Development, Optimization and Validation of a Sandwich ELISA for Detection of Recombinant Human Factor VII

2015 ◽  
Vol 5 (1) ◽  
pp. 26-31
Author(s):  
Sheila Sarial ◽  
Rozhan Sonboli ◽  
Shayan Maleknia ◽  
Fatemeh Ashori ◽  
Sara Rezaeiravesh ◽  
...  
Keyword(s):  
Low Dose ◽  
Human Factor ◽  
Sandwich Elisa ◽  
Factor Vii ◽  
Enzyme Linked Immunosorbent Assay ◽  
P Value ◽  
Limit Of Quantification ◽  
Intermediate Precision ◽  
Downstream Processes

Recombinant human factor VII (rh-FVII) is produced by engineered BHK cells at low dose. Accordingly, establishment of a precise method is crucial to reliably measuring expression level of this protein during manufacturing pro-cesses. We developed and established a reproducible sandwich enzyme-linked immunosorbent assay (ELISA) method for measuring amount of FVII during biopharmaceutical in upstream and downstream processes of Aryo-Seven. A sandwich ELISA was designed using two different high affinity mon-oclonal antibodies (mAb1 and mAb2) against h-FVII. The bounded FVII to the first antibody was revealed by the use of a second mouse anti-FVII monoclo-nal antibody (1F1-B11), labeled with HRP that binds to another antigenic determinant of the FVII. Then, validation was done by determination of spec-ificity, linearity, accuracy, precision, reproducibility, detection and quantifica-tion limit and robustness according to ICH Q2 (R1) guideline. In developed ELISA, no interference was found between FVII and proteins derived from BHK which commonly exist in the supernatant. Linear range of detection was from 25-1.56 ng/mL with P-Value <0.001. In accuracy, spiked samples showed 109±2% recovery. Intra and intermediate precision assays showed %RSD not more than20. Detection limit of this assay was 0.99 ng /mL and limit of quantification was 2.99 ng/ml. The sandwich ELISA was found to be useful tool for measuring FVII/ FVIIa. The ELISA approach was precise, repro-ducible, and accurate. The ELISA therefore, is offered as an assured kit for detection of recombinant human factor.

An Enzyme Immunoassay (ELISA) for the Quantitation of Human Factor VII

1986 ◽  
Vol 56 (03) ◽  
pp. 250-255 ◽  
Author(s):  
C Boyer ◽  
M Wolf ◽  
C Rothschild ◽  
M Migaud ◽  
J Amiral ◽  
...  
Keyword(s):  
Human Factor ◽  
Solid Phase ◽  
Factor Vii ◽  
Enzyme Linked Immunosorbent Assay ◽  
Poor Correlation ◽  
Vein Thrombosis ◽  
Coagulant Activity ◽  
Significant Difference ◽  
Large Populations ◽  
Deep Vein

SummaryA new solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of human Factor VII antigen (F VII Ag), using a monospecific rabbit anti-F VII antiserum. Anti-F VII F(ab′)2 fragments were adsorbed to polystyrene plates. The binding of serial dilutions of control or test plasma, containing F VII, was detected by incubation with peroxidase-labeled anti- FV II IgG followed by the addition of hydrogen peroxyde and O-phenylenediamine. This ELISA is specific, sensitive (detection limit: 0.05%) and accurate (coefficient of variation: 1.5-4% for within- and 1.6-9% for between-assays). F VII coagulant activity (F VII C) and F VII Ag were determined in large populations of controls and patients. In normal plasma (n = 38), F VII Ag ranged from 83 to 117% and the correlation coefficient between F VII Ag and F VII C was 0.94. In patients with severe (F VII C inf. 1%) congenital F VII deficiency (n = 5), F VII Ag was undetectable in two cases (inf. 0.05%) and markedly reduced (0.35 to 5.6%) in the three other cases. In patients with liver cirrhosis (n = 15), F VII Ag ranged from 21 to 59% and was in good correlation with F VII C (r = 0.84). In dicoumarol treated patients (n = 15), the levels of F VII Ag ranged from 51% to 79% and a poor correlation (r = 0.52) with F VIIC was observed. In “compensated” DIC (n = 5), levels of F VII Ag varied from 60 to 186%, with significantly higher F VII C levels (from 143 to 189%). In contrast, in “decompensated” DIC (n = 7), low F VII Ag and F VII C levels were observed (from 7 to 27%). In patients with deep-vein thrombosis (n = 25), high levels of F VII Ag (from 102 to 136%) and F VII C (from 110 to 150%) were demonstrated. In surgical patients, no significant difference was observed before and one day after intervention.

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.

scholarly journals Development and Validation of a Green Analytical Method for the Determination of Aspirin and Domperidone Bulk or Formulation Using UV and HPLC

2020 ◽  
Vol 10 (6) ◽  
pp. 49-56
Author(s):  
Sneha Jagnade ◽  
Pushpendra Soni ◽  
Lavakesh Kumar Omray
Keyword(s):  
Analytical Method ◽  
Method Development ◽  
Limit Of Detection ◽  
Research Work ◽  
Ultra Violet ◽  
Limit Of Quantification ◽  
Intermediate Precision ◽  
Rp Hplc ◽  
Development And Validation

The aim of present study was to investigate the development and validation of a green analytical method for the determination of aspirin and domperidone. Method Development and Validation for Estimation of Domperidone and Aspirin in bulk or formulation by using RP-HPLC. The RP-HPLC method was developed for estimation of Aspirin and Domperidone in synthetic mixture by isocratically using 10 mM KH2PO4: Acetonitrile (20:80) as mobile phase, Prontosil C-18 column (4.6 x 250 mm, 5μparticle size) column as stationary phase and chromatogram was recorded at 231 nm. Then developed method was validated by using various parameters such as, linearity, Range accuracy, precision repeatability, intermediate precision, robustness, limit of detection, limit of quantification. The proposed methods were found to be linear with correlation coefficient close to one. Precision was determined by repeatability, Intermediate precision and reproducibility of the drugs. The robustness of developed method was checked by changing in the deliberate variation in solvent. The result obtained shows the developed methods to be Cost effective, Rapid (Short retention time), Simple, Accurate (the value of SD and % RSD less than 2), Precise and can be successfully employed in the routine analysis of these drugs in bulk drug as well as in tablet dosage form. The Simplicity, Rapidly and Reproducibility of the proposed method completely fulfill the objective of this research work. Keywords: Asprin; Domperidone; HPLC; Ultra Violet; Validation

Lactate Dehydrogenase Polyclonal Antibody Sandwich ELISA for Determination of Endpoint Heating Temperatures of Ground Beef

1996 ◽  
Vol 59 (1) ◽  
pp. 51-55 ◽  
Author(s):  
CHENG-HSIN WANG ◽  
MOHAMED M. ABOUZIED ◽  
JAMES J. PESTKA ◽  
DENISE M. SMITH
Keyword(s):  
Lactate Dehydrogenase ◽  
Polyclonal Antibody ◽  
Heating Temperature ◽  
Polyclonal Antibodies ◽  
Sandwich Elisa ◽  
Ground Beef ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Marker ◽  
Beef Patties

An enzyme-linked immunosorbent assay (ELISA) for the protein marker lactate dehydrogenase (LDH) was developed to determine endpoint heating temperature (EPT) of ground beef. Ground beef was placed in 10 by 75 mm tubes and heated to temperatures between 62 and 74°C at 2°C intervals. Electrophoresis and immunoblotting of meat extracts indicated a decrease in the intensity of the LDH band as temperature was increased. Polyclonal antibodies (PAb) against bovine muscle LDH were produced and a sandwich ELISA devised using biotinylated PAb as the detecting antibody. The LDH content decreased (P &lt; 0.05) in ground beef heated between 66 and 74°C and was less than 4 μg/g of meat at the recommended minimum endpoint of 69°C. The ELISA was used to estimate the EPT of commercially cooked beef patties.

scholarly journals Enzyme-Linked Immunosorbent Assay Kit for the Determination of Soybean Protein in Processed Foods: Interlaboratory Evaluation

2010 ◽  
Vol 93 (1) ◽  
pp. 243-248 ◽  
Author(s):  
Shinobu Sakai ◽  
Reiko Adachi ◽  
Hiroshi Akiyama ◽  
Reiko Teshima ◽  
Naoki Morishita ◽  
...  
Keyword(s):  
Soybean Protein ◽  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Processed Foods ◽  
Adzuki Bean ◽  
Soybean Proteins ◽  
Elisa Kit ◽  
Number Of Patients ◽  
High Level

Abstract The labeling of foods containing ingredients derived from soybean is recommended in Japan because of an increasing number of patients who are allergic to soybeans. To ensure proper labeling, a novel sandwich ELISA kit for the determination of soybean protein in processed foods (FASTKIT Ver. II, Soybean, Nippon Meat Packers, Inc.; soy kit) has been developed. Five types of incurred samples (model processed foods: rice gruel, sausage, sweet adzuki bean soup, sweet potato cake, and tomato sauce) containing 10 g soybean soluble protein/g food were prepared for use in interlaboratory evaluations of the soy kit. The soy kit displayed a sufficient RSDR value (interlaboratory precision: 9.313.4 RSDR) and a high level of recovery (97114) for all the incurred samples. The RSDr value for the incurred samples was mostly &lt;4.8. The results of this interlaboratory evaluation suggest that the soy kit can be used as a precise and reliable tool for the determination of soybean proteins in processed foods.

scholarly journals Structure determination of the major asparagine-linked sugar chain of human factor VII-von Willebrand factor

FEBS Letters ◽  
1983 ◽  
Vol 151 (1) ◽  
pp. 22-26 ◽  
Author(s):  
P. Debeire ◽  
J. Montreuil ◽  
B. Samor ◽  
C. Mazurier ◽  
M. Goudemand ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Structure Determination ◽  
Human Factor ◽  
Factor Vii ◽  
Sugar Chain ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Development of the simultaneous analysis of choline salicylate, lidocaine hydrochloride and preservatives in a new dental gel by HPLC method

Chemija ◽  
2021 ◽  
Vol 32 (2) ◽  
Author(s):  
Yuliia Maslii ◽  
Ivan Bezruk ◽  
Anna Materiienko ◽  
Olena Ruban ◽  
Liudas Ivanauskas ◽  
...  
Keyword(s):  
Quantitative Determination ◽  
High Performance ◽  
Limit Of Detection ◽  
Phosphate Buffer Solution ◽  
Hplc Method ◽  
Lidocaine Hydrochloride ◽  
Buffer Solution ◽  
Limit Of Quantification ◽  
Intermediate Precision

A new high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of active pharmaceutical substances and preservatives in a new dental medication has been developed. The optimization of HPLC method parameters was done through studies of a mobile phase composition and a detection wavelength. Our developing method uses an ACE C18 column (250 × 4.6 mm, 5 µm) and a gradient mode for separation with the acetonitrile and phosphate buffer solution (adjusted to pH 3.0) as mobile phases. The flow rate is 1 ml/ min, and the detection was set at 260 nm (DAD). The method was evaluated according to the ICH guidelines and the State Pharmacopoeia of Ukraine in terms of specificity, accuracy, linearity and precision (repeatability and intermediate precision). The limit of detection and the limit of quantification were also calculated. The developed method was put in place for the analysis of a combined dental gel to a quantitative determination of the APIs (choline salicylate, lidocaine hydrochloride) and preservatives (methylparaben, propylparaben).

scholarly journals Validated UV-Vis Spectrophotometric Determination of Andrographolide in Herbal Nano-Preparation

2019 ◽  
Vol 31 (9) ◽  
pp. 1985-1988
Author(s):  
Indah Hairunisa ◽  
Muhammad Da'i ◽  
Erindyah Retno Wikantyasning ◽  
Andhika Rizky Gilang Mahaputra ◽  
Normaidah Normaidah ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Picric Acid ◽  
Limit Of Quantification ◽  
Intermediate Precision ◽  
Herbal Products ◽  
Relative Standard ◽  
Acid Reagent ◽  
Interday Precision ◽  
Detection And Quantification

Determination of major bioactive compounds in polyphyto-formulation is important for production of standardized herbal products. A fast, simple and inexpensive method for detection and quantification of andrographolide concentration in nanoemulsion preparations containing a combination of Andrographis paniculata (Burm f.) Ness. and Phyllanthus niruri L. has been developed. Detection and quantification were carried out using UV-vis spectrophotometry analysis with picric acid reagent and NaOH (8:2) in methanol solvents, read at maximum wavelength 479 nm with 22 min of incubation time. Validation was done by determine the parameters such as linearity, intra and interday precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ). The results obtained showed linearity with r = 0.9945 (y = 0.0109x − 0.2066) in the range of 30-80 μg/mL. The accuracy (recovery) varied in the range of 97.15 to 104.42 %. Percentage of relative standard deviation (% RSD) for precision and intermediate precision value were 3.23 and 3.02 % with LOD value 211 μg/mL and the LOQ 705 μg/mL. As a conclusion, this method is suitable to detect andrographolide content in herbal nano-preparation.

scholarly journals Determination of Walnut Protein in Processed Foods by Enzyme-Linked Immunosorbent Assay: Interlaboratory Study

2010 ◽  
Vol 93 (4) ◽  
pp. 1255-1261 ◽  
Author(s):  
Shinobu Sakai ◽  
Reiko Adachi ◽  
Hiroshi Akiyama ◽  
Reiko Teshima ◽  
Hirotoshi Doi ◽  
...  
Keyword(s):  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Processed Foods ◽  
Food Allergens ◽  
2S Albumin ◽  
Elisa Kit ◽  
Tree Nuts ◽  
Relative Standard ◽  
High Level

Abstract Because food allergens from tree nuts, including walnuts, are a frequent cause of adverse food reactions for allergic patients, the labeling of foods containing ingredients derived from tree nuts is required in numerous countries. According to Japanese regulations, the labeling of food products containing walnuts is recommended. To ensure proper labeling, a novel sandwich ELISA kit for the determination of walnut protein in processed foods (Walnut Protein [2S-Albumin] Kit; Morinaga Institute of Biological Science, Inc.; walnut kit) has been developed. We prepared seven types of incurred samples (model processed foods: biscuits, bread, sponge cake, orange juice, jelly, chicken meatballs, and rice gruel) containing 10 g walnut soluble protein/g of food for use in interlaboratory evaluations of the walnut kit. The walnut kit displayed sufficient reproducibility relative standard deviations (interlaboratory precision: 5.89.9 RSDR) and a high level of recovery (81119) for all the incurred samples. All the repeatability relative standard deviation (RSDr) values for the incurred samples that were examined were less than 6.0. The results of this interlaboratory evaluation suggested that the walnut kit could be used as a precise and reliable tool for determination of walnut protein in processed foods.

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