scholarly journals A Chemiluminescent Immunoassay for Osteocalcin in Human Serum and a Solution to the “Hook Effect”

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Shuang Han ◽  
Yifeng Xue ◽  
Junlan Zhang ◽  
Jianrong Huang ◽  
Xiuxia Liu ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Human Serum ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Bone Gla Protein ◽  
Serum Osteocalcin ◽  
Established Method ◽  
Clinical Patient ◽  
Hook Effect ◽  
Chemiluminescent Immunoassay

A chemiluminescent immunoassay for human serum osteocalcin, or bone Gla protein, was established using a double-antibody sandwich model. Examination of the hook effect revealed that it was significantly reduced, and no hook effect was observed at an osteocalcin concentration of 4000 ng/mL. The established method showed good analytical performance and thermal stability. The limit of detection was 0.03 ng/mL. The intra-assay coefficient of variation (CV) was 3.22%–5.64%, the interassay CV was 4.42%–7.25%, and the recovery rate was 93.22%–107.99%. Cross-reactivity (CR) was not observed with bovine, rat, mouse, rabbit, or porcine samples. The developed method showed a good correlation with the N-MID product from Roche. In total, 1069 clinical patient samples were measured using the reagent kit developed in this study.

RADIOIMMUNOASSAY OF 3,3′-DI-IODOTHYRONINE IN UNEXTRACTED SERUM: THE EFFECT OF ENDOGENOUS TRI-IODOTHYRONINE

1978 ◽  
Vol 79 (3) ◽  
pp. 357-362 ◽  
Author(s):  
T. J. VISSER ◽  
L. M. KRIEGER-QUIST ◽  
R. DOCTER ◽  
G. HENNEMANN
Keyword(s):  
Human Serum ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Transport Proteins ◽  
Normal Serum ◽  
Serum Concentrations ◽  
Specific Radioimmunoassay ◽  
Highly Sensitive ◽  
The Mean ◽  
Mean Concentration

The development of a highly sensitive and specific radioimmunoassay for 3,3′-di-iodothyronine (3,3′-T2) is described. The assay was applied to the measurement of 3,3′-T2 in unextracted human serum and used 8-anilino-l-naphthalene-sulphonic acid to inhibit the binding of 3,3′-T2 to serum transport proteins. The lower limit of detection of the assay was 2 fmol 3,3′-T2 per tube, which corresponded to 10 pmol 3,3′-T2/l serum. The mean concentration of 3,3′-T2 in normal serum was found to be 23 pmol/l, which is considerably lower than most values reported previously. Evidence is presented which suggests that the cross-reactivity of tri-iodothyronine with the antiserum to 3,3′-T2 is an important factor in the measurement of serum concentrations of 3,3′-T2 by radioimmunoassay.

scholarly journals Development of a PTHrP Chemiluminescent Immunoassay to Assess Humoral Hypercalcemia of Malignancy

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A1015-A1015
Author(s):  
Susan Louise Ashrafzadeh-Kian ◽  
Joshua Bornhorst ◽  
Alicia Algeciras-Schimnich
Keyword(s):  
Limit Of Detection ◽  
Reference Interval ◽  
Cross Reactivity ◽  
Humoral Hypercalcemia Of Malignancy ◽  
Clinical Sensitivity ◽  
Hypercalcemia Of Malignancy ◽  
Humoral Hypercalcemia ◽  
Hook Effect ◽  
Limit Of Quantitation ◽  
Acridinium Ester

Abstract Background: Measurement of parathyroid hormone related peptide (PTHrP) is helpful in the diagnosis and clinical management of patients suspected of humoral hypercalcemia of malignancy (HHM). In these patients uncontrolled release of PTHrP by tumor cells is responsible for the hypercalcemia and PTH concentrations are typically suppressed. Objective: Develop a sensitive and specific assay for quantitation of PTHrP in plasma. Method: Calibrators (PTHrP 1-86) and samples (50uL) were incubated with an anti-PTHrP goat polyclonal acridinium ester labeled antibody. Complexes were transferred and incubated in a microplate coated with an anti-PTHrP polyclonal rabbit antibody. After washing, the acridinium ester generated signal, which is directly proportional to the amount of PTHrP in sample, was quantified. Results: In this assay PTHrp was stable for 24 hours ambient, 3 days refrigerated, 34 days frozen and through 3 freeze/thaws. Intra and inter-assay imprecision in EDTA plasma (~0.16-35.0 pmol/L) ranged from 2.2-8.6% and 5-15%, respectively. The limit of detection was 0.04 pmol/L and the limit of quantitation was 0.16 pmol/L (15% CV). The analytical measuring range was 0.39-50.5 pmol/L (slope of 1.07 and r2 of 0.99). Average spike recovery was 98% (range 85-108%). The assay was not affected by hemoglobin of ≤500 mg/dL, triglycerides of ≤2000 mg/dL, or bilirubin of ≤50mg/dL. No hook effect was noted up to 500 pmol/L. PTH (1-84) did not cross-react in the assay. C-terminal PTHrP(107-139), and N-terminal PTHrP(1-36) had no significant cross-reactivity (≤1.1%). Mid-PTHrP(38-94) had 8.3% cross-reactivity. Comparison with an in-house PTHrP assay (n=267) showed an r2 of 0.96, and slope of 2.25 by Passing-Bablok regression fit. The 97.5% reference interval for PTHrP (n=114) was ≤0.7 pmol/L, however a higher concentration (≤4.2 pmol/L) was identified as a more specific clinical cut-off. A retrospective clinical validation study showed that using ≤4.2 pmol/L resulted in a 91% clinical sensitivity and a 98% clinical specificity. Conclusion: We have developed an analytically and clinically sensitive and specific PTHrP immunoassay. A cutoff of ≤4.2 pmol/L is clinically useful in the evaluation of patients suspected of hypercalcemia of malignancy.

scholarly journals Development of a Chemiluminescence Immunoassay for Quantification of 25-Hydroxyvitamin D in Human Serum

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Shuang Han ◽  
Wuxian Qiu ◽  
Junlan Zhang ◽  
Zhonghu Bai ◽  
Xiao Tong
Keyword(s):  
Human Serum ◽  
Magnetic Beads ◽  
Cross Reactivity ◽  
Free Form ◽  
Chemiluminescence Immunoassay ◽  
Dihydroxyvitamin D3 ◽  
Clinical Patient ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D ◽  
And Performance

In this study, a chemiluminescence immunoassay (CLIA) for human serum 25-hydroxyvitamin D (25(OH)D) was established by a competition model. In serum, more than 99% of total circulating 25(OH)D binds to protein and less than 1% of 25(OH)D is in free form (Jassil et al., 2017). Before measuring concentration of 25(OH)D in serum, a releasing procedure should be conducted. A new reagent is used to release binding 25(OH)D to free form. Streptavidin (SA) was labeled to magnetic beads by a 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) method. Biotinylated VD was used as a competitor of 25(OH)D in samples. Anti-VD antibody (aby) was labeled to horseradish peroxidase (HRP) by EDC to react with 25(OH)D and biotinylated-VD molecules. The pretreated samples or standards were added into the reaction tube with biotin-VD and anti-VD aby-HRP, free 25(OH)D in the sample competes with biotinylated VD for binding to anti-VD aby-HRP, an SA-labeled magnetic particle is added to isolate the signal-generating complex, and the signal is inversely proportional to the 25(OH)D concentration in the sample. The method established shows good thermostability and performance. The limitation of detection (LoD) is 1.43 ng/mL. The intra-assay coefficient of variation (CV) is 3.66%–6.56%, the interassay CV is 4.19%–7.01%, and the recovery rate is 93.22%–107.99%. Cross-reactivity (CR) was remarkably low with vitamin D2, vitamin D3, 1, 25-dihydroxyvitamin D3, and 1, 25-dihydroxyvitamin D2. At the same time, the cross-reaction values with 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 were 97% and 100%, respectively. The developed method shows good correlation with the total VD product from Roche and DiaSorin. 1096 clinical patient samples were measured with developed reagent kit in this study. 7 types of disease were involved, and the concentration of 25(OH)D is less than 30 ng/mL in 94.98% of patients.

scholarly journals Development of a two-site solid-phase immunochemiluminescent assay for measurement of dimeric inhibin-A in human serum and other biological fluids

1996 ◽  
Vol 42 (8) ◽  
pp. 1159-1167 ◽  
Author(s):  
D S McConnell ◽  
V Padmanabhan ◽  
T B Pollak ◽  
N P Groome ◽  
J J Ireland ◽  
...  
Keyword(s):  
Human Serum ◽  
Follicular Fluid ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Biological Fluids ◽  
Cross Reactivity ◽  
Inhibin A ◽  
Vitro Fertilization ◽  
Related Proteins

Abstract Inhibin is a heterodimeric glycoprotein that inhibits the secretion of follitropin from the pituitary and has been isolated in two distinct forms composed of a common alpha subunit and either a beta A or beta B subunit. Utilizing paired monoclonal antibodies specific to the alpha and beta A subunit, we have developed an immunochemiluminescent assay for dimeric inhibin-A. The assay is capable of quantifying free and bound inhibin-A in human serum and follicular fluid. The limit of detection is 10 ng/L. Related proteins exhibit little cross-reactivity or interference. Recovery is excellent. Whereas samples from men and postmenopausal women are near the detection limit of the assay, inhibin-A is higher in the luteal than the follicular phase of normally cycling women, 20-fold higher during in vitro fertilization treatment, and approximately 200-fold greater in pregnancy. The assay measures inhibin-A in follicular fluid from a variety of other species.

Detection of Glucose in Human Serum Based on Silicon Dot Probe

2020 ◽  
Vol 16 (6) ◽  
pp. 744-752
Author(s):  
Kuan Luo ◽  
Xinyu Jiang
Keyword(s):  
Quantum Dots ◽  
Blood Glucose ◽  
Human Serum ◽  
Organic Dyes ◽  
Limit Of Detection ◽  
Fasting Blood Glucose ◽  
Glucose Biosensor ◽  
Metal Nanoclusters ◽  
Glucose Levels ◽  
Highly Sensitive

Background: Diabetes Mellitus (DM) is a major public metabolic disease that influences 366 million people in the world in 2011, and this number is predicted to rise to 552 million in 2030. DM is clinically diagnosed by a fasting blood glucose that is equal or greater than 7 mM. Therefore, the development of effective glucose biosensor has attracted extensive attention worldwide. Fluorescence- based strategies have sparked tremendous interest due to their rapid response, facile operation, and excellent sensitivity. Many fluorescent compounds have been employed for precise analysis of glucose, including quantum dots, noble metal nanoclusters, up-converting nanoparticles, organic dyes, and composite fluorescent microspheres. Silicon dot as promising quantum dots materials have received extensive attention, owing to their distinct advantages such as biocompatibility, low toxicity and high photostability. Methods: MnO2 nanosheets on the Si nanoparticles (NPs) surface serve as a quencher. Si NPs fluorescence can make a recovery by the addition of H2O2, which can reduce MnO2 to Mn2+, and the glucose can thus be monitored based on the enzymatic conversion of glucose by glucose oxidase to generate H2O2. Therefore, the glucose concentration can be derived by recording the fluorescence recovery spectra of the Si NPs. Results: This probe enabled selective detection of glucose with a linear range of 1-100 μg/mL and a limit of detection of 0.98 μg/mL. Compared with the commercial glucometer, this method showed favorable results and convincing reliability. Conclusion: We have developed a novel method based on MnO2 -nanosheet-modified Si NPs for rapid monitoring of blood glucose levels. By combining the highly sensitive H2O2/MnO2 reaction with the excellent photostability of Si NPs, a highly sensitive, selective, and cost-efficient sensing approach for glucose detection has been designed and applied to monitor glucose levels in human serum with satisfactory results.

Calcium/Copper alginate framework doped with CuO nano-particles as a novel adsorbent for micro-extraction of benzodiazepines from human serum

2020 ◽  
Vol 16 ◽  
Author(s):  
Nadereh Rahbar ◽  
Fatemeh Ahmadi ◽  
Zahra Ramezani ◽  
Masoumeh Nourani
Keyword(s):  
Human Serum ◽  
High Performance ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Nano Particles ◽  
Limit Of Quantification ◽  
Analytical Methodology ◽  
Fiber Fabrication ◽  
Relative Standard ◽  
Green Procedure

Background: Sample preparation is one of the most challenging phases in pharmaceutical analysis, especially in biological matrices, affecting the whole analytical methodology. Objective: In this study, a new Ca(II)/Cu(II)/alginate/CuO nanoparticles hydrogel fiber (CCACHF) was synthesized through a simple, green procedure and applied for fiber micro solid phase extraction (FMSPE) of diazepam (DIZ) and oxazepam (OXZ) as model drugs prior to high-performance liquid chromatography-UV detection (HPLC-UV). Methods: Composition and morphology of the prepared fiber were characterized and the effect of main parameters on the fiber fabrication and extraction efficiency have been studied and optimized. Results: In optimal conditions, calibration curves were linear ranging between 0.1–500 µg L−1 with regression coefficients of 0.9938 and 0.9968. Limit of detection (LOD) (S/N=3) and limit of quantification (LOQ) (S/N=10) of the technique for DIZ and OXZ were 0.03 to 0.1 µg L−1. Within-day and between-day relative standard deviations (RSDs) for DIZ and OXZ were 6.0–12.5% and 3.3–9.4%, respectively. Conclusion: The fabricated adsorbent has been substantially employed to extraction of selected benzo-diazepines (BZDs) from human serum real specimens and the obtained recoveries were also satisfactory (82.1-109.7%).

scholarly journals PERFORMANCE CHARACTERISTICS OF A FRET-BASED IMMUNOASSAY FOR QUANTITATION OF INFLIXIMAB ON A POINT-OF-CARE INSTRUMENT SYSTEM

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S57-S57
Author(s):  
Edgar Ong ◽  
Ruo Huang ◽  
Richard Kirkland ◽  
Michael Hale ◽  
Larry Mimms
Keyword(s):  
Whole Blood ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Quantitative Detection ◽  
Therapeutic Drug ◽  
Analytical Sensitivity ◽  
Sample Type ◽  
Hook Effect ◽  
Limit Of Quantitation ◽  
Assay Precision

Abstract Introduction A fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassay was developed for the quantitative detection of infliximab (IFX) and biosimilars for use in therapeutic drug monitoring using only 20 µL of fingerstick whole blood or serum at the point-of-care. The Procise IFX assay and ProciseDx analyzer are CE-marked. Studies were performed to characterize analytical performance of the Procise IFX assay on the ProciseDx analyzer. Methods Analytical testing was performed by spiking known amounts of IFX into negative serum and whole blood specimens. Analytical sensitivity was determined using limiting concentrations of IFX. Linearity was determined by testing IFX across the assay range. Hook effect was assessed at IFX concentrations beyond levels expected to be found within a patient. Testing of assay precision, cross-reactivity and potential interfering substances, and biosimilars was performed. The Procise IFX assay was also compared head-to-head with another CE-marked assay: LISA-TRACKER infliximab ELISA test (Theradiag, France). The accuracy of the Procise IFX assay is established through calibrators and controls traceable to the WHO 1st International Standard for Infliximab (NIBSC code: 16/170). Results The Procise IFX assay shows a Limit of Blank, Limit of Detection, and Lower Limit of Quantitation (LLoQ) of 0.1, 0.2, and 1.1 µg/mL in serum and 0.6, 1.1, and 1.7 µg/mL in whole blood, respectively. The linear assay range was determined to be 1.7 to 77.2 µg/mL in serum and whole blood. No hook effect was observed at an IFX concentration of 200 µg/mL as the value reported as “>ULoQ”. Assay precision testing across 20 days with multiple runs and reagent lots showed an intra-assay coefficient of variation (CV) of 2.7%, an inter-assay CV of <2%, and a total CV of 3.4%. The presence of potentially interfering/cross-reacting substances showed minimal impact on assay specificity with %bias within ±8% of control. Testing of biosimilars (infliximab-dyyb and infliximab-abda) showed good recovery. A good correlation to the Theradiag infliximab ELISA was obtained for both serum (slope=1.01; r=0.99) and whole blood (slope=1.01; r=0.98) samples (Figure 1). Conclusion Results indicate that the Procise IFX assay is sensitive, specific, and precise yielding results within 5 minutes from both whole blood and serum without the operator needing to specify sample type. Additionally, it shows very good correlation to a comparator assay that takes several hours and sample manipulation to yield results. This makes the Procise IFX assay ideal for obtaining fast and accurate IFX quantitation, thus allowing for immediate drug level dosing decisions to be made by the physician during patient treatment.

scholarly journals Siphon-Controlled Automation on a Lab-on-a-Disc Using Event-Triggered Dissolvable Film Valves

Biosensors ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 73
Author(s):  
Brian D. Henderson ◽  
David J. Kinahan ◽  
Jeanne Rio ◽  
Rohit Mishra ◽  
Damien King ◽  
...  
Keyword(s):  
Risk Factor ◽  
Limit Of Detection ◽  
C Reactive Protein ◽  
Cvd Risk ◽  
Low Resource Settings ◽  
Low Resource ◽  
Reactive Protein ◽  
Limit Of Quantitation ◽  
Chemiluminescent Immunoassay ◽  
Event Triggered

Within microfluidic technologies, the centrifugal microfluidic “Lab-on-a-Disc” (LoaD) platform offers great potential for use at the PoC and in low-resource settings due to its robustness and the ability to port and miniaturize ‘wet bench’ laboratory protocols. We present the combination of ‘event-triggered dissolvable film valves’ with a centrifugo-pneumatic siphon structure to enable control and timing, through changes in disc spin-speed, of the release and incubations of eight samples/reagents/wash buffers. Based on these microfluidic techniques, we integrated and automated a chemiluminescent immunoassay for detection of the CVD risk factor marker C-reactive protein displaying a limit of detection (LOD) of 44.87 ng mL−1 and limit of quantitation (LoQ) of 135.87 ng mL−1.

scholarly journals IMMUNOLOGIC STUDIES OF WATER-SOLUBLE HUMAN AMYLOID FIBRILS

1969 ◽  
Vol 130 (4) ◽  
pp. 797-808 ◽  
Author(s):  
Edward C. Franklin ◽  
Mordechai Pras
Keyword(s):  
Human Serum ◽  
Comparative Studies ◽  
Amyloid Fibrils ◽  
Complement Fixation ◽  
Cross Reactivity ◽  
Water Soluble ◽  
Antigenic Determinants ◽  
Normal Tissues ◽  
Absorption Studies ◽  
Normal Human

Eight preparations of soluble amyloid and degraded amyloid (DAM) were compared immunologically. Unlike amyloid fibrils, six of eight preparations of DAM proved to be relatively strong immunogens. Antisera to DAM reacted weakly or not at all with normal human serum or extracts of normal tissues, but were specifically reactive with amyloid fibrils or DAM. Comparative studies of DAM'S from eight different subjects showed some degree of cross-reactivity among them, yet demonstrated that they were not identical. Similar conclusions were obtained by quantitative precipitin and complement fixation analyses. Comparison of the amyloid fibrils with the homologous DAM by complement fixation and absorption studies demonstrated the existence in DAM of antigenic determinants that were lacking or inaccessible in the native fibrils. A search for amyloid precursors and antibodies to amyloid in the sera of 12 patients proved unsuccessful.

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