scholarly journals Quantification of bee-derived peptide defensin-1 in honey by competitive enzyme-linked immunosorbent assay, a new approach in honey quality control

2016 ◽  
Vol 34 (No. 3) ◽  
pp. 233-243 ◽  
Author(s):  
Valachová Ivana ◽  
Bučeková Marcela ◽  
Majtán Juraj
Keyword(s):  
Antibacterial Activity ◽  
Immunosorbent Assay ◽  
Inhibitory Concentration ◽  
Rapid Screening ◽  
Enzyme Linked Immunosorbent Assay ◽  
New Approach ◽  
Relative Standard ◽  
Dependent Correlation ◽  
Sensitive Method ◽  
Significant Concentration

We established and evaluated a polyclonal antibody based competitive enzyme-linked immunosorbent assay for the quantification of defensin-1 in honey. The assay showed an inhibitory concentration (IC<sub>50</sub>) value of 111.5 ± 15.41 ng/ml with a detection limit of 7.8125 ng/ml. The regaining of defensin-1 in spiked ‘artificial honey’ was between 87.05 and 112.96% with relative standard deviation less than 9.2%. Sensitivity and specificity of the test were experimentally validated on a sample of 20 different honeys. The antibacterial activity of these honey samples showed a significant concentration-dependent correlation with the production of defensin-1 (n = 20; r = −0.6598; P = 0.0016). The assay provides a specific and sensitive method for the screening of defensin-1 in honey. The method to detect honeybee-derived proteins in honey is a promising approach to verifying the authenticity of honey. The defensin-1 ELISA could also be used for the rapid screening of honeys suitable for medicinal purposes.

scholarly journals Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. e00128-18 ◽  
Author(s):  
Danka Pavliakova ◽  
Peter C. Giardina ◽  
Soraya Moghazeh ◽  
Shite Sebastian ◽  
Maya Koster ◽  
...  
Keyword(s):  
Human Serum ◽  
Lower Limit ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Correlate Of Protection ◽  
Immune Correlate ◽  
Relative Standard ◽  
Limit Of Quantitation

ABSTRACT A Luminex-based direct immunoassay (dLIA) platform has been developed to replace the standardized pneumococcal enzyme-linked immunosorbent assay platform. The multiplex dLIA simultaneously measures the concentration of serum immunoglobulin G (IgG) antibodies specific for pneumococcal capsular polysaccharide (PnPS) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The assay uses poly-l-lysine (PLL)-conjugated PnPS, chemically coupled to spectrally distinct Luminex microspheres. Assay validation experiments were performed using residual human serum samples obtained from 13-valent pneumococcal conjugate vaccine (13vPnC) clinical studies. Assay results are expressed as IgG antibody concentrations in micrograms per milliliter using the international reference serum, 007sp. The lower limit of quantitation (LLOQ) for all serotypes covered in the 13-plex dLIA fell within the range of 0.002 to 0.038 µg/ml serum IgG. The difference between the lower limit and upper limit of the assay range was >500-fold for all serotypes, and assay variability was <20% relative standard deviation (RSD) for all serotypes. IgG antibody measurements were shown to be serotype-specific (some cross-reactivity was observed only between the structurally related serotypes 6A and 6B as well as 19A and 19F), and no interference was observed between the serotypes when the assay was performed in the 13-plex format compared to the singleplex assays. The 13-plex dLIA platform developed by Pfizer Inc. generates up to 143 test results in a single 96-well plate and is a suitable replacement of the enzyme-linked immunosorbent assay (ELISA) platform for evaluating vaccine clinical trials. IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.

scholarly journals Interlaboratory Evaluation of Two Enzyme-Linked Immunosorbent Assay Kits for the Determination of Crustacean Protein in Processed Foods

2008 ◽  
Vol 91 (1) ◽  
pp. 123-129 ◽  
Author(s):  
Shinobu Sakai ◽  
Rieko Matsuda ◽  
Reiko Adachi ◽  
Hiroshi Akiyama ◽  
Tamio Maitani ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Relative Standard Deviation ◽  
Immunosorbent Assay ◽  
Soluble Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Processed Foods ◽  
Relative Standard ◽  
High Level

Abstract The labeling of foods containing material derived from crustaceans such as shrimp and crab is to become mandatory in Japan because of increases in the number of allergy patients. To ensure proper labeling, 2 novel sandwich enzyme-linked immunosorbent assay (ELISA) kits for the determination of crustacean protein in processed foods, the N kit (Nissui Pharmaceutical Co., Ltd, Ibaraki, Japan) and the M kit (Maruha Nichiro Holdings, Inc., Ibaraki, Japan), have been developed. Five types of model processed foods containing 10 and/or 11.9 g/g crustacean soluble protein were prepared for interlaboratory evaluation of the performance of these kits. The N kit displayed a relatively high level of reproducibility relative standard deviation (interlaboratory precision; 4.08.4 RSDR) and sufficient recovery (6586) for all the model processed foods. The M kit displayed sufficient reproducibility (17.620.5 RSDR) and a reasonably high level of recovery (82103). The repeatability relative standard deviation (RSDr) values regarding the detection of crustacean proteins in the 5 model foods were mostly &lt;5.1 RSDr for the N kit and 9.9 RSDr for the M kit. In conclusion, the results of this interlaboratory evaluation suggest that both these ELISA kits would be very useful for detecting crustacean protein in processed foods.

Determination of Total Aflatoxin Levels in Peanut Butter by Enzyme-Linked Immunosorbent Assay: Collaborative Study

1992 ◽  
Vol 75 (4) ◽  
pp. 693-697 ◽  
Author(s):  
Alan L Patey ◽  
Matthew Sharman ◽  
John Gilbert
Keyword(s):  
Immunosorbent Assay ◽  
Aflatoxin B1 ◽  
Peanut Butter ◽  
Collaborative Study ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Total Aflatoxin ◽  
Aflatoxin Level ◽  
Relative Standard

Abstract Laboratories in Australia, Japan, Spain, the United Kingdom, and the United States participated in a collaborative study to evaluate a commercial enzyme- linked immunosorbent assay for the determination of total aflatoxin. Collaborators were sent 10 randomly numbered samples (5 blind duplicates) of roasted peanut butter. Two pairs were "blank" peanut butters to which aflatoxin B1, B2, G1, and G2 standards had been added. The other 3 pairs of peanut butters were 1 low aflatoxin level sample and 2 naturally contaminated samples. The assay is based on indirect competition. Test samples containing (free) aflatoxin, added to aflatoxin-coated microwells, compete for specific monoclonal rat anti-aflatoxin. As the concentration of aflatoxin in the test samples increases, the amount of rat antiaflatoxin binding to the aflatoxin attached to the well decreases. After a wash step to remove unbound material, the amount of rat anti-aflatoxin bound to the well is determined by its reaction with peroxidase conjugated rabbit anti-rat globulin. Bound peroxidase activity is then determined by the addition of a substrate, whose color development is inversely proportional to the aflatoxin concentration and is measured by absorbance. Coefficients of variation (CV) for total aflatoxin concentrations, for mean levels of 9,30, and 89µg/kg, were between 28 and 37% for the low level and 2 naturally contaminated samples, which contained mainly aflatoxin B1. CVs for the spiked samples were lower (24-25%) for mean levels of 11 and 20 µg/kg; recoveries were 84 and 89%, respectively. Ranges for relative standard deviations for repeatabilty and reproducibility were 9-30% and 25-37%, respectively. The method has been adopted first action by AOAC International.

A new approach of indirect enzyme-linked immunosorbent assay for determination of D-glutamic acid throughin situconjugation

2016 ◽  
Vol 37 (3) ◽  
pp. 296-306
Author(s):  
Seiichi Sakamoto ◽  
Rika Nagamitsu ◽  
Yurino Matsuura ◽  
Yumi Tsuneura ◽  
Hitoshi Kurose ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
New Approach

scholarly journals Determination of Cry9C Protein in Corn-Based Foods by Enzyme-Linked Immunosorbent Assay: Interlaboratory Study

2001 ◽  
Vol 84 (6) ◽  
pp. 1891-1902 ◽  
Author(s):  
Mary W Trucksess ◽  
T Artis ◽  
C Diaz ◽  
C Fernandez ◽  
K Harkin ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Specific Interaction ◽  
Strong Acid ◽  
The United States ◽  
Interlaboratory Study ◽  
Enzyme Linked Immunosorbent Assay ◽  
Corn Flour ◽  
Relative Standard ◽  
Standard Deviations

Abstract The performance of a commercially available enzyme-linked immunosorbent assay kit (Enviro-Logix) was assessed for the determination of Cry9C protein, which is produced by the genetically modified corn StarLink, in 8 types of corn-based foods (starch, refined oil, soft tortillas, tortilla chips, corn flakes, corn puffs, corn muffins, and corn bread) in an interlaboratory study involving 7 laboratories in the United States. The assay kit is a double antibody sandwich and is based on the specific interaction between antibody and antigen. The Cry9C protein analyte is sandwiched between 2 antibodies, one to capture the analyte and the other is conjugated to the enzyme, horseradish peroxidase. The enzyme uses tetramethylbenzidine/peroxide for color development. A strong acid stopping reagent is then used to change the color from blue to a stable yellow. The intensity of the color is proportional to the concentration of the Cry9C protein. In this study blind duplicates of control samples (blank material prepared from non- StarLink corn), spiked samples (blank material with the addition of Cry9C protein), and samples containing incurred analyte (products prepared with StarLink corn) were analyzed. Cry9C protein from 2 different sources was used to spike the food products. Cry9C protein produced and purified from a bacterial host was used to prepare spiked test samples at 2.72 and 6.8 ng/g. Cry9C protein from StarLink corn flour was used to prepare spiked samples at 1.97 ng/g. Average recoveries for samples spiked with corn flour Cry9C protein at 1.97 ng/g ranged from 73 to 122%, within-laboratory relative standard deviations (RSDr) ranged from 6 to 22%, and between-laboratories relative standard deviations (RSDR) ranged from 16 to 56%. Average recoveries for samples spiked with bacterial Cry9C protein at 2.72 and 6.8 ng/g ranged from 27 to 96% and from 32 to 113%, respectively; RSDr values ranged from 10 to 35%and from 7 to 38%, respectively; and the RSDR ranged from 28 to 84%and 15 to 75%, respectively. The incurred test samples were found to contain Cry9C protein at levels ranging from 0.8 to 3187 ng/g depending on the product, RSDr values ranged from 5 to 16% and RSDR values ranged from 11 to 71%. Results of the statistical analysis indicate that this method is applicable to the determination of Cry9C protein in the 8 types of collaboratively studied corn-based products containing Cry9C protein (from StarLink ) at levels of ≥2 ng/g.

scholarly journals Single-Laboratory Validation of the Biosense Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA) for Determination of Domoic Acid Toxins in Shellfish

2007 ◽  
Vol 90 (4) ◽  
pp. 1000-1010 ◽  
Author(s):  
Hans Kleivdal ◽  
Sven-Inge Kristiansen ◽  
Mona V Nilsen ◽  
Lyn Briggs
Keyword(s):  
Immunosorbent Assay ◽  
Domoic Acid ◽  
Matrix Effects ◽  
Limit Of Detection ◽  
Method Comparison ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Shellfish Poisoning ◽  
Relative Standard

Abstract Method validation was conducted for an enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA) toxins, known to give amnesic shellfish poisoning (ASP) symptoms, in shellfish. The calibration curve range of the assay is approximately 10260 pg/mL, with a dynamic working range for DA toxins in shellfish from 0.01 to at least 250 mg/kg. The ASP ELISA showed no significant cross-reactivity to structural analogs, and proved to be robust to deliberate alterations of the optimal running conditions. The shellfish matrix effects observed with mussels, oysters, and scallops were eliminated by diluting shellfish extracts 1:200 prior to analysis, leading to a limit of detection at 0.003 mg/kg. Thirteen blank shellfish homogenates were spiked with certified mussel material containing DA to levels in the range of 0.125 mg DA/kg, and analyzed in quadruplicate on 3 different days. The relative standard deviation (RSD) under intra-assay repeatability conditions ranged from 6.5 to 13.1%, and under interassay repeatability conditions the RSD ranged from 5.7 to 13.4%, with a mean value of 9.3%. The recoveries ranged from 85.5 to 106.6%, with a mean recovery of 102.2%. A method comparison was conducted with liquid chromatography with ultraviolet detection, using naturally contaminated scallop samples (n = 27) with DA levels at 0244 mg/kg. The overall correlation coefficient was 0.960 and the slope of the regression was 1.218, indicating a good agreement between the methods.

scholarly journals Determination of Vegetal Proteins in Milk Powder by Enzyme-Linked Immunosorbent Assay: Interlaboratory Study

2002 ◽  
Vol 85 (6) ◽  
pp. 1390-1397 ◽  
Author(s):  
Lourdes Sánchez ◽  
María D Pérez ◽  
Pilar Puyol ◽  
Miguel Calvo ◽  
Gary Brett ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Polyclonal Antibodies ◽  
Collaborative Study ◽  
Milk Powder ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wheat Proteins ◽  
Blind Test ◽  
Relative Standard ◽  
Standard Deviations ◽  
Phosphate Buffered Saline

Abstract Eight laboratories participated in a collaborative study to evaluate an enzyme-linked immunosorbent assay (ELISA) to determine soy, pea, and wheat proteins in pasteurized or ultra-high temperature (UHT) milk powders. To perform this assay, polyclonal antibodies for soy, pea, and wheat proteins were obtained from rabbit sera. Collaborators received calibration standards composed of milk powder containing 0–8% (w/w) vegetal protein in total protein and blind test samples containing approximately 1, 2, and 5% (w/w) vegetal protein. An indirect competitive ELISA was performed with a kit prepared by a participating laboratory; the kit contained plates coated with soy, pea, or wheat proteins, the corresponding specific antisera, enzyme-labeled second antibody, and substrate solution. Test samples and calibrants were extracted with phosphate-buffered saline, pH 7.4, containing 0.05% Tween and assayed with the ELISA kits. The degree of adulteration was affected by the type of heat treatment applied to the samples. The estimated percentage of vegetal protein addition was close to the theoretical value for pasteurized samples but much lower for UHT samples. For pasteurized samples, intralaboratory relative standard deviations ranged from 5 to 22% and interlaboratory relative standard deviations ranged from 14 to 34%.

Effect of Spacer and the Enzyme-Linked Immunosorbent Assay

2016 ◽  
Vol 66 (5) ◽  
pp. 471
Author(s):  
Manisha Sathe ◽  
Shruti Srivastava ◽  
Sumit Agrawal ◽  
Ramrao Ghorpade
Keyword(s):  
Immunosorbent Assay ◽  
Inhibitory Concentration ◽  
Methylphosphonic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Immunoassay ◽  
Ic50 Value ◽  
Lower Detection ◽  
Enzyme Conjugate ◽  
Enzyme Label ◽  
Free Antigen

The effect of spacers and the enzyme-linked immunosorbent assay (ELISA) formats on the functional parameters of assays such as lower detection limit, inhibitory concentration at 50 per cent (IC50), and specificity were studied. Enzyme conjugates having hydrophobic and hydrophilic spacers were prepared using O-isopropyl methylphosphonic acid (IMPA) and horseradish peroxidase (HRP) as an enzyme label. Comparison was made with reference to enzyme conjugate without any spacer. The present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate significantly improves the sensitivity of assays. An enhanced IC50 value achieved was 0.01 μg mL−1 for free antigen detection by direct immunoassay using hydrophilic spacers and precoating of ELISA plates by secondary antibody. The use of a hydrophilic spacer might have helped in projecting the hapten in the aqueous phase, leading to enhanced antibody binding signal and improved sensitivity of the assay.

Sign in / Sign up

Export Citation Format

Share Document