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%.

scholarly journals Determination of Total Fumonisins in Corn by Competitive Direct Enzyme-Linked Immunosorbent Assay: Collaborative Study

2002 ◽  
Vol 85 (2) ◽  
pp. 404-410 ◽  
Author(s):  
Chuck B Bird ◽  
Bruce Malone ◽  
Larry G Rice ◽  
P Frank Ross ◽  
Robert Eppley ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Fusarium Species ◽  
Collaborative Study ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Portion ◽  
Relative Standard ◽  
Standard Deviations ◽  
Different Levels

Abstract Fumonisins—mycotoxins produced by some Fusarium species—have been shown to be the causative agent of diseases in horses and other domesticated animals as well as possible carcinogens in humans. A collaborative study was conducted to evaluate the effectiveness of a competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for the determination of total fumonisins (B1, B2, and B3) in corn. The test portion was extracted with methanol–water (7 + 3), filtered, diluted, and tested on the CD-ELISA. Naturally and artificially contaminated corn test portions were sent to 13 collaborators in the United States. Naturally contaminated field test portions were prepared at 3 different levels. Artificially contaminated test portions were spiked at 1.0, 3.0, and 5.0 mg/kg total fumonisins (B1, B2, and B3). Average recoveries of total fumonisins were 120, 100, and 90%, respectively. The relative standard deviations for repeatability ranged from 13.3 to 23.3% and the relative standard deviations for reproducibility ranged from 15.8 to 30.3% across all levels tested. HORRAT values, calculated for each individual sample, ranged from 1.24 to 1.94. This method demonstrated acceptable intra- and interlaboratory precision at the levels tested.

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.

scholarly journals Determination of Total Phosphorus in Foods by Colorimetry: Summary of NMKL Collaborative Study

1996 ◽  
Vol 79 (6) ◽  
pp. 1408-1410 ◽  
Author(s):  
T Katrhna Pulliainen ◽  
Harriet C Wallin
Keyword(s):  
Total Phosphorus ◽  
Phosphorus Content ◽  
Collaborative Study ◽  
Colorimetric Method ◽  
Milk Powder ◽  
Molybdenum Blue ◽  
Relative Standard ◽  
Standard Deviations ◽  
Skimmed Milk Powder

Abstract A collaborative study was conducted to validate a spectrophotometric–colorimetric method for determining total phosphorus in foods. The sample was dry-ashed in the presence of zinc oxide, and total phosphorus content was measured colorimetrically as molybdenum blue. Twelve laboratories from the Nordic countries participated in the study. The test materials included potato flour, sausage, cold ham, infant formula powder, cheese, and skimmed milk powder. Participants received 12 randomly coded samples of 2 blind duplicates of each material. Phosphorus contents of materials varied between 0.076 and 0.96 g/100 g. Relative standard deviations for repeatability of the method varied from 1.1% for 0.96 g phosphorus/100 g to 5.4% for 0.29 g phosphorus/100 g. Relative standard deviations for reproducibility varied from 3.6% for 0.96 g phosphorus/100 g to 7.7% for 0.23 g phosphorus/100 g. The colorimetric method for determination of total phosphorus in foods has been adopted first action by AOAC INTERNATIONAL as an NMKUAOAC INTERNATIONAL method.

Enzyme-Linked Immunosorbent Assay of Total Aflatoxins B1, B2, and G1 in Corn: Follow-up Collaborative Study

1994 ◽  
Vol 77 (3) ◽  
pp. 655-658 ◽  
Author(s):  
Mary W Trucksess ◽  
Michael E Stack
Keyword(s):  
Immunosorbent Assay ◽  
Polyclonal Antibodies ◽  
Collaborative Study ◽  
Screening Method ◽  
Test Sample ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Device ◽  
Total Aflatoxin ◽  
Peroxidase Conjugate

Abstract A direct competitive enzyme-linked immunosorbent assay screening method for af latoxins at 20 ng/g in corn was studied by 15 collaborating laboratories. Test samples of corn were extracted by blending with methanol-water (8 + 2). The extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of <30%. Each diluted filtrate was applied to a test device containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin Bi-peroxidase conjugate was added, the test device was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. A test sample was judged to contain ≥20 ng af latoxins/g when, after exactly 1 min, no color was observed on the filter; if a blue or gray color developed, the test sample was judged to contain <20 ng aflatoxins/g. All laboratories correctly identified naturally contaminated corn test samples. Only one false positive was found for controls containing no aflatoxins. The correct responses for positive test samples spiked at levels of 10,20, and 30 ng aflatoxins/g (the ratio of B1:B2:G1 was 15:1:3) were 67,97, and 100%, respectively. This method was adopted first action by AOAC INTERNATIONAL as a change in method for 990.34 for screening for aflatoxins B1, B2, and G1 in corn at total aflatoxin concentrations of ≥20 ng/g.

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.

Enzyme-Linked Immunosorbent Assay for Detection of Zearalenone in Corn, Wheat, and Pig Feed: Collaborative Study

1994 ◽  
Vol 77 (6) ◽  
pp. 1500-1508 ◽  
Author(s):  
Glenn A Bennett ◽  
Terry C Nelsen ◽  
Brjnton M Miller
Keyword(s):  
Immunosorbent Assay ◽  
Collaborative Study ◽  
Screening Method ◽  
The United States ◽  
False Positives ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
False Negatives ◽  
Relative Standard ◽  
Pig Feed

Abstract A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for zearalenone in corn, wheat, and feed at 500 ng/g was evaluated by 23 collaborators (22 laboratories) in an international collaborative study. Eighteen samples of spiked or naturally contaminated corn, wheat, and pig feed were prepared by the sponsoring laboratory and sent for testing with complete test kits to participating collaborators in Canada, Italy, Sweden, The Netherlands, and the United States. Test samples were extracted with methanolwater solution (70 + 30) by shaking on a wrist-action shaker for 3 min. A portion of the extract was mixed with an equal volume of zearalenone-enzyme conjugate, and the mixture was incubated with zearalenone-specific monoclonal antibodies coated onto microtiter wells. All test samples were assayed in duplicate. One of 52 (2%) blanks was reported positive. Thirty-nine of the 52 (75%) samples that were spiked at 500 ng/g were reported as positive. Forty-nine of the 51 (96%) samples with concentrations at or above 1000 ng/g were reported as positive. The overall incidence of false negatives was 6.0% and the incidence of false positives was 22.7% by the ELISA method. Only one (3.4%) false negative was reported for samples containing ≥800 ng/g. In the spectrophotometric method, 8 collaborators determined approximate levels of zearalenone in test samples from standard curves constructed from spiked extracts (0–3000 ng/g of each commodity tested). This method gave and overall incidence of false negatives of 5.7% and false positives of 17.8%. Average relative standard deviations, RSDr (repeatability) and RSDR (reproducibility), were 11.6 and 25.1% for spiked samples and 11.7 and 33.1% for naturally contaminated samples, respectively. Standard curves were constructed with each set of samples assayed. Comparison of absorbance values from these standard curves indicate the performance of reagents and antibody used in the assay. The ELISA method has been adopted first action by AOAC INTERNATIONAL as a screening method for zearalenone at ≥800 ng/g in corn, wheat, and pig feed.

scholarly journals Determination of Magnesium and Calcium in Foods by Atomic Absorption Spectrometry after Microwave Digestion: NMKL Collaborative Study

1998 ◽  
Vol 81 (6) ◽  
pp. 1202-1208 ◽  
Author(s):  
Kaare Julshamn ◽  
Amund Maage ◽  
Harriet C Wallin ◽  
◽  
S Lierhagen ◽  
...  
Keyword(s):  
Atomic Absorption Spectrometry ◽  
Atomic Absorption ◽  
Microwave Digestion ◽  
Collaborative Study ◽  
Milk Powder ◽  
General Meeting ◽  
Absorption Spectrometry ◽  
Relative Standard ◽  
Standard Deviations ◽  
High Concentrations

Abstract On the basis of results of the performed collaborative study, the 49th Annual General Meeting of the Nordic Committee on Food Analysis (NMKL) in The Faroe Islands, August 1995, approved this method to be printed and included in NMKL's collection of methods of analysis of foods. Eleven laboratories participated in an interlaboratory methods-performance (collaborative) study of a method for determining magnesium and calcium in foodstuffs by atomic absorption spectrometry (AAS) after wet microwave digestion. The study was preceded by a practice round of familiarization samples. The method was tested on 7 materials: 5 foods (apple, milk powder, minced fish, wheat bran, and chocolate cake) and 2 composite diets ranging in Mg content from 240 to 3900 mg/kg and in Ca content from 290 to 9300 mg/kg. The materials were presented to study participants as blind duplicates, and participants were asked to perform single determinations on each sample. Repeatability relative standard deviations (RSDr) ranged from 1.9 to 4.9% for Mg and from 2.2 to 8.1 % for Ca. Reproducibility relative standard deviations (RSDR) ranged from 4.0 to 13% for Mg and from 5.9 to 23% for Ca. For Ca, lowest RSDR values were found for samples with high concentrations of Ca (>3800 mg/kg sample) and with nitrate ion residues of <1.3% (w/v).

scholarly journals Measurement of Total Fructan in Foods by Enzymatic/Spectrophotometric Method: Collaborative Study

2000 ◽  
Vol 83 (2) ◽  
pp. 356-364 ◽  
Author(s):  
Barry V McCleary ◽  
Ann Murphy ◽  
David C Mugford ◽  
Rikki Andersen ◽  
John Ashton ◽  
...  
Keyword(s):  
Enzyme Assay ◽  
Reducing Sugars ◽  
Collaborative Study ◽  
Milk Powder ◽  
Acid Hydrazide ◽  
Jerusalem Artichoke ◽  
Hot Water ◽  
Sugar Alcohols ◽  
Relative Standard ◽  
Standard Deviations

Abstract An AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measuring oligofructans and fructan polysaccharide (inulins) in mixed materials and food products. The sample is extracted with hot water, and an aliquot is treated with a mixture of sucrase (a specific sucrose-degrading enzyme), α-amylase, pullulanase, and maltase to hydrolyze sucrose to glucose and fructose, and starch to glucose. These reducing sugars are then reduced to sugar alcohols by treatment with alkaline borohydride solution. The solution is neutralized, and excess borohydride is removed with dilute acetic acid. The fructan is hydrolyzed to fructose and glucose using a mixture of purified exo- and endo-inulinanases (fructanase mixture). The reducing sugars produced (fructose and glucose) are measured with a spectrophotometer after reaction with para-hydroxybenzoic acid hydrazide. The samples analyzed included pure fructan, chocolate, low-fat spread, milk powder, vitamin tablets, onion powder, Jerusalem artichoke flour, wheat stalks, and a sucrose/cellulose control flour. Repeatability relative standard deviations ranged from 2.3 to 7.3%; reproducibility relative standard deviations ranged from 5.0 to 10.8%.

Enzyme-Linked Immunosorbent Assay of Aflatoxins Bi, B2, and Gi in Corn, Cottonseed, Peanuts, Peanut Butter, and Poultry Feed: Collaborative Study

1989 ◽  
Vol 72 (6) ◽  
pp. 957-962 ◽  
Author(s):  
Mary W Trucksess ◽  
Michael E Stack ◽  
Stanley Nesheim ◽  
Douglas L Park ◽  
Albert E Pohland
Keyword(s):  
Immunosorbent Assay ◽  
Polyclonal Antibodies ◽  
Peanut Butter ◽  
Collaborative Study ◽  
Screening Method ◽  
Steam Bath ◽  
Test Sample ◽  
Positive Test ◽  
Poultry Feed ◽  
Enzyme Linked Immunosorbent Assay

Abstract A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for aflatoxins at 20 ng/g was studied by 12 collaborators. Test samples of peanut butter were extracted by blending with methanol-water-hexane (55 + 45 + 100) and heating the test extracts on a steam bath; test samples of the other commodities were extracted by blending with methanol-water (80 + 20). All test extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of <30%. Each diluted filtrate was applied to a cup containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin B1-peroxidase conjugate was added, the cup was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. The test sample was judged to contain >20 ng aflatoxins/g when, after exactly 1 min, no color was observed on the filter; when a blue or gray color developed, the test sample was judged to contain <20 ng aflatoxins/g. All collaborators correctly identified naturally contaminated corn and raw peanut positive test samples. No false positives were found for controls containing <2 ng aflatoxins/g. The correct responses for positive test samples spiked at levels of 10, 20, and >30 ng aflatoxins/g (the ratio of B1, B2, and G1 was 10:1:3) were 52, 86, and 96%, respectively. The method, which is rapid and simple, has been adopted official first action for screening for aflatoxins at >20 ng/g in cottonseed and peanut butter and for aflatoxins at >30 ng/g in corn and raw peanuts. The procedure will require further study for poultry feed. Positive test samples may require reanalysis by an official, quantitative method.

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.

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