nM-1 cm-2, respectively. Ease of production, low cost and high electron transfer rate of CeO2-MWCNTs/CCE promise it as a novel electro-analytical tool for determination of important species in real samples.

scholarly journals Preconcentration of Copper with Solid Phase Extraction and its Determination by Flame Atomic Absorption Spectrometry

2008 ◽  
Vol 5 (4) ◽  
pp. 878-883 ◽  
Author(s):  
Ayob Parchehbaf Jadid ◽  
Habibollah Eskandari
Keyword(s):  
Detection Limit ◽  
Solid Phase Extraction ◽  
Solid Phase ◽  
Copper Ions ◽  
Absorption Spectrometry ◽  
Phase Extraction ◽  
Flame Atomic Absorption ◽  
Low Levels ◽  
Determination Of Copper

A new method for the solid phase extraction (SPE) and determination of copper ions at low levels is presented. Extraction percent and the effects of some factors were evaluated. The detection limit was in the range of 2.26 µg·L-1. This procedure has been successfully applied to determination of copper in water samples.

A solid-phase enzymeimmunoassay for the determination of progesterone in bovine, porcine and ovine plasma

1995 ◽  
Vol 75 (4) ◽  
pp. 525-529 ◽  
Author(s):  
R. P. Del Vecchio ◽  
W. D. Sutherland ◽  
M. L. Connor
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Petroleum Ether ◽  
Bovine Serum ◽  
Solid Phase ◽  
Plasma Samples ◽  
Assay Sensitivity ◽  
Coefficients Of Variation ◽  
Rabbit Igg

The purpose of this project was to develop a valid quantitative enzymeimmunoassay (EIA) for progesterone in blood plasma of cattle, pigs and sheep. Rabbit anti-progesterone, mouse monoclonal anti-rabbit IgG, authentic progesterone, and acetylcholine esterase bound covalently to progesterone were the principal reagents used to develop the EIA. Ninety-six well microliter plates were coated with mouse monoclonal anti-rabbit IgG and saturated with bovine serum albumin before use. Rabbit anti-progesterone was diluted to a working dilution of 1:2.0 × 106. Standard curves were linear and ranged from 1.56 to 400 pg of progesterone per well which allowed for the measurement of 0.03125 to 8.0 ng mL−1. Assay sensitivity averaged 1.56 pg well−1. Progesterone was extracted from plasma samples with petroleum ether. Plasma samples (n = 3 or 4 from each species) with unknown amounts of progesterone that were extracted and serially diluted with EIA buffer did not deviate from parallelism with progesterone standard curves in buffer. The correlation between EIA and radioimmunoassay (RIA) measurements of progesterone in the same plasma samples was high (P < 0.0001) for all three species (r = 0.96 for bovine; r = 0.96 for porcine; r = 0.94 for ovine). The regression of EIA data on RIA data produced the following equations:[Formula: see text]The intra- and inter-assay coefficients of variation were 5.4 and 10.6% for bovine, 5.8 and 11.0% for porcine and, 6.1 and 12.3% for ovine, respectively. These data show that this EIA is a valid and reliable memod for quantitating progesterone in extracts of bovine, porcine and ovine plasma. Key words: Enzymeimmunoassay, progesterone, plasma, bovine, porcine, ovine

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