scholarly journals Development of a Sensitive Chemiluminescence Immunoassay for the Quantification of Folic Acid in Human Serum

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Xiang Chen ◽  
Qiyang Zhou ◽  
Ting Zhang ◽  
ChunXin Wang ◽  
Zheng Yu ◽  
...  
Keyword(s):  
Folic Acid ◽  
Horseradish Peroxidase ◽  
Human Serum ◽  
Magnetic Beads ◽  
Megaloblastic Anemia ◽  
Human Growth ◽  
Cross Reactivity ◽  
Alkaline Condition ◽  
Folate Binding Protein ◽  
Chemiluminescence Immunoassay

Folic acid (FA) is an important vitamin for human growth, especially for pregnant women. FA deficiency is associated with megaloblastic anemia, neural tube defects, cardiovascular diseases, irritability, diarrhea, and psychiatric disorders. Normally, FA molecules bind to folate-binding protein (FBP) in the serum as complex. Before quantify the FA concentration, a releasing procedure should be conducted. Alkaline condition and tris(2-carboxyethyl)phosphine (TCEP) are used to release binding FA to freeing state. In this work, a chemiluminescence immunoassay (CLIA) for human serum FA was established by competition model. Streptavidin (SA) was labeled to magnetic beads by an 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDAC/NHS) method. Activated biotin molecules were labeled to FBP molecules purified from milk. FA was labeled to horseradish peroxidase (HRP) by EDAC to activate the FA molecules. The pretreated samples or standards were added into the reaction tube with biotin-FBP and FA-horseradish peroxidase (HRP), FA in the sample compete with FA-HRP for binding to biotin-FBP, the signal is inversely proportional to the FA concentration. The method established shows good thermostability and performance. The limitation of detection (LOD) is 0.44 ng/mL. The intra-assay coefficient of variation (CV) is 3.6%–7.1%, the interassay CV is 4.2%–7.5%, and the recovery rate is 92.1%–103.5%. Cross reactivity (CR) was remarkably low with aminopterin, folinic acid, and methotrexate. The method shows good correlation with the FA CLIA product from Beckman Coulter; the equation is y = 0.9618x−0.1434 while the R2 value is 0.9224. The established method is sensitive, rapid, and accurate which can fully satisfy for the clinical requirement.

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.

Reticulocyte Folate Concentration: A Tool To Monitor Immediate Folate Availability

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4655-4655
Author(s):  
Peter J. Miller ◽  
Elizabeth B. Agnew ◽  
Mary Ann Knovich ◽  
Andrew M. Farland ◽  
Johanna Waldron ◽  
...  
Keyword(s):  
Folic Acid ◽  
Magnetic Beads ◽  
Conflicts Of Interest ◽  
Binding Protein ◽  
Megaloblastic Anemia ◽  
Folate Deficiency ◽  
Serum Folate ◽  
Red Cell ◽  
Folate Binding Protein ◽  
Folate Concentration

The utilization of folic acid during erythropoiesis is widely known and accepted in medical literature and deficiency is known to cause a characteristic megaloblastic anemia resulting from the inhibition or ineffective synthesis of DNA. Although the resultant megaloblastic anemia may take considerable time before evidence or symptoms present, there are more acute changes visualized on peripheral smear that are representative of a functional folate deficiency. In addition to erythrocyte macrocytosis, hypersegmentation of neutrophils can also be seen. Often times, despite these visualized changes measurement of serum folate and/or total red cell folate yields a result within the accepted “normal” range of the assay. It has been demonstrated that these visualized characteristics represent a functional folate deficiency and can be overcome with folic acid supplementation regardless of the measured folic acid levels indicating a yet to be understood mechanism of folate utilization. Here, we sought to measure the folic acid levels in the earliest erythrocyte progenitors in peripheral circulation, the reticulocytes. Methods Reticulocytes were isolated using anti-CD71 (the transferrin receptor) coated magnetic beads. After separation, a sample slide was made utilizing methylene blue for visual confirmation of reticulocyte isolation. The reticulocytes were then lysed with citric acid and a Nanodrop-1000 spectrophotometer was used to determine absorbance at 413 nanometers. This absorbance was used to determine the sample hemoglobin concentration from a simple calibration curve. The sample folic acid level was then determined using the lysing method utilizing mouse monoclonal anti-folate binding protein, paramagnetic particles coated in anti-mouse IgG, human serum albumin and milk folate binding protein. Results were calculated as nanogram of folate per gram of hemoglobin. Results Twenty-five samples from normal individuals, not taking folate supplements, were analyzed. The range of results was 2.51 to 17.38 with a mean level of 9.61. Conclusion This protocol effectively and efficiently allows for isolation of reticulocytes in numbers high enough for measurement of folate in nanogram per gram of hemoglobin. By this method we show the normal reticulocyte folate level to be approximately 3-15 ng/g of Hgb. This figure is consistent with the normal red cell folate concentration. Further analysis is planned for comparing these results in patients with a suspected functional folate deficiency but “normal” red cell folate levels. Efficient isolation of reticulocytes and measurement of folate levels will allow us to probe the underlying cause of acute folate deficiency seen in very sick patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of the Diagnostic Performance of a Novel COVID-19 PETIA in Comparison to Four Routine N-, S- and RBD-Antigen Based Immunoassays

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1332
Author(s):  
Alexander Spaeth ◽  
Thomas Masetto ◽  
Jessica Brehm ◽  
Leoni Wey ◽  
Christian Kochem ◽  
...  
Keyword(s):  
Immune Response ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chemiluminescence Immunoassay ◽  
Face To Face ◽  
Comprehensive Comparison ◽  
Broad Variety ◽  
Viral Responses ◽  
Novel Coronavirus ◽  
High Consistency

In 2019, a novel coronavirus emerged in Wuhan in the province of Hubei, China. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) quickly spread across the globe, causing the neoteric COVID-19 pandemic. SARS-CoV-2 is commonly transmitted by droplet infection and aerosols when coughing or sneezing, as well as high-risk exposures to infected individuals by face-to-face contact without protective gear. To date, a broad variety of techniques have emerged to assess and quantify the specific antibody response of a patient towards a SARS-CoV-2 infection. Here, we report the first comprehensive comparison of five different assay systems: Enzyme-Linked Immunosorbent Assay (ELISA), Chemiluminescence Immunoassay (CLIA), Electro-Chemiluminescence Immunoassay (ECLIA), and a new Particle-Enhanced Turbidimetric Immunoassay (PETIA) for SARS-CoV-2. Furthermore, we also evaluated the suitability of N-, S1- and RBD-antigens for quantifying the SARS-CoV-2 specific immune response. Linearity and precision, overall sensitivity and specificity of the assays, stability of samples, and cross-reactivity of general viral responses, as well as common coronaviruses, were assessed. Moreover, the reactivity of all tests to seroconversion and different sample matrices was quantified. All five assays showed good overall agreement, with 76% and 87% similarity for negative and positive samples, respectively. In conclusion, all evaluated methods showed a high consistency of results and suitability for the robust quantification of the SARS-CoV-2-derived immune response.

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.

A Novel Chemiluminescence Immunoassay Using Solid-Phase Antigen for Free 17β-Estradiol in Human Serum

2011 ◽  
Vol 29 (11) ◽  
pp. 2520-2524 ◽  
Author(s):  
Yuanyuan Qi ◽  
Hui Chen ◽  
Zhen Lin ◽  
Guonan Chen ◽  
Jinming Lin
Keyword(s):  
Human Serum ◽  
Solid Phase ◽  
Chemiluminescence Immunoassay ◽  
17Β Estradiol

Renal folate absorption and the kidney folate binding protein. II. Microinfusion studies

1987 ◽  
Vol 252 (4) ◽  
pp. F757-F760 ◽  
Author(s):  
J. Selhub ◽  
S. Nakamura ◽  
F. A. Carone
Keyword(s):  
Folic Acid ◽  
Brush Border ◽  
Half Life ◽  
Binding Protein ◽  
Membrane Surface ◽  
Rapid Change ◽  
Acid Uptake ◽  
Folate Binding Protein ◽  
Acid Absorption ◽  
Folic Acid Absorption

Surface proximal convoluted tubules (PCT) in rats were microinfused in situ with [3H]folic acid to study the role of folate binding protein (FBP) in the kidney brush-border membrane for renal conservation and transport of folate [3H]folic acid absorption was linearly related to tubular length of PCT and occurred largely in this segment of the tubule. Unlabeled folate derivatives inhibited [3H]folic acid absorption, the extent of which was dependent on the type of unlabeled folate used and its concentration. At equivalent concentrations, inhibition was most effective with unlabeled folic acid, slightly lower than with 5-methyltetrahydrofolate and least effective with methotrexate. Comparisons between [3H]folic acid absorption before and after infusion of a saturating dose of unlabeled folic acid or repetitive injections of [3H]folic acid into the same tubular site revealed continuous and rapid regeneration of unsaturated folic acid uptake sites with an apparent half-life of 28.75 +/- 8.75 s. Determination of [3H] retained in the tubule at various periods after microinfusion of [3H]folic acid revealed slow cellular disappearance with an apparent half-life of 47.3 +/- 5.4 min. It is proposed that the brush-border FBP functions as a receptor of infused folic acid and that following the binding of the ligand the folic acid/FBP complex undergoes a rapid change that results in the internalization of folic acid and regeneration of unsaturated binding sites at the membrane surface. Internalized folic acid is slowly released into renal capillaries.

The Effect of Small Doses of Folic Acid in Nutritional Megaloblastic Anemia

1963 ◽  
Vol 13 (6) ◽  
pp. 369-377 ◽  
Author(s):  
G. IZAK ◽  
M. RACHMILEWITZ ◽  
SHWE ZAN ◽  
N. GROSSOWICZ
Keyword(s):  
Folic Acid ◽  
Megaloblastic Anemia ◽  
Small Doses
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