bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epitope Analysis of a Prostate-specific Antigen (PSA) C-Terminal-specific Monoclonal Antibody and New Aspects for the Discrepancy between Equimolar and Skewed PSA Assays
Authors: Nagasaki Hiroshi , Watanabe Motoyuki , Komatsu Naoki , Kaneko Takashi , Dubé Jean Y , Kajita Tadahiro , Saitoh Yoshihiro , Ohta Yohsuke 
Published: January 2020

Abstract
Background: Immunoassays to measure prostate-specific antigen (PSA) often give different values for the same patient samples, and the calibrators among commercial immunoassays are not interchangeable. We developed three novel assays to quantify the free and complexed forms of PSA in serum.
Methods: We synthesized 46 peptides, which encompassed the entire PSA molecule, and determined the interactions between selected monoclonal antibodies (MAbs) and those peptides or the intact PSA molecule.
Results: MAb PA313 did not cross-react with human glandular kallikrein (hK2), which has 78% amino acid homology to PSA. This MAb bound with KD = 40 nmol/L to the C-terminal peptide of PSA and distinguished between a synthetic peptide derived from PSA (PSA46A: NH2-C-R226KWIKDTIVANP237-COOH) that differed from one derived from hK2 (PSA46B: NH2-C-R226KWIKDTAANP237-COOH) by a single amino acid. Only the MAb combination of PA313/PA121 showed equimolar reactivity with PSA and with PSA complexed with α1-antichymotrypsin (PSA-ACT). The free form of PSA (F-PSA) was determined by MAbs PA313/FPA503, and the amount of complexed PSA (C-PSA) in PSA-ACT was determined by αACT/PA313. The total PSA (T-PSA) measured by either of the equimolar assays (PA313/PA121 or Tandem-R) was consistent with the sum of F-PSA and C-PSA. In contrast, T-PSA by a skewed assay (IMx) was higher than F-PSA + C-PSA when the ratio of F-PSA to T-PSA (F/T) was &gt;0.15. T-PSA measured by IMx was nearly equal to F-PSA/0.55 + C-PSA. The coefficient 0.55 reflected different reactivities of the IMx assay with PSA-ACT and PSA.
Conclusion: The discrepancy between the values measured by equimolar and skewed assays depends on the ratio of free to total PSA in the sample.
Apolipoprotein E in Cerebrospinal Fluid: Relation to Phenotype and Plasma Apolipoprotein E Concentrations
Authors: Yamauchi Kazuyoshi , Tozuka Minoru , Nakabayashi Tetsuo , Sugano Mitsutoshi , Hidaka Hiroya , Kondo Yoshiyuki , Katsuyama Tsutomu 
Published: January 2020

AbstractBackground: Apolipoprotein (apo) E may be related to the development of Alzheimer disease, but data on apoE in cerebrospinal fluid (CSF) are limited. The aim of the present study was to measure apoE in CSF and relate its concentrations to apoE phenotype and CSF lipids.Methods: We adapted an assay for CSF apoE sensitivity using an ELISA. It allowed us to measure CSF apoE with sufficient reproducibility and precision.Results: The within- and between-run CVs were &lt;7%, and the detection limit was 0.025 mg/L. No cross-reaction was found for other apolipoproteins. No significant differences related to sex or apoE phenotype were observed in the CSF apoE concentration. The mean CSF apoE concentration was significantly higher in the 0–5 year group (n = 6; 18.47 ± 1.14 mg/L, mean ± SD) than in the &gt;5 year group (n = 34; 8.82 ± 3.31 mg/L). The mean concentrations of total cholesterol (TC) and phospholipid (PL) in CSF were 2.68 ± 2.16 and 6.50 ± 2.84 mg/L (n = 52), respectively. Although no significant differences in TC or PL in the CSF were found with respect to sex or age, the concentrations in subjects with the apoE phenotype E4/E3 were significantly lower than in those with E3/E3 and E3/E2. The concentrations of apoE, TC, and PL in CSF did not correlate with those in plasma. The time-related fluctuations in CSF apoE were independent of those in total protein and IgG. CSF apoE was significantly correlated with TC and PL concentrations in the CSF, but not with the number of cells in the CSF.Conclusions: These findings support the idea that apoE and lipids are unable to cross the blood-brain barrier and that their concentrations in CSF may reflect production in central nervous tissue.
A Practical Approach to Determine Cutoff Concentrations for Opiate Testing with Simultaneous Detection of Codeine, Morphine, and 6-Acetylmorphine in Urine
Authors: Paul Buddha D , Shimomura Eric T , Smith Michael L 
Published: January 2020

AbstractBackground: Both the Department of Defense (DoD) and the Department of Health and Human Services (DHHS) currently require two confirmation tests to verify use of heroin, one test for total morphine and a separate test for 6-acetylmorphine (6-AM). Our aim was to determine appropriate free-codeine, free-morphine, and 6-AM cutoff concentrations that could be substituted for total-morphine, total-codeine, and 6-AM cutoff concentrations and to develop a less labor-intensive method for measuring codeine, morphine, and 6-AM.Methods: Urine samples containing opiates were extracted, derivatized, and analyzed using gas chromatography–mass spectrometry with selective ion monitoring.Results: The limits of detection for codeine, morphine, and 6-AM were 6, 5, and 0.5 μg/L, respectively. Recoveries were &gt;90%. Quantification was linear over the concentration range of 6–1000 μg/L for codeine, 5–5000 μg/L for morphine, and 0.5–800 μg/L for 6-AM. Cutoff concentrations for confirmation of opiates were 100, 100, and 10 μg/L for free codeine, free morphine, and 6-AM.Conclusion: The proposed cutoff concentrations for free morphine and 6-AM provide better detection windows for morphine and heroin use than the cutoff concentrations for total morphine and 6-AM used at present. Detection of free codeine, instead of total codeine, simplifies interpretation of codeine use. The single-extraction method enables simultaneous, less labor-intensive analysis of morphine, codeine, and 6-AM.
Library of Sequence-specific Radioimmunoassays for Human Chromogranin A
Authors: Børglum Jensen Tine , Hilsted Linda , Rehfeld Jens F 
Published: January 2020

AbstractBackground: Human chromogranin A (CgA) is an acidic protein widely expressed in neuroendocrine tissue and tumors. The extensive tissue- and tumor-specific cleavages of CgA at basic cleavage sites produce multiple peptides.Methods: We have developed a library of RIAs specific for different epitopes, including the NH2 and COOH termini and three sequences adjacent to dibasic sites in the remaining part of CgA.Results: The antisera raised against CgA(210–222) and CgA(340–348) required a free NH2 terminus for binding. All antisera displayed high titers, high indexes of heterogeneity (∼1.0), and high binding affinities (Keff0 ∼ 0.1 × 1012 to 1.0 × 1012 L/mol), implying that the RIAs were monospecific and sensitive. The concentration of CgA in different tissues varied with the assay used. Hence, in a carcinoid tumor the concentration varied from 0.5 to 34.0 nmol/g tissue depending on the specificity of the CgA assay. The lowest concentration in all tumors was measured with the assay specific for the NH2 terminus of CgA. This is consistent with the relatively low concentrations measured in plasma from carcinoid tumor patients by the N-terminal assay, whereas the assays using antisera raised against CgA(210–222) and CgA(340–348) measured increased concentrations.Conclusion: Only some CgA assays appear useful for diagnosis of neuroendocrine tumors, but the entire library is valuable for studies of the expression and processing of human CgA.
Affinity Chromatography: A Review of Clinical Applications
Authors: Hage David S 
Published: January 2020

AbstractAffinity chromatography is a type of liquid chromatography that makes use of biological-like interactions for the separation and specific analysis of sample components. This review describes the basic principles of affinity chromatography and examines its use in the testing of clinical samples, with an emphasis on HPLC-based methods. Some traditional applications of this approach include the use of boronate, lectin, protein A or protein G, and immunoaffinity supports for the direct quantification of solutes. Newer techniques that use antibody-based columns for on- or off-line sample extraction are examined in detail, as are methods that use affinity chromatography in combination with other analytical methods, such as reversed-phase liquid chromatography, gas chromatography, and capillary electrophoresis. Indirect analyte detection methods are also described in which immunoaffinity chromatography is used to perform flow-based immunoassays. Other applications that are reviewed include affinity-based chiral separations and the use of affinity chromatography for the study of drug or hormone interactions with binding proteins. Some areas of possible future developments are then considered, such as tandem affinity methods and the use of synthetic dyes, immobilized metal ions, molecular imprints, or aptamers as affinity ligands for clinical analytes.
When Is a Heterophile Antibody Not a Heterophile Antibody? When It Is an Antibody against a Specific Immunogen
Authors: Kaplan Irina V , Levinson Stanley S 
Published: January 2020

AbstractHeterophile antibodies are antibodies produced against poorly defined antigens. These are generally weak antibodies with multispecific activities. Human anti-animal antibodies that develop as a result of treatments with animal immunoglobulins are antibodies with strong avidities, produced against well-defined antigens. Although heterophile antibodies and human anti-animal antibodies interfere with immunological assays by similar mechanisms, modes for identifying the sources of the antibodies and for circumventing or retarding the interference may differ. Unfortunately, there has not been a well-organized attempt to encourage correct definition of these antibodies. This problem of inexact definition is highlighted by recent articles in this Journal. In the present discussion, we examine the history leading to this problem and discuss the origins and the reasons that the nature of the antibody is important for rectifying the problem. We propose a simple nomenclature for general usage that should appropriately characterize these antibodies in most cases.
Single-Nucleotide Polymorphisms in Intron 2 of CYP21P: Evidence for a Higher Rate of Mutation at CpG Dinucleotides in the Functional Steroid 21-Hydroxylase Gene and Application to Segregation Analysis in Congenital Adrenal Hyperplasia
Authors: Jiddou Renée R , Wei Wan-Li , Sane Kumud S , Killeen Anthony A 
Published: January 2020

Abstract
Background: Intron 2 of CYP21, the functional steroid 21-hydroxylase gene contains several single-nucleotide polymorphisms (SNPs). We tested the hypothesis that intron 2 of the pseudogene, CYP21P, might also be polymorphic and provide markers for segregation analysis of this region of the genome, including observable markers for segregation analysis of CYP21 gene deletions. A comparison of SNPs in both genes might provide insights into the rates of mutation in these duplicated genes.
Methods: After amplification with PCR, we examined restriction site polymorphisms in intron 2 of CYP21P in 24 members of the parental generation of the Centre d’Étude du Polymorphisme Humain families and selected offspring.
Results: Intron 2 of CYP21P contains frequent SNPs around nucleotide 398 and nucleotide 509, which can be typed by PCR/restriction enzyme digestion with HaeIII. Of the 48 CYP21P alleles examined, 44 could be characterized unambiguously. Of these 44 alleles, 4 were deleted, and the frequencies of restriction at the polymorphic HaeIII sites were 20 of 40 at nucleotide 398 and 30 of 40 at nucleotide 509. Both polymorphisms result from C→T transitions that occur at CpG dinucleotides. The frequencies of C at these nucleotides in CYP21P are significantly higher than at the corresponding nucleotides in CYP21 of the same individuals (P &lt;0.01).
Conclusion: These data suggest that these CpG dinucleotides are more frequently mutated in CYP21 than in CYP21P, and that several mutations at CpG dinucleotides in the coding regions of CYP21 might result from CpG instability rather than the more usually proposed mechanism of gene conversion. These frequent SNPs provide useful markers for studying both allelic segregation of CYP21, particularly for chromosomes with known CYP21 deletions, and for investigating the origin of these polymorphisms.
Anti-Free Prostate-specific Antigen Monoclonal Antibody Epitopes Defined by Mimotopes and Molecular Modeling
Authors: Michel Sandrine , Deléage Gilbert , Charrier Jean-Philippe , Passagot Jacques , Battail-Poirot Nicole , Sibai Geneviève , Jolivet Michel , Jolivet-Reynaud Colette 
Published: January 2020

AbstractBackground: Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer, and the free PSA/total PSA ratio has been shown to be efficient for distinguishing prostate cancer from benign prostatic hyperplasia. We report here the characterization of seven mouse monoclonal antibodies (mAbs) and the partial localization of two conformational epitopes identified by anti-free PSA mAbs.Methods: The mAbs were studied by competition and sandwich assays, and the epitope localization of the two anti-free PSA mAbs (6C8D8 and 5D3D11) was performed using phage displayed peptide libraries and molecular modeling.Results: The seven mAbs were classified into three groups according to their recognition specificities and their ability to inhibit the enzymatic activity of PSA and the formation of PSA-α1-antichymotrypsin (ACT) complex. Among the anti-free PSA mAb group, 6C8D8 recognized the phage displayed peptide RKLRPHWLHFHPVAV, two parts of which presented similarities with two regions distant on the PSA sequence but joined in the tridimensional structure. mAb 5D3D11 recognized the peptide DTPYPWGWLLDEGYD, which is similar to a PSA region located on the board of the groove containing the PSA enzymatic site. Both epitopes were located in the theoretical ACT binding site described previously. Moreover, these mAbs were able to inhibit the enzymatic activity of PSA.Conclusions: These epitope localizations are in agreement with the ability of both mAbs to inhibit enzymatic activity and ACT fixation. The results presented here could bring information for the generation of clinically relevant PSA assays.
Combined Inhibin and CA125 Assays in the Detection of Ovarian Cancer
Authors: Robertson David M , Cahir Nicholas , Burger Henry G , Mamers Pamela , McCloud Philip I , Pettersson Kim , McGuckin Michael 
Published: January 2020

Abstract
Background: The reproductive hormone inhibin has been used as a diagnostic marker of ovarian mucinous and granulosa cell cancers. The aims of this study were to develop a new inhibin immunofluorometric assay (αC IFMA) to replace an inhibin RIA as a diagnostic marker of these ovarian cancers and to assess whether the αC IFMA in combination with CA125, which detects serous cancers, leads to an improved biochemical diagnosis of all ovarian cancers.
Methods: Serum inhibin concentrations were determined in healthy postmenopausal women (n = 165) and women with ovarian cancers (n = 154), using an inhibin RIA and an αC IFMA, which detects inhibin forms containing the αC subunit as well as the free αC subunit.
Results: The αC IFMA gave a similar or better discrimination of mucinous (90% vs 71%) and granulosa cell (100% vs 100%) cancers compared with the inhibin RIA. Combination of CA125 and αC IFMA values by canonical variate analysis or by multiROC analysis showed that the percentage of all ovarian cancers detected was significantly increased compared with either CA125 or αC IFMA alone.
Conclusions: The αC IFMA shows a similar or better specificity compared with the RIA, but with increased sensitivity. In combination with CA125, the αC IFMA provides an effective dual test for the detection of the majority (90%) of ovarian cancers.
Detection of Human Serum Tumor Necrosis Factor-α in Healthy Donors, Using a Highly Sensitive Immuno-PCR Assay
Authors: Saito Kaori , Kobayashi Daisuke , Sasaki Masateru , Araake Hiroshi , Kida Takashi , Yagihashi Atsuhito , Yajima Tomomi , Kameshima Hidekazu , Watanabe Naoki 
Published: January 2020

AbstractBackground: Tumor necrosis factor-α (TNFα) is an important mediator of inflammatory and autoimmune diseases. Analysis of its pathophysiologic roles has been difficult because low concentrations of TNFα, including those in healthy controls, cannot be measured by existing methods.Methods: We developed a sensitive immuno-PCR assay for the detection of TNFα in human serum. The DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNFα sandwiched by antibodies was detected by amplification of the DNA label using PCR.Results: The limit of detection of the assay was 0.001 ng/L, an ∼5 × 104-fold improvement compared with a conventional ELISA. The mean serum TNFα concentration (± SD) in healthy donors was 0.021 ± 0.044 ng/L in men (n = 29) and 0.033 ± 0.065 ng/L in women (n = 25).Conclusion: This method may be useful for analyzing the significance of TNFα concentration in various diseases.
Serum Galactosyl Hydroxylysine as a Biochemical Marker of Bone Resorption
Authors: Al-Dehaimi Abdul Wahed , Blumsohn Aubrey , Eastell Richard 
Published: January 2020

AbstractBackground: Serum-based biochemical markers of bone resorption may provide better clinical information than urinary markers because direct comparison with serum markers of bone formation is possible and because the within-subject variability of serum markers may be lower. We describe a method for the measurement of free β-1-galactosyl-O-hydroxylysine (Gal-Hyl) in serum.Methods: The assay used preliminary ultrafiltration of serum, dansylation, and separation by reversed-phase HPLC with fluorescence detection. Healthy subjects were recruited from population-based studies of bone turnover.Results: The within-run (n = 15) and between-run (n = 15) CVs were 7% and 14%, respectively, at a mean value of 48 nmol/L. In women and pubertal girls, serum free Gal-Hyl correlated with urine free Gal-Hyl (r = 0.84; P &lt;0.001). Serum Gal-Hyl was higher during puberty and increased after menopause. The fractional renal clearance of free Gal-Hyl relative to that of creatinine was 0.90 (95% confidence interval, 0.82–0.98). Serum free Gal-Hyl decreased by 36% (SE = 4%) in 14 patients with mild Paget disease treated with an oral bisphosphonate, and this decrease was significantly (P &lt;0.001) greater than that seen for either serum tartrate-resistant acid phosphatase (9%; SE = 4%) or serum C-terminal telopeptide of collagen I (19%; SE = 8%).Conclusion: Serum free Gal-Hyl may be useful as a serum marker of bone resorption.
The Role of Food and Drug Administration Regulation of In Vitro Diagnostic Devices—Applications to Genetics Testing
Authors: Gutman Steven 
Published: January 2020

Abstract
The Food and Drug Administration (FDA) has been involved in the regulation of in vitro diagnostic devices (IVDs or laboratory tests) since the introduction of the Medical Device Amendments of 1976. IVDs developed as kits or systems intended for use in multiple laboratories require review by the FDA before being marketed to ensure appropriate performance and labeling. IVDs developed as in-house, or so-called “home-brew”, tests or laboratory test services are considered medical devices, but historically have not been subject to premarket review as a matter of enforcement discretion. FDA recently established a new regulatory paradigm for in-house tests based on classification of the active ingredients or building blocks of these tests as analyte-specific reagents (ASRs). ASRs are exempt from premarket review but subject to both manufacturing and labeling controls. Currently, genetic tests are received and reviewed by the FDA in the same manner as other in vitro diagnostic tests. The FDA currently is in the process of chartering a new genetics advisory panel to provide the agency with outside expertise to deal with genetic testing issues. We are also continuing to work with other agencies within the Department of Health and Human Services to determine how we can cooperatively help foster this important new area of testing.
Measurement of the Complex between Prostate-specific Antigen and α1-Protease Inhibitor in Serum
Authors: Zhang Wan-Ming , Finne Patrik , Leinonen Jari , Vesalainen Satu , Nordling Stig , Stenman Ulf-HÅkan 
Published: January 2020

Abstract
Background: Prostate-specific antigen (PSA) occurs in serum both free and in complex with protease inhibitors. The complex with α1-antichymotrypsin (ACT) is the major form in serum, and the proportion of PSA-ACT is higher in prostate cancer (PCa) than in benign prostatic hyperplasia (BPH). PSA also forms a complex with α1-protease inhibitor (API) in vitro, and the PSA-ACT complex has been detected in serum from patients with prostate cancer. The aim of the present study was to develop a quantitative method for the determination of PSA-API and to determine the serum concentrations in patients with PCa and BPH.
Methods: The assay for PSA-API utilizes a monoclonal antibody to PSA as capture and a polyclonal antibody to API labeled with a Eu-chelate as a tracer. For calibrators, PSA-API formed in vitro was used. Serum samples were obtained before treatment from 82 patients with PCa, from 66 patients with BPH, and from 22 healthy females.
Results: The concentrations of PSA-API are proportional to the concentrations of total PSA. PSA-API comprises 1.0–7.9% (median, 2.4%) of total immunoreactive PSA in PCa and 1.3–12.2% (median, 3.6%) in BPH patients with serum PSA concentrations &gt;4 μg/L. In patients with 4–20 μg/L total PSA, the proportion of PSA-API serum is significantly higher in BPH (median, 4.1%) than in PCa (median, 3.2%; P = 0.02).
Conclusions: The proportion of PSA-API in serum is lower in patients with PCa than in those with BPH. These results suggest that PSA-API is a potential adjunct to total and free PSA in the diagnosis of prostate cancer.
Impact of Antibody Specificity and Calibration Material on the Measure of Agreement between Methods for Cardiac Troponin I
Authors: Newman David J , Olabiran Yemi , Bedzyk William D , Chance Suzette , Gorman Eileen G , Price Christopher P 
Published: January 2020

AbstractBackground: Available assays for cardiac troponin I (cTnI) yield numerically different results. The aim of this study was to compare patient values obtained from four cTnI immunoassays.Methods: We studied the Stratus® II assay, the Opus® II assay, the Access® assay, and a research-only cTnI heterogeneous immunoassay that uses the Dade Behring aca® plus immunoassay system equipped with two new noncommercial monoclonal antibodies. Because the aca plus cTnI assay is for research only, we first evaluated and analytically validated it for serum and citrated plasma. Initially, each method was calibrated using the method-specific calibrator supplied by each manufacturer; however, the aca plus cTnI assay was calibrated using patient serum pools containing cTnI and selected on the basis of increased creatine kinase MB isoenzyme and with values assigned by use of the Stratus cTnI assay. For method comparisons, individual patient sample cTnI values were determined and compared with the Stratus II assay.Results: Passing and Bablock regression analysis yielded slopes of 1.44 (r = 0.96; n = 72) for the Opus II vs Stratus II assays; 0.07 (r = 0.91; n = 72) for the Access vs Stratus II assays; and 0.90 (r = 0.91, n = 72) for the aca plus vs Stratus II assays. The recalibration of each method with a Stratus II-assigned serum pool improved, but did not entirely eliminate, the slope differences between the different assays (range, 1.00–1.16). The observed scatter in the correlation curves remained.Conclusion: There is a need to further explore the specificities of these assays with respect to the different circulating forms of cTnI.