Differentiating iron-loading anemias using a newly developed and analytically validated ELISA for human serum erythroferrone

Erythroferrone (ERFE), the erythroid regulator of iron metabolism, inhibits hepcidin to increase iron availability for erythropoiesis. ERFE plays a pathological role during ineffective erythropoiesis as occurs in X-linked sideroblastic anemia (XLSA) and β-thalassemia. Its measurement might serve as an indicator of severity for these diseases. However, for reliable quantification of ERFE analytical characterization is indispensable to determine the assay’s limitations and define proper methodology. We developed a sandwich ELISA for human serum ERFE using polyclonal antibodies and report its extensive analytical validation. This new assay showed, for the first time, the differentiation of XLSA and β-thalassemia major patients from healthy controls (p = 0.03) and from each other (p<0.01), showing the assay provides biological plausible results. Despite poor dilution linearity, parallelism and recovery in patient serum matrix, which indicated presence of a matrix effect and/or different immunoreactivity of the antibodies to the recombinant standard and the endogenous analyte, our assay correlated well with two other existing ERFE ELISAs (both R2 = 0.83). Nevertheless, employment of one optimal dilution of all serum samples is warranted to obtain reliable results. When adequately performed, the assay can be used to further unravel the human erythropoiesis-hepcidin-iron axis in various disorders and assess the added diagnostic value of ERFE.

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Polyclonal pancreatic elastase assay is superior to monoclonal assay for diagnosis of acute pancreatitis

Clinical Chemistry ◽

10.1093/clinchem/43.12.2339 ◽

1997 ◽

Vol 43 (12) ◽

pp. 2339-2344 ◽

Cited By ~ 5

Author(s):

Volker Keim ◽

Niels Teich ◽

Andrea Reich ◽

Fritz Fiedler ◽

Joachim Mössner

Keyword(s):

Acute Pancreatitis ◽

Abdominal Pain ◽

Polyclonal Antibodies ◽

Roc Curves ◽

Diagnostic Value ◽

Serum Concentrations ◽

Clinical Value ◽

Serum Samples ◽

Pancreatic Elastase ◽

Serum Elisa

Abstract We compared the clinical values for diagnosis of acute pancreatitis of two commercial assays for pancreatic elastase: an ELISA procedure with monoclonal antibodies and a RIA technique with polyclonal antibodies. In 14 patients with acute pancreatitis, serum concentrations of elastase determined by ELISA (ELISA-elastase) decreased much faster (half-life 0.4 days) than those of elastase determined by RIA (RIA-elastase) (2.2 days), amylase (0.8 days), or lipase (0.9 days). Serum samples from 253 additional patients with abdominal pain (32 of these with acute pancreatitis) were analyzed. In sera collected up to 48 h after the onset of disease, the ROC curves showed a slightly higher diagnostic value of RIA-elastase. In samples taken later, at a sensitivity of 90% the specificity of RIA-elastase was 95% (ELISA-elastase 40%). We conclude that serum ELISA-elastase is of much lower clinical value than RIA-elastase for diagnosis of acute pancreatitis.

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Adalimumab and anti-adalimumab LISA-TRACKER immunoassays performance criteria for therapeutic drug monitoring of adalimumab-amgen biosimilar (ABP501)

BMC Immunology ◽

10.1186/s12865-021-00473-1 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Fabien Francois ◽

Loubna Naimi ◽

Xavier Roblin ◽

Anne-Emmanuelle Berger ◽

Stephane Paul

Keyword(s):

Human Serum ◽

Dynamic Range ◽

Polyclonal Antibodies ◽

Performance Criteria ◽

Clinical Samples ◽

Therapeutic Drug ◽

Serum Samples ◽

Acceptance Criteria ◽

Sample Stability ◽

Validation Parameters

Abstract Background ABP501 is a biosimilar to Reference Adalimumab (HUMIRA®) produced by AMGEN. Adalimumab (ADA) has a marketing authorization for Crohn's disease, ulcerative colitis and other inflammatory or autoimmune diseases. The aim of this study was to evaluate the LISA-TRACKER assays developed by Theradiag (France), for the monitoring of ABP501 and anti-ABP501 antibodies in human serum. Results 68 ABP501 clinical samples were measured with the LISA TRACKER Duo Adalimumab assay. LISA TRACKER has been validated as suitable for quantification of ABP501 in human serum samples. Accuracy of the LISA-TRACKER was measured using 3 human serum matrices spiked with known levels of biosimilar, 3 levels spanning the dynamic range. Percentages of recovery were ranged from 90 to 120% for biosimilar batch1, and between 93 and 105% for biosimilar batch2. The acceptance criteria (CV < 20%) were met for intra-run (from 3.8 to 16.5%) and inter-run imprecision (from 4.4 to 13.9%) including the two batches. All results were comprised within ± 20% from results, obtained with the kit and sample unexposed in order to evaluate stability of the sample, stability of the kit and consistency of the results. In any case, but two, all percentages of inhibition were > 50% for specificity. Specificity was tested with Biosimilar spiked samples, Biosimilar with Humira® spiked samples, and clinical samples from patients treated with adalimumab biosimilar. All of these samples were spiked with polyclonal antibodies directed against Humira®. Specificity inhibition and specificity detection steps were also part of the validation parameters. Reagents made with ABP501 gave similar results than reagents made with Humira® meeting acceptance criteria. Conclusions LISA-TRACKER ADA and anti-ADA assays are reliable for the monitoring of patients treated with ABP501.

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Discrimination of cysteamine from mercapto amino acids through isoelectric point-mediated surface ligand exchange of β-cyclodextrin-modified gold nanoparticles

Journal of Materials Chemistry B ◽

10.1039/d0tb00462f ◽

2020 ◽

Vol 8 (18) ◽

pp. 4039-4045

Author(s):

Quanbao Ma ◽

Xun Fang ◽

Junting Zhang ◽

Lili Zhu ◽

Xiabing Rao ◽

...

Keyword(s):

Amino Acids ◽

Gold Nanoparticles ◽

Human Serum ◽

Isoelectric Point ◽

Ligand Exchange ◽

Serum Samples ◽

Human Serum Samples ◽

Surface Ligand ◽

First Time ◽

Surface Ligand Exchange

A pI-mediated R6G-β-CD@AuNPs system was designed for the first time for the discrimination of CA from GSH/Cys/Hcy in human serum samples.

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Adalimumab and Anti-adalimumab Lisa-tracker Immunoassays Performance Criteria for Therapeutic Drug Monitoring of Adalimumab-amgen Biosimilar (Abp501)

10.21203/rs.3.rs-44850/v1 ◽

2020 ◽

Author(s):

Fabien FRANCOIS ◽

Loubna NAIMI ◽

Xavier ROBLIN ◽

Anne-Emmanuelle Berger ◽

Stephane Paul

Keyword(s):

Human Serum ◽

Dynamic Range ◽

Polyclonal Antibodies ◽

Performance Criteria ◽

Clinical Samples ◽

Therapeutic Drug ◽

Serum Samples ◽

Acceptance Criteria ◽

Sample Stability ◽

Validation Parameters

Abstract Background. ABP501 is a biosimilar to Reference Adalimumab (HUMIRA®) produced by AMGEN. Adalimumab (ADA) has marketing authorization for Crohn's disease, ulcerative colitis and other inflammatory or autoimmune diseases. The aim of this study was to evaluate the LISA-TRACKER assays developed by Theradiag (France), for the monitoring of ABP501 and anti-ABP501 antibodies in human serum. Methods. Accuracy of the LISA-TRACKER was measured using 3 human serum matrices spiked with known levels of biosimilar, 3 levels spanning the dynamic range. Specificity was tested with Biosimilar spiked samples, Biosimilar with Humira® spiked samples, and clinical samples from patients treated with adalimumab biosimilar. All of these samples were spiked with polyclonal antibodies directed against Humira®. Intra-run, inter-run imprecision, inhibition, kit’s stability, specificity inhibition and specificity detection steps were also part of the LISA-TRACKER Duo Adalimumab assay validation parameters. Results. 68 ABP501 clinical samples were measured with the LISA TRACKER Duo Adalimumab assay. LISA TRACKER has been validated as suitable for quantification of ABP501 in human serum samples. Concerning accuracy, percentages of recovery were ranged from 90–120% for biosimilar batch1, and between 93% and 105% for biosimilar batch2. The acceptance criteria (CV < 20%) were met for intra-run (from 3.8–16.5%) and inter-run imprecision (from 4.4–13.9%) including the two batches. All results were comprised within +/-20% from results, obtained with the kit and sample unexposed in order to evaluate stability of the sample, stability of the kit and consistency of the results. In any case but two, all percentages of inhibition were > 50% for specificity. Reagents made with ABP501 gave similar results than reagents made with Humira® meeting acceptance criteria. Conclusions. LISA-TRACKER ADA and anti-ADA assays are reliable for the monitoring of patients treated with ABP501.

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Use of polyclonal IgY antibodies to detect serum immune complexes in patients with active hookworm infection

Parasitology ◽

10.1017/s0031182020000220 ◽

2020 ◽

Vol 147 (6) ◽

pp. 715-720

Author(s):

Dayane Costa de Souza ◽

Lucas Silva de Faria ◽

José Eduardo Neto de Sousa ◽

Camila de Almeida Lopes ◽

Vanessa da Silva Ribeiro ◽

...

Keyword(s):

Immune Complexes ◽

Protein Complexes ◽

Polyacrylamide Gel Electrophoresis ◽

Sandwich Elisa ◽

Sodium Dodecyl ◽

Egg Yolk ◽

Diagnostic Value ◽

Enzyme Linked Immunosorbent Assay ◽

Hookworm Infection ◽

Serum Samples

AbstractDefinitive diagnosis of hookworm infection is usually based on the microscopic detection of eggs in a stool sample; however, several cases display a low or irregular egg output. Serodiagnosis can be a useful tool to identify these cases, but conventional tests do not differentiate past from active infections. The aim of this study was to obtain and apply egg yolk polyclonal immunoglobulin (IgY) antibodies to detect immune complexes (ICs) in serum samples from patients infected with hookworm. Hens were immunized with Ancylostoma ceylanicum saline extract, their eggs were collected and then IgY antibodies were extracted and purified. Antibody purity was tested by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and specificity was assessed by immunoblotting and immunofluorescence. IgY production was evaluated by kinetics enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA tested the ability of IgY to detect ICs in serum samples, from which diagnostic parameters were calculated. Antibody responses increased steadily from day 7 to 42. In the immunoblotting assay, IgY recognized two protein complexes. The immunofluorescence assay showed no staining in control samples. The sandwich ELISA presented a very high diagnostic value, with a sensitivity of 90% and a specificity of 86.7%. Our pioneer strategy highlights the potential use of egg yolk IgY as a diagnostic test to detect active hookworm infection.

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The Novel Apolipoprotein A5 Is Present in Human Serum, Is Associated with VLDL, HDL, and Chylomicrons, and Circulates at Very Low Concentrations Compared with Other Apolipoproteins

Clinical Chemistry ◽

10.1373/clinchem.2004.040824 ◽

2005 ◽

Vol 51 (2) ◽

pp. 351-359 ◽

Cited By ~ 139

Author(s):

Peter J O’Brien ◽

William E Alborn ◽

John H Sloan ◽

Maverick Ulmer ◽

Amechand Boodhoo ◽

...

Keyword(s):

Human Serum ◽

Particle Distribution ◽

Sandwich Elisa ◽

Serum Triglyceride ◽

Mouse Serum ◽

Peroxisome Proliferator ◽

Serum Triglycerides ◽

Apolipoprotein A5 ◽

Lipoprotein Particle ◽

First Time

Abstract Background: The recently discovered apolipoprotein A5 (ApoA5) is fast gaining attention as a key regulator of serum triglyceride concentrations. An ApoA5 mouse knock-out model produced an approximately fourfold increase in serum triglycerides, whereas a knock-in model with human ApoA5 produced 50–70% lower concentrations of mouse serum triglycerides. In addition, peroxisome proliferator-activated receptor-α agonists, which are used clinically to lower serum triglyceride concentrations, cause increased ApoA5 mRNA expression. Despite these compelling molecular biology data, relatively little is known about ApoA5 protein in human serum. Methods: To better understand circulating concentrations and lipoprotein particle distribution of ApoA5, we expressed the recombinant human ApoA5 protein and raised antibodies against both the NH2 and COOH termini. Results: Using the above reagents, we demonstrate for the first time that ApoA5 is present in human serum, although at much lower concentrations than other apolipoproteins such as ApoA1. Using a dual-antibody sandwich ELISA that we developed, we observed ApoA5 concentrations in human serum ranging from 24 to 406 μg/L compared with ∼1 g/L for ApoA1. We also examined the lipoprotein particle distribution of ApoA5 and found that ApoA5 was detectable in VLDL, HDL, and chylomicrons, but not LDL. Conclusions: These data demonstrate for the first time that ApoA5 is a secreted protein present in human serum and is associated with specific lipoprotein particles. In addition, our data indicate that the circulating concentration of human ApoA5 is very low compared with other apolipoproteins.

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Simultaneous Multianalyte ELISA Performed on a Microarray Platform

Clinical Chemistry ◽

10.1093/clinchem/47.8.1451 ◽

2001 ◽

Vol 47 (8) ◽

pp. 1451-1457 ◽

Cited By ~ 128

Author(s):

Rick Wiese ◽

Yuri Belosludtsev ◽

Tom Powdrill ◽

Patricia Thompson ◽

Mike Hogan

Keyword(s):

Human Serum ◽

Calibration Curve ◽

Sandwich Elisa ◽

Specific Antigen ◽

Simultaneous Detection ◽

Microarray Platform ◽

Microtiter Plate ◽

Serum Samples ◽

Human Serum Samples ◽

Calibration Curves

Abstract Background: A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Here we describe the construction and use of such a multiplex ELISA to measure prostate-specific antigen (PSA), α1-antichymotrypsin-bound PSA (PSA-ACT), and interleukin-6 (IL-6). Methods: We silanized glass plates and printed the appropriate capture antibodies to allow for the construction of “sandwich” ELISA quantification assays. We examined specificity of the assay for appropriate antigen, assembled calibration curves, and obtained PSA concentrations for 14 human serum samples. We compared the serum PSA concentrations derived through the use of our array with values obtained independently using a standard ELISA method. Results: R 2 values generated by our microarray for the PSA and PSA-ACT calibration curves were 0.989 and 0.979, respectively. Analyte concentrations used for the construction of these curves were 0.31–20 μg of protein/L of diluent. IL-6 calibration curve concentrations were 4.9–300 ng of IL-6/L of diluent. The R2 value for the IL-6 calibration curve was 0.983. The 14 human serum samples screened by this micro-ELISA technique for PSA concentrations generated a regression equation (linear) with a slope of 0.83 ± 0.10 and intercept of 0.74 ± 0.70 (R2 = 0.88). Conclusions: Multiplexed ELISA arrays are a feasible option for analyte quantification in complex biologic samples.

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Serum Proprotein Convertase Subtilisin Kexin Type 9 Is Correlated Directly with Serum LDL Cholesterol

Clinical Chemistry ◽

10.1373/clinchem.2007.091280 ◽

2007 ◽

Vol 53 (10) ◽

pp. 1814-1819 ◽

Cited By ~ 112

Author(s):

William E Alborn ◽

Guoqing Cao ◽

Holly E Careskey ◽

Yue-Wei Qian ◽

Danise R Subramaniam ◽

...

Keyword(s):

Human Serum ◽

Total Cholesterol ◽

Ldl Cholesterol ◽

Sandwich Elisa ◽

Hdl Cholesterol ◽

Serum Concentrations ◽

Loss Of Function ◽

Proprotein Convertase ◽

Serum Samples ◽

Lipoprotein Particle

Abstract Background: Proprotein convertase subtilisin kexin type 9 (PCSK9) is gaining attention as a key regulator of serum LDL-cholesterol (LDLC). This novel serine protease causes the degradation of hepatic LDL receptors by an unknown mechanism. In humans, gain-of-function mutations in the PCSK9 gene cause a form of familial hypercholesterolemia, whereas loss-of-function mutations result in significantly decreased LDLC and decreased cardiovascular risk. Relatively little is known about PCSK9 in human serum. Methods: We used recombinant human PCSK9 protein and 2 different anti-PCSK9 monoclonal antibodies to build a sandwich ELISA. We measured PCSK9 and lipids in 55 human serum samples and correlated the results. We used the anti-PCSK9 antibodies to assay lipoprotein particle fractions separated by sequential flotation ultracentrifugation. Results: Serum concentrations of PCSK9 ranged from 11 to 115 μg/L and were directly correlated with serum concentrations of LDLC (r = 0.45, P = 0.001) and total cholesterol (r = 0.50, P = 0.0003), but not with triglycerides (r = 0.15, P = 0.28) or HDL cholesterol concentrations (r = 0.13, P = 0.36). PCSK9 was not detectable in any lipoprotein particle fraction, including LDL. Conclusions: PCSK9 is present in human serum, likely not associated with specific lipoprotein particles. The circulating concentrations of human PCSK9 are directly correlated with LDL and total cholesterol concentrations.

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TBE in Sweden

Tick-borne encephalitis - The Book ◽

10.33442/26613980_12b32-3 ◽

2020 ◽

Keyword(s):

Genetic Characterization ◽

Immunoglobulin M ◽

Enzyme Linked Immunosorbent Assay ◽

Second Phase ◽

Serum Samples ◽

Tick Borne Encephalitis ◽

Specific Immunoglobulin ◽

Tick Borne Encephalitis Virus ◽

First Time

Tick-borne encephalitis virus (TBEV) was isolated for the first time in Sweden in 1958 (from ticks and from 1 tick-borne encephalitis [TBE] patient).1 In 2003, Haglund and colleagues reported the isolation and antigenic and genetic characterization of 14 TBEV strains from Swedish patients (samples collected 1991–1994).2 The first serum sample, from which TBEV was isolated, was obtained 2–10 days after onset of disease and found to be negative for anti-TBEV immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA), whereas TBEV-specific IgM (and TBEV-specific immunoglobulin G/cerebrospinal fluid [IgG/CSF] activity) was demonstrated in later serum samples taken during the second phase of the disease.

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Simultaneous Monitoring of Febuxostat and Uric Acid in Human Serum Samples Using the Direct Square-Wave Voltammetric Method

Current Analytical Chemistry ◽

10.2174/1573411014666180730112905 ◽

2019 ◽

Vol 15 (6) ◽

pp. 678-684

Author(s):

Biljana Nigović ◽

Jakov Vlak

Keyword(s):

Uric Acid ◽

Human Serum ◽

Oxidase Inhibitor ◽

Serum Samples ◽

Boron Doped Diamond ◽

Fast Analysis ◽

Xanthine Oxidase Inhibitor ◽

Voltammetric Method ◽

Square Wave ◽

Human Serum Samples

Background: High uric acid serum level, hyperuricemia, is now associated with many diseases such as gout, chronic kidney disease, hypertension, coronary artery disease and diabetes. Febuxostat is a novel selective xanthine oxidase inhibitor approved for the treatment of hyperuricemia. Objective: The aim of this study was to develop a first analytical method for the simultaneous determination of febuxostat and uric acid. Methods: An unmodified boron-doped diamond electrode provided concurrent quantitation of drug at low levels and uric acid, which has clinical significance in the diagnosis and therapy of hyperuricemia, at relatively high concentrations. The direct square-wave voltammetric method was applied to the analysis of both analytes in human serum samples. Results: Under the optimized conditions, the linear response of peak current on febuxostat concentration was achieved in the range from 7.5 × 10-7 to 3 × 10-5 M, while uric acid showed two linear ranges of 5 × 10-6 - 5 × 10-5 M and 5 × 10-5 - 2 × 10-4 M. The method was successfully utilised for quantification of both analytes in human serum samples. Good recoveries were obtained without interference from common inorganic cations and anions as well as glucose, dopamine, ascorbic and folic acids at concentrations expected in physiological conditions. Conclusion: The great benefits of developed method are fast analysis (only 7.5 s for run), low cost and simplicity of performance.

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