scholarly journals Detection of the Ovarian Cancer Biomarker Lysophosphatidic Acid in Serum

Biosensors ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 13 ◽  
Author(s):  
Brian De La Franier ◽  
Michael Thompson
Keyword(s):  
Ovarian Cancer ◽  
Lysophosphatidic Acid ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Medical Condition ◽  
Large Population ◽  
Rapid Screening ◽  
Phase System ◽  
Serum Samples ◽  
Early Results

Lysophosphatidic acid (LPA) is present during the medical condition of ovarian cancer at all stages of the disease, and, therefore possesses considerable potential as a biomarker for screening its presence in female patients. Unfortunately, there is currently no clinically employable assay for this biomarker. In the present work, we introduce a test based on the duel protein system of actin and gelsolin that could allow the quantitative measurement of LPA in serum samples in a biosensing format. In order to evaluate this possibility, actin protein was dye-modified and complexed with gelsolin protein, followed by surface deposition onto silica nanoparticles. This solid-phase system was exposed to serum samples containing various concentrations of LPA and analyzed by fluorescence microscopy. Measurements conducted for the LPA-containing serum samples were higher after exposure to the developed test than samples without LPA. Early results suggest a limit of detection of 5 μM LPA in serum. The eventual goal is to employ the chemistry described here in a biosensor configuration for the large population-scale, rapid screening of women for the potential occurrence of ovarian cancer.

scholarly journals Development of a Sensitive Bead-Based Assay for Enhanced Monoclonal Antibody Detection

2011 ◽  
Vol 1346 ◽  
Author(s):  
Manuel E. Ruidíaz ◽  
Natalie Mendez ◽  
Ana B. Sanchez ◽  
Bradley T. Messmer ◽  
Andrew C. Kummel
Keyword(s):  
Monoclonal Antibody ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Fluorescent Labeling ◽  
Antibody Detection ◽  
Serum Samples ◽  
Mimetic Peptide ◽  
Mimetic Peptides ◽  
Zero Response

ABSTRACTMonoclonal antibodies are increasingly used in the treatment of cancer due to their enhanced targeting and immune system stimulation properties. Dosage guidelines typically do not account for personal cancer load or metabolism, thereby possibly affecting treatment outcome or causing unwanted side effects. The requirement for an assay that can quickly and precisely measure the concentration of the monoclonal antibody in a serum sample of a patient during therapy is unmet. A bead-based assay with peptide antigen mimetics has been developed to rapidly determine the concentration of antibody drug present in serum specimens with high sensitivity. Alemtuzumab (anti-CD52) and rituximab (anti-CD20) antigen mimetic peptides, as discovered by phage display, were synthesized on 10 um TentaGel resin beads using conventional solid phase peptide synthesis techniques. The beads were modified to allow for multiplexing and microfluidic handling via fluorescent labeling and magnetic functionalization. The antigen-displaying fluoromagnetic particles were incubated with spiked serum samples which allowed free antibody to be captured. Primary antibody detection was performed on alemtuzumab while rituximab detection was used to compensate for non-specific serum binding to the beads. After washing, the beads were incubated with a fluorescently tagged secondary label for detection by flow cytometry. (Results) A fast, low cost, specific assay has been developed with several key techniques which allows detection at low concentration (0.1ug/ml) of spiked samples. Primary to achieving this detection limit was the implementation of a compensation scheme where two antigen mimetic peptides behave linearly (R2=0.996) which enables the calculation of the zero response of the antigen mimetic peptide of interest (alemtuzumab antigen mimetic) while measuring the zero response of the compensatory antigen mimetic peptide (rituximab antigen mimetic) during primary assay measurement. This reduces fluorescence response variation due to variations present due to sample preparation, storage and different patients because of the equivalent interactions these effects have on the compensatory beads. The developed assay is therefore robust against serum variation and enables a lower limit of detection.

scholarly journals Multicenter study on TGPO autoantibody prevalence in various thyroid and non-thyroid diseases; relationships with thyroglobulin and thyroperoxidase autoantibody parameters

1999 ◽  
pp. 563-569 ◽  
Author(s):  
V Estienne ◽  
C Duthoit ◽  
VD Costanzo ◽  
PJ Lejeune ◽  
M Rotondi ◽  
...  
Keyword(s):  
Thyroid Nodules ◽  
Solid Phase ◽  
Post Partum ◽  
Large Population ◽  
Collaborative Study ◽  
Normal Subjects ◽  
Thyroid Diseases ◽  
Serum Samples ◽  
Autoimmune Thyroid Diseases ◽  
Autoimmune Thyroid

OBJECTIVE: TGPO autoantibodies (aAbs) that bind simultaneously to thyroglobulin (Tg) and thyroperoxidase (TPO) are present in the serum of patients with autoimmune thyroid diseases (AITD) and have been found to differ from monospecific Tg and TPO aAbs. To obtain further insights on the prevalence defined as the rate of occurrence and significance of TGPO aAbs in a large population, we carried out a collaborative study involving 15 European teams. METHODS: Serum samples from 3122 patients with various thyroid and non-thyroid diseases and normal subjects were assayed using a novel TGPO aAb detection kit. This test was designed so that TGPO aAbs are trapped between the Tg-coated solid phase and the soluble TPO labeled with a radioiodinated monoclonal antibody. RESULTS: Only three out of the 220 normal subjects (prevalence of 1.4%) were found to have positive TGPO aAb levels, which were mainly observed in the patients with AITD: the group of patients suffering from Hashimoto's thyroiditis had a TGPO aAb prevalence of 40.5% (n=437 patients), those with Graves' disease, a prevalence of 34.6% (n=645) and those with post-partum thyroiditis, 16.0% (n=243). Among the non-AITD patients with positive TGPO aAb levels, the TGPO aAb prevalence ranged from 20.7% among those with thyroid cancer (n=246) to 0% among those with toxic thyroid nodules (n=47). Among the patients with non-thyroid diseases, the TGPO aAb prevalence ranged from 9.8% in the case of Biermer's pernicious anemia (n=78) to 0% in that of premature ovarian failure (n=44). It is worth noting that the groups showing the highest TGPO aAb prevalence also contained the patients with the highest TGPO aAb titers. Statistical comparisons between the TGPO aAb prevalences in the various groups showed that TGPO aAb could be used as a parameter to distinguish between the groups of Hashimoto's and Graves' patients and between the women with post-partum thyroiditis and the post-partum women with only Tg and/or TPO aAb established during early pregnancy. Unexpectedly, the correlations between TGPO aAbs and Tg and TPO aAbs were found to depend mainly on the assay kit used. CONCLUSION: High TGPO aAb titers are consistently associated with AITD but the reverse was not found to be true. TGPO aAbs are a potentially useful tool, however, for establishing Hashimoto's diagnosis, and would be worth testing in this respect with a view to using them for routine AITD investigations.

Detecting and quantifying marijuana metabolites in serum and urine of 19 dogs affected by marijuana toxicity

2021 ◽  
pp. 104063872110272
Author(s):  
Alyson H. Fitzgerald ◽  
Yuntao Zhang ◽  
Scott Fritz ◽  
William H. Whitehouse ◽  
Tamera Brabson ◽  
...  
Keyword(s):  
Human Urine ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Clinical Signs ◽  
The United States ◽  
Urine Samples ◽  
Drug Screen ◽  
Urine Drug Screen ◽  
Serum Samples ◽  
Urine Drug

Veterinarians diagnose marijuana toxicity based on clinical signs and history, or in conjunction with an over-the-counter (OTC) human urine drug screen. With the legalization of recreational marijuana use becoming more prevalent in the United States, a more accurate test to aid in the diagnosis of canine marijuana toxicity is needed. We collected urine and serum samples from 19 dogs with confirmed or suspected marijuana toxicosis from multiple veterinary hospitals and analyzed them with a novel UPLC-MS/MS method. Calibrations from 0.1 to 100 ng/mL and QC materials were prepared. Samples were extracted, purified, and eluted with solid-phase extraction. Urine samples were tested with an OTC human urine drug screen. The limit of detection (LOD) and lower limit of quantification (LLOQ) ranges for marijuana metabolites in serum were 0.05–0.25 ng/mL and 0.1–0.5 ng/mL, respectively. In urine, the LOD and LLOQ ranges for the metabolites were 0.05–0.1 ng/mL and 0.1–0.5 ng/mL, respectively. In serum, median and range of metabolite concentrations (ng/mL) detected included: THC, 65.0 (0.14–160); 11-OH-Δ9-THC, 4.78 (1.15–17.8); 11-nor-9-carboxy-Δ9-THC, 2.18 (0.71–7.79); CBD, 0.28 (0.11–82.5); and THC-glucuronide, 2.05 (0.72–18.3). In the 19 urine samples, metabolite: creatinine (ng: mg) values detected included: THC, 0.22 (0.05–0.74); 11-OH-Δ9-THC, 0; 11-nor-9-carboxy-Δ9-THC, 1.32 (0.16–11.2); CBD, 0.19 (0.12–0.26); THC-COOH-glucuronide, 0.08 (0.04–0.11); and THC-glucuronide, 0.98 (0.25–10.7). Twenty of 21 urine samples tested negative for THC on the urine drug screen. All 19 serum samples contained quantifiable concentrations of THC using our novel UPLC-MS/MS method. Utilizing a UPLC-MS/MS method can be a useful aid in the diagnosis of marijuana toxicosis in dogs, whereas using an OTC human urine drug test is not a useful test for confirming marijuana exposure in dogs because of the low concentration of THC-COOH in urine.

scholarly journals Determination of flumazenil in serum by liquid chromatography-mass spectrometry: Application to kinetics study in acute diazepam overdose

2016 ◽  
Vol 73 (2) ◽  
pp. 146-151
Author(s):  
Snezana Djordjevic ◽  
Jasmina Jovic-Stosic ◽  
Vesna Kilibarda ◽  
Zoran Segrt ◽  
Natasa Perkovic-Vukcevic
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Half Life ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Serum Samples ◽  
Elimination Half Life ◽  
Elimination Half ◽  
Limit Of Quantitation

Backgound/Aim. Flumazenil is benzodiazepine receptor antagonist. It has been studied for a various indications, including reversal of sedation after surgery or diagnostic procedures, awakening of comatose patients in benzodiazepine overdose, or for symptomatic treatment of hepatic encephalopathy. Some drugs, like theophylline, may prolong its elimination half-life. Considering the long half-life of diazepam and its metabolites, concomitant use of theophylline may reduce the need for repeated dosing of flumazenil in patients with acute diazepam poisoning. The aim of this study was to introduce a reliable and accurate method for determining the concentration of flumazenil after therapeutic application in patients with acute poisoning, and using that method to assess whether the kinetics of flumazenil change in the presence of aminophylline (combination of theophylline and ethylenediamine in a 2 : 1 ratio) applied as concomitant therapy. Methods. Blood samples from patients with acute diazepam poisoning that received flumazenil at the dose of 0.5 mg, or the same dose with 3 mg/kg of body weight of aminophylline, were collected 1, 3, 10, 30, 60, 120 and 240 min after its intravenous administration. Samples were prepared by solid-phase extraction on Oasis HLB cartridges with ethylacetate as extracting agens. Flumazenil was determined by liquid chromatography with mass spectrometry (LC-MS) in single ion monitoring mode at m/z 304. Separation of flumazenil from matrix compound was performed on Lichrospher RP-8 column using the mixture of acidic acetonitrile and 20 mM of ammonium acetate in water (55 : 45) as a mobile phase. Results. The applied analitycal method showed excellent recovery (94.65%). The obtained extracts were much cleaner than the extracts obtained by the same extractant in the process of liquid-liquid extraction. The limit of detection of the LC-MS method described in this paper was 0.5 ng/mL and the limit of quantitation was 1 ng/mL. In the patients treated with both flumazenil and aminophylline, the elimination constant for flumazenil was significantly lower and the elimination half-life was longer (p < 0.05) in comparison with the same parameters in the patients who received flumazenil alone. Conclusion. The applied LC-MS method for the determination of flumazenil in serum samples of patients with acute diazepam poisoning is rapid, sensitive, precise and specific. Concomitant use with theophylline significantly prolonged elimination of flumazenil during the treatment of acute poisonings with diazepam.

scholarly journals Efficient Matrix Cleanup of Soft-Gel-Type Dietary Supplements for Rapid Screening of 92 Illegal Adulterants Using EMR-Lipid dSPE and UHPLC-Q/TOF-MS

2021 ◽  
Vol 14 (6) ◽  
pp. 570
Author(s):  
Beomhee Kim ◽  
Wonwoong Lee ◽  
Youlee Kim ◽  
Jihyun Lee ◽  
Jongki Hong
Keyword(s):  
Dietary Supplements ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Multiple Reaction Monitoring ◽  
Rapid Screening ◽  
Wide Range ◽  
Matrix Removal ◽  
Soft Gels ◽  
Tof Ms

An efficient matrix cleanup method was developed for the rapid screening of 92 illegal adulterants (25 erectile dysfunction drugs, 15 steroids, seven anabolic steroids, 12 antihistamines, 12 nonsteroidal anti-inflammatory drugs (NSAIDs), four diuretics, and 17 weight-loss drugs) in soft-gel-type supplements by ultra-high performance liquid chromatography-quadrupole/time of flight-mass spectrometry (UHPLC-Q/TOF-MS). As representative green chemistry methods, three sample preparation methods (dispersive liquid-liquid microextraction (DLLME), “quick, easy, cheap, effective, rugged, and safe” dispersive solid-phase extraction (QuEChERS-dSPE), and enhanced matrix removal-lipid (EMR-Lipid) dSPE) were evaluated for matrix removal efficiency, recovery rate, and matrix effect. In this study, EMR-Lipid dSPE was shown to effectively remove complicated matrix contents in soft-gels, compared to DLLME and QuEChERS-dSPE. For the rapid screening of a wide range of adulterants, extracted common ion chromatogram (ECIC) and neutral loss scan (NLS) based on specific common MS/MS fragments were applied to randomly collected soft-gel-type dietary supplement samples using UHPLC-Q/TOF-MS. Both ECICs and NLSs enabled rapid and simple screening of multi-class adulterants and could be an alternative to the multiple reaction monitoring (MRM) method. The developed method was validated in terms of limit of detection (LOD), precision, accuracy, recovery, and matrix effects. The range of LODs was 0.1–16 ng/g. The overall precision values were within 0.09–14.65%. The accuracy ranged from 81.6% to 116.6%. The recoveries and matrix effects of 92 illegal adulterants ranged within 16.9–119.4% and 69.8–114.8%, respectively. The established method was successfully applied to screen and identify 92 illegal adulterants in soft-gels. This method can be a promising tool for the high-throughput screening of various adulterants in dietary supplements and could be used as a more environmentally friendly routine analytical method for screening dietary supplements illegally adulterated with multi-class drug substances.

Multicenter evaluation of human placental alkaline phosphatase as a possible tumor-associated antigen in serum.

1988 ◽  
Vol 34 (10) ◽  
pp. 1995-1999 ◽  
Author(s):  
M E De Broe ◽  
D E Pollet
Keyword(s):  
Ovarian Cancer ◽  
Alkaline Phosphatase ◽  
Testicular Cancer ◽  
Cancer Patients ◽  
Solid Phase ◽  
Ca 125 ◽  
Placental Alkaline Phosphatase ◽  
Serum Samples ◽  
Heavy Smoking ◽  
Human Placental Alkaline Phosphatase

Abstract Using a solid-phase monoclonal antibody enzyme immuno-assay, we evaluated in a multicenter study (18 laboratories) the utility of evaluating catalytic activity of human placental alkaline phosphatase (hPLAP, EC 3.1.3.1) in serum as a potential tumor marker. We determined hPLAP in serum samples from 130 patients with ovarian cancer, 79 patients with testicular cancer (53 seminoma testis, 26 nonseminoma testis), 537 patients with various other malignant diseases (95 lung, 39 gastrointestinal, 195 breast, 208 others), 291 patients with benign diseases, and 213 healthy controls. To assess the influence of smoking on hPLAP activity in serum, we evaluated 79 serum samples from patients with noncancerous diseases for whom smoking habits had been recorded. Our main findings are: (a) hPLAP activity is frequently increased (greater than 100 mU/L) in pre-operative serum samples from ovarian cancer patients (49%) and from testicular cancer patients (59% overall; 72% seminoma, 35% nonseminoma); (b) heavy smoking may increase hPLAP activity; (c) excluding heavy smokers, a 96% specificity for cancerous lesions was observed; (d) in patients with ovarian malignancies, CA 125 and hPLAP may behave as independent markers; and (e) in patients with seminoma, hPLAP is clearly more frequently increased than is beta-choriogonadotropin.

Analysis for Fully Oxidized Neopterin in Serum by High-Performance Liquid Chromatography

1992 ◽  
Vol 38 (9) ◽  
pp. 1722-1724 ◽  
Author(s):  
J J Rippin
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Extraction Procedure ◽  
Internal Standard ◽  
Marrow Transplant ◽  
Ammonia Solution ◽  
Ferric Nitrate ◽  
Serum Samples ◽  
The Mean

Abstract Fully oxidized D-neopterin in serum can be measured by HPLC. Serum samples were preincubated with ferric nitrate/EDTA solution to remove any dihydroneopterin, which is unstable and may give spuriously high results because of its conversion to D-neopterin. Pterins were extracted onto solid-phase propylbenzenesulfonic acid minicolumns and eluted with a 1:5 (by vol) mixture of ammonia solution (308 g/L) in acetonitrile. Extracts were evaporated and then reconstituted in mobile phase (50 mL of methanol per liter of 50 mmol/L phosphate buffer, pH 6.2) before injection. Separation was performed with a 25-cm ODS2 column (particle size, 5 microns) at 32 degrees C with fluorescence detection (lambda ex 360 nm, lambda em 440 nm). The between-batch CV was 7.1% and 5.6% for neopterin concentrations of 21.7 and 67.3 nmol/L, respectively. The limit of detection was 0.75 nmol/L, and the mean recovery of the extraction procedure was 90% for neopterin and internal standard. Correlation with a radioimmunoassay (x) gave y = 0.99x + 0.64 (r = 0.970, Sy/x = 2.75). The method allows daily analysis of serum D-neopterin in small batches and is currently used to monitor patients undergoing bone-marrow transplant.

scholarly journals Determination of Tilmicosin in Bovine and Porcine Sera by Liquid Chromatography

1997 ◽  
Vol 80 (6) ◽  
pp. 1183-1190 ◽  
Author(s):  
John W Moran ◽  
James M Turner ◽  
Mark R Coleman
Keyword(s):  
Limit Of Detection ◽  
Solid Phase ◽  
Reversed Phase ◽  
Gradient System ◽  
Serum Samples ◽  
Porcine Serum ◽  
Microbiological Methods ◽  
Control Serum ◽  
Relative Standard ◽  
Limit Of Quantitation

Abstract A liquid chromatographic (LC) assay is described for determining tilmicosin in bovine and porcine blood sera. Tilmicosin is isolated from the serum matrix and purified by solid-phase extraction with C18 sorbent. Sample is analyzed by LC using a gradient system with a phenyl reversed-phase column that separates tilmicosin from the matrix in 30 min. Tilmicosin is measured by UV absorbance at 280 nm. Validation of assay included evaluation of accuracy, precision, linearity, specificity, sensitivity, range, and sample stability. The method has a limit of quantitation of 0.1 ppm and a validated range of 0.1 to 10.0 ppm. Recoveries were 91–95% for bovine serum and 85–93% porcine serum. The limit of detection was 0.05 (μg/mL. Limits of detection and quantitation were based on 3 and 6 times the baseline noise of control serum samples, respectively. Relative standard deviations of precision samples (n = 6) were 2% or less for both sera. The method has better specificity and analysis time than previous microbiological methods for tilmicosin in sera.

Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers

1991 ◽  
Vol 37 (9) ◽  
pp. 1639-1644 ◽  
Author(s):  
I Nishizono ◽  
S Iida ◽  
N Suzuki ◽  
H Kawada ◽  
H Murakami ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Carcinoembryonic Antigen ◽  
Enzyme Immunoassay ◽  
Tumor Markers ◽  
Lower Limit ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Serum Samples ◽  
Coated Particles ◽  
Chemiluminescent Enzyme Immunoassay

Abstract We developed a chemiluminescent enzyme immunoassay (CLEIA) to quantify such tumor markers as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA19-9, and CA125. We used a novel chemiluminescent substrate, a derivative of 1,2-dioxetane phosphate (AMPPD), to measure alkaline phosphatase as a labeling enzyme to Fab' fragments of antibody. Regardless of the solid phase, i.e., polystyrene beads (6 mm diameter) or ferrite-coated particles (0.3 microns diameter), the standard curves within the dynamic ranges of the conventional RIA or enzyme immunoassay (EIA) were linear in all cases except for those with AFP. Use of the ferrite particles further shortens the immunoreaction, so the assay can be performed in 30 min. In addition, the relationships between concentrations of the marker and chemiluminescent signals for CA19-9, CA125, and CEA were linear up to concentrations about 10-fold greater than the ordinary dynamic ranges. Intra- and interassay CVs (averages for individual analyte) were 2.2%-4.9% and 2.0%-5.8%, respectively. In an analysis of serum samples, results of the CLEIA correlated reasonably well with those of RIA or EIA. The lower limit of detection by CLEIA with ferrite particles was 390 arb. units/L for CA19-9, 990 arb. units/L for CA125, 0.06 micrograms/L for CEA, and 0.03 micrograms/L for AFP. Thus, the sensitivity increased to between two- and 10-fold that of RIA or colorimetric EIA, depending on the analytes.

scholarly journals SPME-LC/MS-based serum metabolomic phenotyping for distinguishing ovarian cancer histologic subtypes: a pilot study

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mariola Olkowicz ◽  
Hernando Rosales-Solano ◽  
Vathany Kulasingam ◽  
Janusz Pawliszyn
Keyword(s):  
Ovarian Cancer ◽  
Operating Characteristic ◽  
Solid Phase ◽  
Gynecological Cancer ◽  
Serum Samples ◽  
Metabolomic Profiling ◽  
Metabolomic Data ◽  
Wide Range ◽  
Auc Value ◽  
Comprehensive Mapping

AbstractEpithelial ovarian cancer (EOC) is the most common cause of death from gynecological cancer. The outcomes of EOC are complicated, as it is often diagnosed late and comprises several heterogenous subtypes. As such, upfront treatment can be highly challenging. Although many significant advances in EOC management have been made over the past several decades, further work must be done to develop early detection tools capable of distinguishing between the various EOC subtypes. In this paper, we present a sophisticated analytical pipeline based on solid-phase microextraction (SPME) and three orthogonal LC/MS acquisition modes that facilitates the comprehensive mapping of a wide range of analytes in serum samples from patients with EOC. PLS-DA multivariate analysis of the metabolomic data was able to provide clear discrimination between all four main EOC subtypes: serous, endometrioid, clear cell, and mucinous carcinomas. The prognostic performance of discriminative metabolites and lipids was confirmed via multivariate receiver operating characteristic (ROC) analysis (AUC value > 88% with 20 features). Further pathway analysis using the top 57 dysregulated metabolic features showed distinct differences in amino acid, lipid, and steroids metabolism among the four EOC subtypes. Thus, metabolomic profiling can serve as a powerful tool for complementing histology in classifying EOC subtypes.

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