scholarly journals Elisa Method For Serum Hepcidin Quantification In Bulgarian Population

2014 ◽  
Vol 41 (1) ◽  
pp. 22-29 ◽  
Author(s):  
V. Manolov ◽  
B. Atanasova ◽  
V. Vasilev ◽  
K. Tzatchev ◽  
M. Velizarova
Keyword(s):  
Monoclonal Antibodies ◽  
Iron Homeostasis ◽  
Sandwich Elisa ◽  
Active Form ◽  
Hemodialysis Patients ◽  
Healthy Population ◽  
Elisa Method ◽  
Limit Of Quantification ◽  
Elisa Format ◽  
Bulgarian Population

Summary Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Hepcidin quantification in human blood may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes ELISA immunoassay for hepcidin quantification in human serum. We used a sandwich ELISA method from USCN Life Science inc., that consists of ready to use, pre-coated 96-well strip plate with 2 antihepcidin-25 monoclonal antibodies. A recombinant hepcidin in 16 μg/l concentration is used as a standard; it reconstitutes with 1.0 ml standard diluent to prepare a stock solution. We correlated ELISA results of hepcidin-25 measurements in healthy population with hemodialysis patients. The sandwich ELISA was highly specific for hepcidin-25, having a low limit of quantification of 0.020 μg/l. Hepcidin- 25 concentrations were increased in hemodialysis patients (median 33.05 μg/l, range 22.31 -60.98 μg/l, n = 10) compared with healthy individuals (median 12.41 μg/l, range 6.05-18.53 μg/l, n = 40). The use of 2 monoclonal antibodies in a sandwich ELISA format provides a robust, convenient and not very expensive method for measuring concentrations of the active form of hepcidin. It should help to improve our understanding of the role of hepcidin in regulating iron metabolism.

scholarly journals A Dual-Monoclonal Sandwich ELISA Specific for Hepcidin-25

2010 ◽  
Vol 56 (11) ◽  
pp. 1725-1732 ◽  
Author(s):  
Anthony M Butterfield ◽  
Peng Luan ◽  
Derrick R Witcher ◽  
Joseph Manetta ◽  
Anthony T Murphy ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Monoclonal Antibodies ◽  
Iron Metabolism ◽  
Sandwich Elisa ◽  
Active Form ◽  
Serum Concentrations ◽  
Limit Of Quantification ◽  
Normal Individuals ◽  
Elisa Format ◽  
Serum Hepcidin

BACKGROUND Hepcidin, a key regulator of iron metabolism, binds to the iron transporter ferroportin to cause its degradation. In humans, hepcidin deficiency has been linked to hemochromatosis and iron overload, whereas increased concentrations have been reported in anemia of cancer and chronic disease. There is currently an unmet clinical need for a specific immunoassay with a low limit of quantification to measure serum concentrations of hepcidin-25, the active form of the protein. METHODS We generated 2 antihepcidin-25 monoclonal antibodies and used them to build a sandwich ELISA. We correlated ELISA results to hepcidin-25 measurements by LC-MS and used ELISA to measure serum hepcidin-25 concentrations in normal individuals, cancer patients, and patients with rheumatoid arthritis. RESULTS The sandwich ELISA was highly specific for hepcidin-25, having a limit of quantification of 0.01 μg/L (10 pg/mL). Serum concentrations of hepcidin-25 measured by ELISA correlated with hepcidin-25 concentrations measured by using an independent LC-MS assay (r = 0.98, P < 0.001). Hepcidin-25 concentrations were increased in patients with cancer (median 54.8 μg/L, 25%–75% range 23.2–93.5 μg/L, n = 34) and rheumatoid arthritis (median 10.6 μg/L, 25%–75% range 5.9–18.4 μg/L, n = 76) compared with healthy individuals (median 1.20 μg/L, 25%–75% range 0.42–3.07 μg/L, n = 100). CONCLUSIONS The use of 2 monoclonal antibodies in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of the active form of hepcidin. This ELISA should help to improve our understanding of the role of hepcidin in regulating iron metabolism.

Rapid Detection of Salmonella spp. in Foods by Combination of a New Selective Enrichment and a Sandwich ELISA Using Two Monoclonal Antibodies against Dulcitol 1-Phosphate Dehydrogenase

1996 ◽  
Vol 59 (11) ◽  
pp. 1158-1163 ◽  
Author(s):  
HUAIZE TIAN ◽  
TAKAHISA MIYAMOTO ◽  
TAKASHI OKABE ◽  
YOICHIRO KURAMITSU ◽  
KEN-ICHI HONJOH ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Rapid Detection ◽  
Enrichment Culture ◽  
Sandwich Elisa ◽  
False Negative ◽  
Detection Sensitivity ◽  
Elisa Method ◽  
Salmonella Spp ◽  
Selective Enrichment ◽  
Raw Foods

A rapid-detection method was developed for food-borne dulcitol-positive Salmonella spp. in foods that involves a new preenrichment and selective enrichment system and a sandwich ELISA using two monoclonal antibodies against dulcitol 1-phosphate dehydrogenase. Preenrichment and selective enrichment were in Enterobacteriaceae enrichment mannitol (EEM) broth at 42°C for 6 h and in a new dulcitol-magnesium chloride-pyridinesulfonic acid brilliant green-novobiocin (DMPBN) medium at 42°C for 27 h, respectively. The cells were collected from the selective enrichment culture and suspended in 0.1 ml of 1 N NaOH for 2 min. The solution was neutralized with 0.1 ml of 2 M Tris-HCl buffer (pH 7.5) and the mixture was used as a sample for ELISA. The detection sensitivity of the ELISA was 105 CFU of Salmonella spp. per ml of culture. Competing non-Salmonella organisms in raw food did not interfere with the detection of Salmonella cells even when present at 107: 1 (non-Salmonella: Salmonella ratio) in food. Nonmotile Salmonella gallinarum was detected by the ELISA. The minimum detectable number of initial inoculum of Salmonella typhimurium was 0.69 CFU/25 g of raw chicken after the preenrichment in EEM broth and the selective enrichment in DMPBN medium. The present ELISA method required a total analysis time of 36 h including the preenrichment and selective enrichment periods. The ELISA method was compared with a conventional cultural method for the detection of Salmonella cells in 130 samples of raw foods. Of the samples tested, 16 were Salmonella-positive and 114 samples were negative by both methods. False-positive and false-negative results were not encountered.

Comparative study of assays detecting circulating immune complexes and specific antibodies in patients infected with Toxocara canis

1987 ◽  
Vol 61 (3) ◽  
pp. 196-202 ◽  
Author(s):  
C. Aguila ◽  
C. Cuéllar ◽  
S. Fenoy ◽  
J. L. Guillén
Keyword(s):  
Monoclonal Antibodies ◽  
Comparative Study ◽  
Immune Complexes ◽  
Sandwich Elisa ◽  
Toxocara Canis ◽  
Circulating Immune Complexes ◽  
Soluble Antigen ◽  
Elisa Method ◽  
Active Infection ◽  
Specific Antibodies

ABSTRACTA sandwich ELISA method using previously described E/S antigen-specific monoclonal antibodies has been developed to detect circulating immune complexes in patients infected with Toxocara canis. This technique could be used for the study of the dynamics of the parasite-host relationship, as we believe the detection of immune complexes and/or soluble antigen to be an improvement over detection of antibodies only. In this parasitosis, antibodies may be present in residual levels for prolonged periods after active infection.

scholarly journals Preparation, Characterization and Diagnostic Valuation of Two Novel Anti-HPV16 E7 Oncoprotein Monoclonal Antibodies

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 333
Author(s):  
Renjian Hu ◽  
Zhen Dong ◽  
Kui Zhang ◽  
Guangzhao Pan ◽  
Chongyang Li ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Detection System ◽  
Early Stage ◽  
Sandwich Elisa ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Cervical Cancers ◽  
E7 Oncoprotein ◽  
Oncogenic Protein

At present, the clinical detection method of human papillomavirus (HPV) is mainly based on the PCR method. However, this method can only be used to detect HPV DNA and HPV types, and cannot be used to accurately predict cervical cancer. HPV16 E7 is an oncoprotein selectively expressed in cervical cancers. In this study, we prepared an HPV16 E7-histidine (HIS) fusion oncoprotein by using a prokaryotic expression and gained several mouse anti-HPV16 E7-HIS fusion oncoprotein monoclonal antibodies (mAbs) by using hybridoma technology. Two mAbs, 69E2 (IgG2a) and 79A11 (IgM), were identified. Immunocytochemistry, immunofluorescence, immunohistochemistry, and Western blot were used to characterize the specificity of these mAbs. The sequences of the nucleotide bases and predicted amino acids of the 69E2 and 79A11 antibodies showed that they were novel antibodies. Indirect enzyme-linked immunosorbent assay (ELISA) with overlapping peptides, indirect competitive ELISA, and 3D structural modeling showed that mAbs 69E2 and 79A11 specifically bound to the three exposed peptides of the HPV16 E7 (HPV16 E749–66, HPV16 E773–85, and HPV16 E791–97). We used these two antibodies (79A11 as a capture antibody and 69E2 as a detection antibody) to establish a double-antibody sandwich ELISA based on a horseradish peroxidase (HRP)-labeled mAb and tetramethylbenzidine (TMB) detection system for quantitative detection of the HPV16 E7-HIS fusion oncoprotein, however, it was not ideal. Then we established a chemiluminescence immunoassay based on a labeled streptavidin-biotin (LSAB)-ELISA method and luminol detection system—this was sufficient for quantitative detection of the HPV16 E7-HIS fusion oncogenic protein in ng levels and was suitable for the detection of HPV16-positive cervical carcinoma tissues. Collectively, we obtained two novel mouse anti-HPV16 E7 oncoprotein mAbs and established an LSAB-lumino-dual-antibody sandwich ELISA method for the detection of the HPV16 E7-HIS fusion oncogenic protein, which might be a promising method for the diagnosis of HPV16-type cervical cancers in the early stage.

scholarly journals Efficient detection of pre-proinsulin by double antibody sandwich ELISA

2020 ◽  
Author(s):  
Zhu Zhu ◽  
Guoliang Ma ◽  
Li Wang ◽  
Han Wang ◽  
Hengfang Tang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Insulin ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Insulin Production ◽  
Recombinant Human Insulin ◽  
Elisa Method ◽  
Efficient Detection ◽  
Sds Page ◽  
Conventional Cell

Abstract ObjectiveTo generate monoclonal antibodies against pre-proinsulin (PPI), and establish sandwich ELISA method to provide a basis for PPI detection in recombinant human insulin production.MethodsThe Balb /c mice were immunized with PPI, and the hybridomas secreting anti-PPI monoclonal antibodies were obtained by conventional cell fusion technique and ELISA screening.The antibody was purified using a Protein G gel column and identified for purity by SDS-PAGE. Pairing effect was found by the sandwich ELISA, and the specificity of the paired antibody was determined. A paired antibody with better specificity was selected to establish sandwich ELISA, and construct a quantitative curve, the accuracy and sensitivity of the method were evaluated.ResultsSix anti-PPI monoclonal antibodies were obtained, named P1, P2, P3, P4, P5 and P6, of which P5 had the highest titer. The sandwich ELISA method was established with P5 for plating and P2 for detection antibodie. The linear range of the quantitative curve of PPI by sandwich ELISA was 0. 645-82.5 pg/mL, the recovery was 89%–95%, and the limit of detection was 3.06 pg/mL.ConclusionSix monoclonal antibodies against PPI were generated and the sandwich ELISA method was established to detect PPI in process control and product release control for recombinant human insulin production.

Preparation of monoclonal antibodies against KHV and establishment of an antigen sandwich ELISA for KHV detection

2019 ◽  
Vol 128 ◽  
pp. 36-40 ◽  
Author(s):  
Yingying Li ◽  
Qing Wang ◽  
Sven M. Bergmann ◽  
Weiwei Zeng ◽  
Yingying Wang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa

scholarly journals Fascin-1 and its role as a serological marker in prostate cancer: a prospective case–control study

2021 ◽  
pp. FSO745
Author(s):  
Octavian Sabin Tătaru ◽  
Orsolya Martha ◽  
Felice Crocetto ◽  
Biagio Barone ◽  
Septimiu Voidazan ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Case Control Study ◽  
Sandwich Elisa ◽  
Case Control ◽  
Elisa Method ◽  
Diagnostic Role ◽  
The Difference ◽  
Blood Specimens ◽  
Control Study

Aim: This study aims to investigate any modification of serological FSCN1 in prostate cancer patients compared with patients without neoplasia. Material & methods: Clinical data and blood specimens from patients with and without prostate cancer were obtained. A quantitative sandwich ELISA method was used to determine serological values of FSCN1. Results: Although serum values of FSCN1 were dissimilar in the two cohorts of patients (6.90 vs 7.33 ng/ml), the difference was not statistically significant (p = 0.20). Serum values of FSCN1 stratified for Gleason score groups were not significantly distinguishable (p = 0.65). A negative correlation (rho = -0.331; p = 0.009) was reported between FSCN1 and age. Conclusion: Further studies are required to evaluate a possible diagnostic role of FSCN1 in prostate cancer.

scholarly journals Development of a Sandwich Enzyme-Linked Immunosorbent Assay for Detection and Quantification of Clam Residues in Food Products

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Stef J. Koppelman ◽  
Ashley L. Lardizabal ◽  
Lynn Niemann ◽  
Joe L. Baumert ◽  
Steve L. Taylor
Keyword(s):  
Sandwich Elisa ◽  
Food Product ◽  
Food Products ◽  
Elisa Test ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Allergic Reactions ◽  
Arctica Islandica ◽  
Limit Of Quantification ◽  
Detection And Quantification

Seafood is a frequent cause of allergic reactions to food globally. The presence of undeclared trace amounts of clam can cause allergic reactions in sensitive individuals. Limited tools are available to test food products for the presence of traces of clam. We report on the development of a sandwich ELISA that can detect and quantify clam protein in food. Antisera against a mix of two commercially important clam species, Atlantic Surf (Spisula solidissima) and ocean quahog (Arctica islandica), were raised in rabbit and sheep. A sandwich ELISA was constructed with this antisera, and sensitivity and specificity were evaluated. Also, model food products spiked with clam protein were analyzed to assess the performance of the ELISA. Comparison was made with a commercially available ELISA for crustacea. The lower limit of quantification of the sandwich ELISA is 2.5 ppm clam protein in food samples, allowing the detection of low amounts of clam that may trigger a reaction in clam allergic patients. The sandwich ELISA was highly specific with cross-reactivity only noted for other molluscan shellfish (mussel and scallop). Clam protein in tomato juice and potato cream soup was detected well with recoveries ranging from 65 to 74% and from 74 to 113%, respectively. However when potato cream soup was retorted, the recover fell to 20%, imposing the risk of underestimating the clam content of a food product. A commercially available crustacean ELISA test was not suitable to detect clam protein. The sandwich ELISA described here is suitable for detection and quantification of clam protein in food products. Care should be taken with food products that have been retorted as the results may be underestimated.

Production of monoclonal antibodies for measuring Avastin and its biosimilar by Sandwich ELISA

2019 ◽  
Vol 469 ◽  
pp. 42-46 ◽  
Author(s):  
Maohua Li ◽  
Wenqi An ◽  
Lan Wang ◽  
Feng Zhang ◽  
Jianli Li ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa

scholarly journals Oxidative Balance and Inflammation in Hemodialysis Patients: Biomarkers of Cardiovascular Risk?

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Daniele La Russa ◽  
Daniela Pellegrino ◽  
Alberto Montesanto ◽  
Paolo Gigliotti ◽  
Anna Perri ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Chronic Kidney Disease ◽  
Renal Function ◽  
Cardiovascular Risk ◽  
Kidney Disease ◽  
Cardiovascular Events ◽  
Cardiovascular Complications ◽  
Hemodialysis Patients ◽  
Healthy Population ◽  
Oxidative Balance

During chronic kidney disease, the progressive deterioration of renal function induces several biological/clinical dysfunctions, including enhancement of synthesis of inflammation/oxidative stress mediators. Impaired renal function is an independent cardiovascular risk factor; indeed, cardiovascular complications dominate the landscape of both chronic kidney disease and end-stage renal disease. The aim of this study is to explore the correlation between the global oxidative balance in hemodialysis patients and both inflammatory markers and cardiovascular events. Using photometric tests, this study explored plasmatic oxidative balance in 97 hemodialysis patients compared to a healthy population. In the hemodialysis patients, we showed that oxidative stress values were significantly lower than in controls while effectiveness in the antioxidant barrier was significantly increased in the hemodialysis group. Furthermore, we highlighted a strong correlation between oxidative index and blood levels of C-reactive protein. When patients were divided into two groups based on previous cardiovascular events, we found that subjects with previous cardiovascular events had higher values of both oxidative stress and antioxidant barrier than patients without cardiovascular events. Our results indicated that in hemodialysis patients, the clinical and prognostic significance of oxidative status is very different from general population. As cardiovascular complications represent a strong negative factor for survival of hemodialysis patients, the research of new cardiovascular risk biomarkers in these patients takes on particular importance in order to translate them into clinical practice/primary care.

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