The Validation of Neogen Reveal® Q+ for DON.

BACKGROUND: Reveal® Q+ for DON is an immunochromatographic test for quantitative determination of deoxynivalenol (DON) in grains. OBJECTIVE: A study was conducted to validate performance of this method for determination of DON in naturally contaminated corn and wheat and in DON-spiked corn/soy blend, soybeans, barley, malted barley, buckwheat, brown rice, sorghum, and distillers dried grain. METHODS: In addition to matrix testing, LOD, linearity, selectivity, robustness, and stability/lot-to-lot consistency testing were performed. RESULTS: The LOD was determined to be 0.014 ppm in corn and 0.037 ppm in wheat, and the LOQ 0.042 ppm in corn and 0.11 ppm in wheat. Recovery ranged from 90 to 104% across a range of reference values of 0.5 to 34.5 ppm. Linearity calculation comparing test results with reference values produced R2 values of 0.999 in both matrixes. Internal results with corn and wheat were corroborated in independent laboratory testing. For DON-spiked commodities, mean recovery across a range of DON concentration from 0.5 to 30 ppm ranged from 90 to 109%. Results of selectivity testing showed no cross-reactivity with other mycotoxins and no interference in detection of DON. Reagent lot-to-lot consistency and stability studies showed consistent results across a range of DON levels and established expiration dating for the test of 18 months after manufacture when stored under specified conditions. Conclusions and Highlights: The Reveal Q+ for DON test offers reliable performance as well as the advantages of aqueous sample extraction, procedural simplicity, minimal labor and equipment requirements, and rapid results. CONCLUSIONS: The Reveal Q+ for DON test is validated as a Performace Tested Method in Corn, Wheat, and a variety of other grains. HIGHLIGHTS: The test provides rapid results from a simple aqueous extraction and requires minimal labor and equipment.

Reveal Q+ MAX® for Detection of Total Af latoxin in Corn, Almonds, Pistachios, Walnuts, and Peanuts.

Background: The Reveal Q+ MAX for Aflatoxin is a lateral flow immunochromatographic test intended for quantitative analysis within 6 min after aqueous extraction. Objective: Work was conducted to validate the performance of the Reveal Q+ MAX for Aflatoxin method in selected corn and nut matrixes. Methods: This method was validated under the requirements of the AOAC Research Institute Performance Tested MethodSM program. Five matrixes, including corn naturally contaminated with aflatoxin at 0, 5.2, 21.0, 51.6, 103.6, and 282 ppb as well as peanuts, pistachios, walnuts, and almonds spiked at 0, 5, 20, 50, and 300 ppb were analyzed. Results: Average percentage recoveries of the added aflatoxin from the matrixes ranged from 80.8 to 116.9%. Average LOD for all matrixes is 2 ppb and LOQ is 7 ppb. With the exception of sample size for almonds, robustness trials demonstrated that deliberate changes to the assay parameters minimally affected the Reveal Q+ MAX assay performance. Finally, stability results from three independently manufactured lots support Reveal Q+ MAX for Aflatoxin performance consistency and shelf-life of 18 months when stored at room temperature. Conclusions: This study appropriately validates the Performance Tested MethodSM claim for corn and selected nut matrixes on Reveal Q+ MAX for Aflatoxin, an aqueous lateral flow test kit. Highlights: Aqueous lateral flow test kit detects total aflatoxin between 80 to 120% yield with an LOD of 2 ppb.

Validation of the BetaStar® Advanced for Beta-lactams Test Kit for the Screening of Bulk Tank and Tanker Truck Milks for the Presence of Beta-lactam Drug Residues.

A validation study was conducted for an immunochromatographic method (BetaStar® Advanced for Beta-lactams) for the detection of beta-lactam residues in raw, commingled bovine milk. The assay detected amoxicillin, ampicillin, cloxacillin, penicillin, cephapirin, and ceftiofur below the U.S. Food and Drug Administration tolerance levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. The results of internal and independent laboratory dose-response studies employing spiked samples were in agreement. The test detected all six drugs at the approximate 90/95% sensitivity levels in milk from cows treated with each drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing 1148 control milk samples. Testing the estimated 90/95% sensitivity level for amoxicillin (8.5 ppb), ampicillin (6.9 ppb), cloxacillin (8.9 ppb), penicillin (4.2 ppb), and cephapirin (17.6 ppb), and at 100 ppb for each antibiotic, resulted in 94-100% positive tests for each of the beta-lactam drugs. The results of ruggedness experiments established the operating parameter tolerances for the assay. Cross-reactivity testing established that the assay detects other certain beta-lactam drugs, but it does not cross-react with any of 30 drugs belonging to seven different drug classes. Abnormally high bacterial or somatic cell counts in raw milk produced no assay interference.

Validation of the BetaStar® Advanced for Tetracyclines Test Kit for the Screening of Bulk Tank and Tanker Truck Milks for the Presence of Tetracycline Drug Residues.

A validation study was conducted for an immunochromatographic method (BetaStar® Advanced for Tetracyclines) for detection of tetracycline antibiotic residues in raw, commingled bovine milk. The assay was demonstrated to detect tetracycline, chlortetracycline, and oxytetracycline at levels below the FDA tolerance levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. Results of internal and independent laboratory dose-response studies employing spiked samples were in agreement. All three drugs at the approximate 90/95% sensitivity levels were detected in milk collected from cows that had been treated with the specific drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing 881 control milk samples. Testing the estimated 90/95 sensitivity level for tetracycline (213 ppb), chlortetracycline (272 ppb), and oxytetracycline (180 ppb) and at 1000 ppb for each antibiotic resulted in 100% positive tests for each tetracycline. Results of ruggedness experiments established the operating parameter tolerances for the test. Results of cross-reactivity testing established that the assay detects certain other tetracycline drugs but does not cross-react with any of 32 drugs belonging to seven different drug classes. Abnormally high bacterial or somatic cell counts (SCC) in raw milk produced no assay interference.

Development and Validation of a Quantitative ELISA for the Detection of Almond Residues in Foods.

Neogen Corp. has developed Veratox for Almond Allergen for use in the quantitative analysis and screening of almond protein residues in food products, such as cookies, crackers, chocolate bars, cereals, beverages, and clean-in-place rinses. Quantitation with Veratox for Almond Allergen ranges from 2.5 to 25 ppm and, with dilution, it can be extended for highly positive samples. This paper describes the findings of internal testing and validation studies designed to establish product claims for the assay of Veratox for Almond Allergen.

Validation of MAX Aqueous Extraction on Veratox® for Total Aflatoxin ELISA Test Kit.

Neogen Corp. (Lansing, MI) has developed a common aqueous extraction method for the detection of mycotoxins in the ELISA or lateral flow format. The Veratox® for Total Aflatoxin ELISA extraction method uses a MAX 2 extraction packet and water in replacement of traditionally used organic solvents. Veratox for Total Aflatoxin has a detection range of 5-50 ppb neat or up to 300 ppb with dilution. The kit development focused on superior cross-reactivity, ability to accurately detect naturally contaminated samples, and utilization of an aqueous extraction method. In two separate validation studies, the Veratox for Total Aflatoxin test kit resulted in average yields of 91-114% in naturally contaminated mycotoxin reference material corn. The cross-reactivity profiles for aflatoxins B1, B2, G1, and G2 were 100, 113, 103, and 93%, respectively. This kit is approved by the Grain Inspection, Packers, and Stockyards Administration.

Free Radical Threshold Value: A New Universal Body Constant.

Three evolutionary sources create 'primary' reactive oxygen species (ROS) and 'secondary' lipid oxygen species (LOS), forming the human body's 'free radical ground state'. We present evidence for the existence of a universal free radical threshold value (FRTV), defining the borderline between advantageous and adverse effects of free radicals observed above the free radical ground state. Based on standard vitamin D doses, the calculated amount of ∼3.5 × 10(12) rad/mg ROS/LOS tissue represents the tolerated number of free radicals in skin tissue - defined as FRTV. By means of quantitative ESR x-band spectroscopy, the FRTV was experimentally verified using ex vivo human skin irradiated with ultraviolet + visible (UV + VIS), UVB + UVA and VIS light. In addition, we investigated whether this threshold is also existent in internal organs by extending our experiment to fresh porcine liver. Based on the determination of ROS/LOS below and above the FRTV, ROS > LOS was characterized as beneficial and LOS > ROS as deleterious to the organism, respectively. Results of the experiments using porcine liver confirmed the appearance of the FRTV at radical generation ∼3.5 × 10(12) rad/mg. The relation ROS/LOS before and after the FRTV was consistent with the results determined for the skin. We conclude that the FRTV, theoretically calculated and experimentally confirmed, should be considered as a new 'universal body constant'.

Three-body nature of N(*) and Δ(*) resonances from sequential decay chains.

The Nπ^{0}π^{0} decays of positive-parity N^{*} and Δ^{*} resonances at about 2 GeV are studied at ELSA by photoproduction of two neutral pions off protons. The data reveal clear evidence for several intermediate resonances: Δ(1232), N(1520)3/2^{-}, and N(1680)5/2^{+}, with spin parities J^{P}=3/2^{+}, 3/2^{-}, and 5/2^{+}. The partial wave analysis (within the Bonn-Gatchina approach) identifies N(1440)1/2^{+} and the N(ππ)_{S wave} (abbreviated as Nσ here) as further isobars and assigns the final states to the formation of nucleon and Δ resonances and to nonresonant contributions. We observe the known Δ(1232)π decays of Δ(1910)1/2^{+}, Δ(1920)3/2^{+}, Δ(1905)5/2^{+}, Δ(1950)7/2^{+}, and of the corresponding spin-parity series in the nucleon sector, N(1880)1/2^{+}, N(1900)3/2^{+}, N(2000)5/2^{+}, and N(1990)7/2^{+}. For the nucleon resonances, these decay modes are reported here for the first time. Further new decay modes proceed via N(1440)1/2^{+}π, N(1520)3/2^{-}π, N(1680)5/2^{+}π, and Nσ. The latter decay modes are observed in the decay of N^{*} resonances and at most weakly in Δ^{*} decays. It is argued that these decay modes provide evidence for a 3-quark nature of N^{*} resonances rather than a quark-diquark structure.

Development of a rapid multiplexed assay for the direct screening of antimicrobial residues in raw milk.

Antimicrobial residues found to be present in milk can have both health and economic impacts. For these reasons, the widespread routine testing of milk is required. Due to delays with sample handling and test scheduling, laboratory-based tests are not always suited for making decisions about raw material intake and product release, especially when samples require shipping to a central testing facility. Therefore, rapid on-site screening tests that can produce results within a matter of minutes are required to facilitate rapid intake and product release processes. Such tests must be simple for use by non-technical staff. There is increasing momentum towards the development and implementation of multiplexing tests that can detect a range of important antimicrobial residues simultaneously. A simple in situ multiplexed planar waveguide device that can simultaneously detect chloramphenicol, streptomycin and desfuroylceftiofur in raw dairy milk, without sample preparation, has been developed. Samples are simply mixed with antibody prior to an aliquot being passed through the detection cartridge for 5 min before reading on a field-deployable portable instrument. Multiplexed calibration curves were produced in both buffer and raw milk. Buffer curves, for chloramphenicol, streptomycin and desfuroylceftiofur, showed linear ranges (inhibitory concentration (IC)20-IC80) of 0.1-0.9, 3-129 and 12-26 ng/ml, whilst linear range in milk was 0.13-0.74, 11-376 and 2-12 ng/ml, respectively, thus meeting European legislated concentration requirements for both chloramphenicol and streptomycin, in milk, without the need for any sample preparation. Desfuroylceftiofur-contaminated samples require only simple sample dilution to bring positive samples within the range of quantification. Assay repeatability and reproducibility were lower than 12 coefficient of variation (%CV), whilst blank raw milk samples (n = 9) showed repeatability ranging between 4.2 and 8.1%CV when measured on all three calibration curves. Graphical Abstract MBio SnapEsi reader and cartridge.

Validation study of the BetaStar plus lateral flow assay for detection of beta-lactam antibiotics in milk.

A validation study designed to meet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration, Center for Veterinary Medicine (FDA/CVM) was conducted for a receptor and antibody-based, immunochromatographic method (BetaStar Plus) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, ceftiofur, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels, but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments (NCIMS). Results of the part I (internal) and part II (independent laboratory) dose-response studies employing spiked samples were in close agreement. The test was able to detect all six drugs at the approximate 90/95% sensitivity levels when presented as incurred residues in milk collected from cows that had been treated with the specific drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing of 1031 control milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar Plus assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in raw milk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels.

Reveal Salmonella 2.0 test for detection of Salmonella spp. in foods and environmental samples. Performance Tested Method 960801.

Reveal Salmonella 2.0 is an improved version of the original Reveal Salmonella lateral flow immunoassay and is applicable to the detection of Salmonella enterica serogroups A-E in a variety of food and environmental samples. A Performance Tested Method validation study was conducted to compare performance of the Reveal 2.0 method with that of the U.S. Department of Agriculture-Food Safety and Inspection Service or U.S. Food and Drug Administration/Bacteriological Analytical Manual reference culture methods for detection of Salmonella spp. in chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, raw shrimp, a ready-to-eat meal product, dry pet food, ice cream, spinach, cantaloupe, peanut butter, stainless steel surface, and sprout irrigation water. In a total of 17 trials performed internally and four trials performed in an independent laboratory, there were no statistically significant differences in performance of the Reveal 2.0 and reference culture procedures as determined by Chi-square analysis, with the exception of one trial with stainless steel surface and one trial with sprout irrigation water where there were significantly more positive results by the Reveal 2.0 method. Considering all data generated in testing food samples using enrichment procedures specifically designed for the Reveal method, overall sensitivity of the Reveal method relative to the reference culture methods was 99%. In testing environmental samples, sensitivity of the Reveal method relative to the reference culture method was 164%. For select foods, use of the Reveal test in conjunction with reference method enrichment resulted in overall sensitivity of 92%. There were no unconfirmed positive results on uninoculated control samples in any trials for specificity of 100%. In inclusivity testing, 102 different Salmonella serovars belonging to serogroups A-E were tested and 99 were consistently positive in the Reveal test. In exclusivity testing of 33 strains of non-salmonellae representing 14 genera, 32 were negative when tested with Reveal following nonselective enrichment, and the remaining strain was found to be substantially inhibited by the enrichment media used with the Reveal method. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device development time.

Reveal E. coli 2.0 method for detection of Escherichia coli O157:H7 in raw beef.

Reveal E. coli 2.0 is a new lateral-flow immunodiagnostic test for detection of E. coli O157:H7 and O157:NM in raw beef trim and ground beef. Compared with the original Reveal E. coli O157:H7 assay, the new test utilizes a unique antibody combination resulting in improved test specificity. The device architecture and test procedure have also been modified, and a single enrichment protocol was developed which allows the test to be performed at any point during an enrichment period of 12 to 20 h. Results of inclusivity and exclusivity testing showed that the test is specific for E. coli serotypes O157:H7 and O157:NM, with the exception of two strains of O157:H38 and one strain of O157:H43 which produced positive reactions. In internal and independent laboratory trials comparing the Reveal 2.0 method to the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of E. coli O157:H7 in 65 and 375 g raw beef trim and ground beef samples, there were no statistically significant differences in method performance with the exception of a single internal trial with 375 g ground beef samples in which the Reveal method produced significantly more positive results. There were no unconfirmed positive results by the Reveal assay, for specificity of 100%. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device incubation time or temperature. However, addition of the promoter reagent to the test sample prior to introducing the test device is essential to proper test performance.

Cytocompatibility testing of cell culture modules fabricated from specific candidate biomaterials using injection molding.

Most polymers used in clinical applications today are materials that have been developed originally for application areas other than biomedicine. Testing the cell- and tissue-compatibility of novel materials in vitro and in vivo is of key importance for the approval of medical devices and is regulated according to the Council Directive 93/42/EEC of the European communities concerning medical devices. In the standardized testing methods the testing sample is placed in commercially available cell culture plates, which are often made from polystyrene. Thus not only the testing sample itself influences cell behavior but also the culture vessel material. In order to exclude this influence, a new system for cell testing will be presented allowing a more precise and systematic investigation by preparing tailored inserts which are made of the testing material. Inserts prepared from polystyrene, polycarbonate and poly(ether imide) were tested for their cytotoxity and cell adherence. Furthermore a proof of principle concerning the preparation of inserts with a membrane-like surface structure and its surface modification was established. Physicochemical investigations revealed a similar morphology and showed to be very similar to the findings to analogous preparations and modifications of flat-sheet membranes.

Quasifree photoproduction of eta mesons off the neutron.

Quasifree photoproduction of eta mesons off nucleons bound in the deuteron has been measured with the CBELSA/TAPS detector for incident photon energies up to 2.5 GeV at the Bonn ELSA accelerator. The eta mesons have been detected in coincidence with recoil protons and recoil neutrons, which allows a detailed comparison of the quasifree n(gamma,eta)n and p(gamma,eta)p reactions. The excitation function for eta production off the neutron shows a pronounced bumplike structure at W=1.68 GeV (E{gamma} approximately 1 GeV), which is absent for the proton.

Modification of the omega-meson lifetime in nuclear matter.

Information on hadron properties in the nuclear medium has been derived from the photoproduction of omega mesons on the nuclei C, Ca, Nb, and Pb using the Crystal Barrel/TAPS detector at the ELSA tagged photon facility in Bonn. The dependence of the omega-meson cross section on the nuclear mass number has been compared with three different types of models: a Glauber analysis, a Boltzmann-Uehling-Uhlenbeck analysis of the Giessen theory group, and a calculation by the Valencia theory group. In all three cases, the inelastic omega width is found to be 130-150 MeV/c(2) at normal nuclear matter density for an average 3-momentum of 1.1 GeV/c. In the rest frame of the omega meson, this inelastic omega width corresponds to a reduction of the omega lifetime by a factor approximately 30. For the first time, the momentum dependent omegaN cross section has been extracted from the experiment and is in the range of 70 mb.

Formation of poly(epsilon-caprolactone) scaffolds loaded with small molecules by integrated processes.

Cell stimulation by bioactive molecules has become an important tool in tissue engineering. The homogeneous incorporation of such molecules within the bulk of a polymer-based scaffold compared to surface coating is considered advantageous for most applications and minimizes a burst effect. An efficient way of bulk loading is the incorporation of these molecules during the scaffold formation process. In this paper, two different integrated processes for the preparation of scaffolds from poly(epsilon-caprolactone) (PCL) loaded with a small molecule are investigated. Both formation and loading of the scaffold is carried out in a single-step process. Sudan Red G was selected as a model compound for lipophilic small molecules. A freeze drying and pressure quench (PQ) formation process was selected, and the influence of the small molecule on the formation processes and on the morphology of the obtained scaffold was evaluated and compared. It could be shown for both processes that the formation of loaded scaffolds is possible, and that the small molecule has a very high impact on the foam morphology. In case of the freeze-drying (FD) method, only a load of 1 wt% Sudan Red G was incorporated within the bulk and showed no influence on the foam morphology. In the case of PQ foaming, an incorporation of 43 wt% Sudan Red G was achieved (although tiny crystal needles of the small molecule were found on the surface) and a strong effect on the foam morphology was found. This paper presents an efficient method of incorporating small molecules by integrated processes.

Observation of in-medium modifications of the omega meson.

The photoproduction of omega mesons on nuclei has been investigated using the Crystal Barrel/TAPS experiment at the ELSA tagged photon facility in Bonn. The aim is to study possible in-medium modifications of the omega meson via the reaction gamma + A --> omega + X --> pi(0)gamma + X('). Results obtained for Nb are compared to a reference measurement on a LH2 target. While for recoiling, long-lived mesons (pi(0), eta, and eta;(')), which decay outside of the nucleus, a difference in the line shape for the two data samples is not observed, we find a significant enhancement towards lower masses for omega mesons produced on the Nb target. For momenta less than 500 MeV/c an in-medium omega meson mass of M(medium) = [722(+4)(-4)(stat)+35-5(syst)] MeV/c(2) has been deduced at an estimated average nuclear density of 0.6rho(0).

Validation study of a lateral-flow immunoassay for detection of ruminant by-product material in animal feeds and feed ingredients.

An immunoassay with a lateral flow format has been developed for the detection of ruminant by-product material in animal feeds and feed ingredients. The test is designed for the analysis of animal feeds destined for feeding to ruminants to ensure that they do not contain ruminant by-products in violation of the ruminant-to-ruminant feed ban established by the U.S. Food and Drug Administration in 1997. This feed ban was established as a firewall against exposure of ruminant livestock animals to the prion agents responsible for neurological diseases such as bovine spongiform encephalopathy and scrapie. The test is designed for field use, e.g., at a feed mill, and yields a qualitative (presence/absence) result in 15-20 min. The objective of the study was to validate the lateral-flow test for detection of ruminant by-product material in a variety of finished animal feeds and feed ingredients. Results indicate that the test is specific for ruminant material and can detect as little as 1% ruminant material in these commodities.