scholarly journals Evaluation of Analytical Performances of Magnetic Force-Assisted Electrochemical Sandwich Immunoassay for the Quantification of Carcinoembryonic Antigen

2022 ◽  
Vol 9 ◽  
Author(s):  
Boo Young Hwang ◽  
Eunsoo Kim ◽  
Seung-ha Kim ◽  
Hyundoo Hwang
Keyword(s):  
Carcinoembryonic Antigen ◽  
Magnetic Force ◽  
Method Comparison ◽  
Cross Reactivity ◽  
Sandwich Immunoassay ◽  
Dose Effect ◽  
Clinical Practices ◽  
Hook Effect ◽  
Reference Device ◽  
Limit Of Quantitation

Carcinoembryonic antigen (CEA) is a biomarker indicated in different cancers, targeted for quantitative analysis via immunoassay. Here we introduce a new technique called magnetic force-assisted electrochemical sandwich immunoassay (MESIA) for determination of CEA level in a drop of human serum using a fully automated point-of-care testing (POCT) device. The analytical performances of the assay are assessed based on precision, accuracy, limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ), linearity, Hook effect, interference, cross-reactivity, and method comparison following the guidelines of the Clinical Laboratory Standards Institute (CLSI). The LoD is 0.50 ng/ml. A linear relationship is shown in the range of 0.5–200 ng/ml. A high dose effect is not seen up to approximately 500,000 ng/ml. The recovery range is from 94.7 to 108.9%. The %CV of run-to-run and within-lab variations are less than 2.04 and 4.41% across the CEA concentrations, respectively, whereas reproducibility is 4.45–6.24%. Method comparison shows that the assay correlates well with the reference device (R2 = 0.9884). The assay demonstrates acceptable precision, accuracy, LoB, LoD and LoQ, hook effect, linearity, interference, cross-reactivity, and high correlation with its reference device. Thus, the system is suitable for the quantification of CEA in clinical practices with a POCT manner.

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 Vitamin D testing: Comparison and limitations of currently employed immunoassay with a novel liquid chromatography and tandem mass spectrometry (LCMS/MS) technique.

2021 ◽  
Vol 28 (7) ◽  
pp. 1053-1057
Author(s):  
Sehrish Naz ◽  
◽  
Muhammad Aamir ◽  
Zujaja Hina Haroon ◽  
Sobia Irum ◽  
...  
Keyword(s):  
Vitamin D ◽  
Liquid Chromatography ◽  
Limit Of Detection ◽  
Tertiary Care ◽  
Method Comparison ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Cross Sectional ◽  
Comparison Results ◽  
Limit Of Quantitation

Objective: To compare analytical method for 25 hydroxy vitamin D2 and D3 on LC/MS-MS and with routine vitamin D Immunoassay method. Study Design: Cross Sectional study. Setting: Department of Chemical Pathology and Endocrinology, Pakistan. Period: March 2019 to March 2020. Material & Methods: Samples were extracted and a mass spectrometer coupled to high performance liquid chromatography was adopted for quantitation of 25-hydroxyvitamin D2 and D3 in human samples (serum). After validation it was then applied to 120 serum samples from healthy individuals for method comparison. Results: The method was validated in terms of accuracy, precision, linearity on calibration curve, Limit of Detection and Limit of Quantitation. Our study showed a statistically insignificant difference in results among both the methods (p=0.715). Limit of detection (LOD) was 2.49 ng/ml and limit of quantitation (LOQ) was 3.9 ng/ml for both the metabolites. Percentage RSD was 0.8% and 1.3% for D2 and D3 respectively. This method has an advantage of minimal cross-reactivity with 24,25 hydroxy vit D and 25,26 di- hydroxy vit D metabolite than the routinely used assays. Conclusion: This methodology will be helpful in guiding patient management and assess possibility of malabsorption syndrome in patients on D2 therapy. It can give highly cost effective reliable results of Vitamin D at a tertiary care setting which has an already installed LCMS/MS with huge workload, as compared to costly Immunoassay method.

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 Direct Determination of Free Methionine in Soy-Based Infant Formula

2004 ◽  
Vol 87 (1) ◽  
pp. 123-128
Author(s):  
Paul Johns ◽  
Rosalyn Phillips ◽  
Lobat Dowlat
Keyword(s):  
Infant Formula ◽  
Mobile Phase ◽  
Method Comparison ◽  
Water System ◽  
Direct Determination ◽  
Reversed Phase ◽  
Intermediate Precision ◽  
Relative Standard ◽  
Limit Of Quantitation

Abstract A method was developed for the direct determination of free methionine in soy-based infant formula, with analyte separation and quantitation by reversed-phase liquid chromatography (LC), and UV absorbance at 214 nm, respectively. Sample preparation required only dilution with mobile phase and syringe filtration. Using a 0.02M KH 2 PO 4 mobile phase (pH adjusted to 2.9 with 85% o-phosphoric acid) and 0.7 mL/min flow rate, methionine eluted at approximately 8 min, and total run time was 14 min after column regeneration with acetonitrile–water. System linearity was demonstrated as peak area versus analyte concentration, ranging from 80 to 120% of the formula specification for free methionine (r > 0.999, and all residuals <0.45%). Intermediate precision relative standard deviation values were <1.5% for ready-to-feed and reconstituted powder samples, and recoveries ranged from 98.0 to 103.5% for inter-method comparison with an amino acid analyzer method. The limit of quantitation was 3 mg methionine/L in the “as fed” infant formula. Despite the relatively weak UV absorptivity of methionine, the 214 nm signal was sufficiently intense in the 30–65 mg/L (201–436 μM) range to afford quantitation by peak area proportionation versus a 2-point external standard calibration. This direct UV detection after reversed-phase LC separation provides a simple and accurate method for determining free methionine without derivatization.

scholarly journals A Comparative Analysis of Methods for Quantitation of Sugars during the Corn-to-Ethanol Fermentation Process

2020 ◽  
Vol 25 (5) ◽  
pp. 494-504
Author(s):  
Sarah R. Bilskey ◽  
Samantha A. Olendorff ◽  
Karolina Chmielewska ◽  
Kevin R. Tucker
Keyword(s):  
Liquid Chromatography ◽  
Ethanol Fermentation ◽  
Fermentation Process ◽  
Dynamic Range ◽  
Fermentation Broth ◽  
Standard Procedure ◽  
Method Comparison ◽  
Selected Ion Monitoring ◽  
Comparison Results ◽  
Limit Of Quantitation

The quantitation of sugars, including glucose, the primary fermentable sugar; maltose (DP2); and maltotriose (DP3), is a standard procedure during the corn-to-ethanol fermentation process. The quantitation of glucose by the Megazyme Assay utilizing glucose oxidase and peroxidase enzymes (GOPOD) and UV-Vis detection, high-performance liquid chromatography with refractive index detection (HPLC-RID), and liquid chromatography mass spectrometry (LC-MS) with electrospray ionization (ESI) and selected ion monitoring (SIM) was studied. Three biological flask fermentation replicates were analyzed every 12 h beginning at 14 h of fermentation (T14) until near completion of fermentation (T62). The method comparison results for glucose quantitation showed that the LC-MS SIM analysis had the lowest limit of quantitation (LOQ) at 2 ppm and the widest dynamic range of 2.7 orders of magnitude. The HPLC-RID analysis had a linear dynamic range (LDR) of 1.5 orders of magnitude with an LOQ of 1500 ppm. The Megazyme GOPOD analysis had an LDR of 0.9 orders of magnitude with an LOQ of 120 ppm. The HPLC-RID method was ideal for glucose quantitation when it was present in high concentrations. In contrast, maltose and maltotriose components were found to be present in lower concentrations, such that simultaneous quantitation of the three analytes is difficult during fermentation. The LC-MS method was the only method able to quantify the concentration of glucose successfully and simultaneously with DP2 and DP3 in all the fermentation broth samples collected from T14 through T62 during the corn-to-ethanol fermentation process.

Automated Erythropoietin Testing on Beckman Coulter’s Family of Access® Immunoassay Systems.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3761-3761
Author(s):  
Susan Retka ◽  
Judy Haning ◽  
Richard Fuerstenberg ◽  
Laurent Drai ◽  
Ravindra Mylvaganam
Keyword(s):  
Bone Marrow ◽  
Cancer Patients ◽  
Dynamic Range ◽  
Bone Marrow Cells ◽  
Method Comparison ◽  
The United States ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Analytical Sensitivity ◽  
Glycoprotein Hormone

Abstract Background: Erythropoietin (EPO) is a ~30,400 dalton glycoprotein hormone produced primarily by the kidneys. EPO is the principal factor stimulating bone marrow cells to differentiate into red blood cells (RBCs). Normally, EPO levels increase when oxygen or RBC levels become abnormally low, or decrease if RBC concentrations become abnormally high. The evaluation of EPO is useful in distinguishing between disease etiologies in erythrocytosis or polycythemia (overproduction of RBCs). Primary polycythemia or polycythemia vera is caused by EPO-independent growth of erythrocytic progenitors from neoplastic bone marrow stem cells, and in most cases decreased levels of EPO are found in the serum of affected patients. Various types of secondary polycythemias are associated with the production of elevated levels of EPO. EPO evaluation has been utilized recently to aid in predicting the responsiveness of anemic cancer patients to recombinant human EPO (rhEPO) therapy. An elevated EPO level (>200 mIU/mL) in an anemic patient may predict a low response to rhEPO therapy. Objectives and Methods: This study was designed to determine the performance characteristics of the EPO assay on Beckman Coulter’s family of Access Immunoassay Systems. This automated sandwich assay combines a polyclonal anti-EPO alkaline phosphatase conjugate and 75 microliters of sample (serum or plasma). Following a 10-minute incubation, paramagnetic particles coated with goat anti-mouse IgG / mouse anti-rhEPO monoclonal antibody are added. A second 20-minute incubation allows for antigen-antibody complex formation and is followed by wash and substrate addition steps. Light generated by the reaction is measured with a luminometer. The light production is directly proportional to the concentration of EPO in the sample. The EPO concentration is determined from a stored multipoint calibration curve. The time to first result is < 45 minutes. Results: The dynamic range of the EPO assay is 0.6–750 mIU/mL with an analytical sensitivity of less than or equal to 0.6 mIU/mL. When evaluated using control material in a total of 20 tests with 2 replicates per test, the assay demonstrated a total imprecision of less than or equal to 10% at concentrations from approximately 9.5–475 mIU/mL. Method comparison to a commercially available EPO microtiter assay with 103 samples (range approximately 3–200 mIU/mL) yielded a correlation coefficient of 0.988 and a slope of 1.051. The expected range of 2.59–18.50 mIU/mL (range 1.48–31.88 mIU/mL) was based on a 95% non-parametric analysis of 122 normal samples. When tested using related compounds and common serum and plasma components, no significant cross reactivity or interference was observed. Conclusion: With the expanding uses for EPO testing, including the evaluation of cancer patients who may be candidates for rhEPO therapy, the rapid turnaround time provided by this automated assay for EPO on the Access Immunoassay Systems provides the laboratory with a rapid, sensitive and precise assay for measuring EPO. Pending submission to and clearance by the United States FDA; not yet available for diagnostic use.

Rapid 5-fluorouracil plasma quantification by immunoassay: Validation with FOLFOX6 clinical samples.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 563-563 ◽  
Author(s):  
S. J. Salamone ◽  
C. N. Benfield ◽  
J. B. Courtney ◽  
R. L. Harney ◽  
D. R. Kozo ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Limit Of Detection ◽  
Plasma Concentrations ◽  
Method Comparison ◽  
Medical Decision ◽  
Clinical Samples ◽  
Fast Time ◽  
Good Precision ◽  
Limit Of Quantitation ◽  
Modified Folfox6

563 Background: 5-fluorouracil (5-FU) is one of the most widely used chemotherapy agents in the treatment of colorectal cancer. Studies over the last 30 years have demonstrated wide pharmacokinetic variability of 5-FU which can lead to undue toxicity and suboptimal treatment. Recent clinical studies have demonstrated that managing plasma 5-FU levels and adjusting doses to target steady state concentrations effectively minimizes toxicity and improves outcome. Methods: An existing immunoassay using a novel antibody to 5-FU was modified to directly quantify 5-FU in patient plasma samples without any sample pretreatment. The assay was adapted to Beckman Coulter AU analyzers and to Roche COBAS c 111 analyzer. Method comparison to LC-MS/MS, limit of detection (LoD), lower limit of quantitation (LLoQ), precision, and linearity of the assay were evaluated. The assay was also validated by testing samples of patients being treated by modified FOLFOX6 from an on-going clinical trial ( NCT00943137 ). Results: The modified immunoassay was confirmed to have a linear reportable range from 85 to 18,000ng/mL and a LoD of 52ng/mL. The immunoassay was established to have good precision (CV<6%) around the medical decision points for FOLFOX and FOLFIRI regimens. Method comparison of the immunoassay obtained by testing 58 clinical samples from patients under 5-FU treatment provides excellent correlation to LC- MS/MS: Deming slope 1 ± 0.05, R > 0.98. Another 82 samples of patients on modified FOLFOX6 regimen from clinical trial (( NCT00943137 ) were tested using the immunoassay and range of these sample were reported to be from 95 to 2,970 ng/mL. Conclusions: The assay provided the performance required to rapidly quantify 5-FU plasma concentrations. It was fast (time to 1st result < 10 minutes), precise, correlated well to a physical method, required only 100uL sample on the analyzer and 7ul for actual testing per assay, and was easily adapted to a variety of clinical analyzers. Clinical and pharmacokinetic laboratories could use this assay to introduce an evidence-based approach to optimize 5-FU dosing that would have higher throughput, simpler methodology, and require less labor, space or expense than the physical methods. [Table: see text]

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