Validation of the Reveal® 3-D for Gluten Assay for Detection of Gluten in Clean-in-Place Rinses and Stainless Steel Environmental Surfaces: AOAC Performance Tested MethodSM 122201.

BACKGROUND: Reveal® 3-D for Gluten is an immunochromatographic assay for the qualitative detection of gluten in environmental samples. The test uses monoclonal antibodies reactive to prolamins in wheat. OBJECTIVE: The objective of the study was to validate the Reveal 3-D test for detection of gluten in clean-in-place rinse and swabs from a stainless steel surface. METHODS: Elements of the study included food selectivity and interference testing, matrix testing, an assay robustness study, and reagent stability/lot-to-lot consistency testing. Wheat flour was used as the spiking material for all matrixes. RESULTS: In selectivity and interference testing, nine target matrixes all tested positive and 36 of 39 non-target matrixes tested negative. Almond flour, sesame flour, and cornstarch produced positive results as 100% commodities; reactivity can be eliminated with dilution or by testing without use of food extraction buffer, which is not a standard part of the environmental testing method. With a gluten spike at 9.3 mg/kg, chestnut flour, guar gum, and xanthan gum as 100% commodities inhibited the ability of the assay to detect gluten when tested without dilution. In quaternary ammonium clean-in-place rinse and swabs from stainless steel, 100% positive results were obtained at levels of 2.8 mg/kg and 4.7 µg/100 cm2, respectively. Results of independent laboratory testing of swabs from stainless steel supported those of internal trials. Robustness testing showed that introducing variations to three operating parameters simultaneously had no adverse effect on assay performance. In the reagent stability study, data supported kit expiration dating of 11 months. CONCLUSION: Results of the current study show that the Reveal test is an accurate and reliable method for qualitative detection of gluten in select clean-in-place rinse and environmental samples. HIGHLIGHTS: The Reveal test was able to detect gluten at levels of 2.8 ppm in clean-in-place rinse and 4.7 µg/100 cm2 in swabs from stainless steel.

Validation of the Soleris® Coliform Method for Detection of Coliform Bacteria in Dried Cannabis Flower: AOAC Performance Tested MethodSM 010302.

BACKGROUND: The Soleris® Coliform Vial is a growth-based, automated method for detection of coliform bacteria in foods and other consumer products such as nutraceuticals and cosmetics. The method was granted AOAC Performance Tested MethodSM certification for select foods after successful completion of a validation study. OBJECTIVE: The objective of the current study was to validate the Soleris coliform method for use with dried cannabis flower (>0.3% delta 9-tetrahydrocannabinol). METHODS: A comparative matrix study was performed in which naturally contaminated dried cannabis flower was tested with the Soleris coliform method and with the U.S. Food and Drug Administration Bacteriological Analytical Manual solid medium method. Multiple lots of dried cannabis flower were obtained, pre-screened for coliforms, and blended to produce test materials at four different contamination levels ranging from 4.5 to 1600 CFU/g. Each material was tested at three different Soleris detection threshold levels determined by the dilution used to inoculate the Soleris vials. Probability of detection analysis was performed to determine if differences in the number of positive results obtained with the two methods were significant. RESULTS: For all four dried cannabis flower materials, at all three Soleris test thresholds, there were no significant differences in the number of positive results obtained with the Soleris and cultural plating methods as determined by probability of detection analysis at P < 0.05. CONCLUSION: The Soleris coliform test is an accurate method for detection of coliform bacteria in dried cannabis flower. HIGHLIGHTS: The Soleris coliform method provides cannabis industry QC personnel with an effective method for analysis of dried cannabis flower and produces results in 18-24 h.

Validation of the Soleris® NF-TVC Method for Detection of Aerobic, Mesophilic Microorganisms in Dried Cannabis Flower: AOAC Performance Tested MethodSM 071203.

BACKGROUND: Soleris® Non-Fermenting Total Viable Count (NF-TVC) is a growth-based, automated method for semiquantitative detection of aerobic, mesophilic microorganisms in foods and other consumer products such as nutraceuticals and cosmetics. The method was granted AOAC Performance Tested MethodSM status for select foods after successful completion of a validation study. OBJECTIVE: The objective of the current study was to validate the Soleris NF-TVC method for use with dried cannabis flower [>0.3% delta 9-tetrahydrocannabinol (THC)]. METHODS: The validation consisted of a comparative matrix study in which naturally contaminated dried cannabis flower was tested with the Soleris NF-TVC method and with the AOAC Official Methods of AnalysisSM  966.23 dilution plating method. Multiple lots of dried cannabis flower were obtained, pre-screened for total aerobic, mesophilic viable count levels, and blended to produce test materials at four different levels of contamination ranging from 1.0 × 103 to 2.2 × 105 CFU/g. Each material was tested at three different Soleris detection threshold levels determined by the dilution used to inoculate the Soleris vials. Probability of detection analysis was performed to determine if differences in the number of positive results obtained with the two methods were significant. RESULTS: For all four dried cannabis flower materials, at all three Soleris test thresholds, there were no significant differences in performance comparing the Soleris and reference dilution plating methods as determined by probability of detection analysis at P < 0.05. CONCLUSIONS: It is concluded that the Soleris NF-TVC method is an accurate and effective method for detection of aerobic, mesophilic microorganisms in dried cannabis flower. HIGHLIGHTS: The Soleris NF-TVC method provides cannabis industry quality control personnel with an effective method for analysis of dried cannabis flower and produces results in 24-48 h.

Validation of the Soleris®Enterobacteriaceae Method for Detection of Enterobacteriaceae in Dried Cannabis Flower: AOAC Performance Tested MethodSM 121901.

BACKGROUND: The Soleris®Enterobacteriaceae vial is a growth-based, automated method for detection of bacteria of the family Enterobacteriaceae in foods and other sample types including nutraceuticals and cosmetics. The Soleris method is used in a "dilute-to-specification" or threshold manner, in which a result is scored as positive or negative around a predetermined cutoff (in CFU/g) established by the dilution and volume of sample homogenate tested. The Soleris method was granted AOAC Performance Tested MethodSM (PTM) status for select foods after successful completion of a validation study (PTM 121901). OBJECTIVE: The objective of this study was to validate the method for the detection of Enterobacteriaceae in dried cannabis flower [>0.3% delta-9-tetrahydrocannabinol (THC)]. METHODS: The matrix study included comparison of Soleris method presumptive results to confirmation from the Soleris vials, and comparison of the Soleris confirmed results to those of the ISO 21528-2:2017 colony count method. Test materials at four different levels of contamination ranging from 7.8 to 3500 CFU/g were tested at three dilutions, corresponding to test thresholds. RESULTS: Probability of detection analysis at P < 0.05 showed there were no significant differences between Soleris presumptive and confirmed results, and no significant differences between Soleris confirmed and ISO 21528-2:2017 results. CONCLUSION: The results provided evidence that the Soleris Enterobacteriaceae test is an accurate method for detection of Enterobacteriaceae in dried cannabis flower. HIGHLIGHTS: The Soleris Enterobacteriaceae method provides cannabis industry QC personnel with an effective method for analysis of dried cannabis flower and produces results in 20-24 h.

Emergency Response Validation of the Soleris® Direct Yeast and Mold Method for Detection of Yeast and Mold in Dried Cannabis Flower: AOAC Performance Tested MethodSM 051301.

BACKGROUND: Soleris® Direct Yeast and Mold (DYM) is a growth-based, automated method for detection of yeast and mold in select foods and other sample types including nutraceuticals and cosmetics. The Soleris method is used in a "dilute-to-specification" or threshold manner in which a result is scored as positive or negative around a predetermined cutoff (in CFU/g) established by the dilution and volume of sample homogenate tested. OBJECTIVE: The objective of this study was to validate the method for testing of dried cannabis flower. The validation was conducted under the Emergency Response Validation program of the AOAC Research Institute. METHOD: The study included inclusivity and exclusivity testing, in particular testing of yeast and mold species associated with cannabis, and a matrix study in which Soleris method presumptive results were compared with Soleris confirmed results using Dichloran Rose-Bengal Chloramphenicol (DRBC) agar for confirmation. Samples at four different levels of natural yeast and mold contamination were tested at two test thresholds. RESULTS: In inclusivity testing, all 63 yeast and mold strains tested produced positive results within the specified test duration of 72 h. In exclusivity testing, 36 of 37 strains tested produced no detection within 72 h. In matrix testing, there were no significant differences between Soleris presumptive and confirmed results for any contamination level or test threshold as determined by probability of detection analysis. CONCLUSIONS: Results indicate that the Soleris method is an effective procedure for detection of yeast and mold in dried cannabis flower. HIGHLIGHTS: With the Soleris method, results are available within 72 h compared with the 5-7 days required for microbiological culture methods.

Validation of Modifications to the Soleris®E. coli Method for Detection and Threshold Determination of Escherichia coli in Select Foods: Level 3 Modification to AOAC Performance Tested MethodSM 101101.

BACKGROUND: Soleris®E. coli is an automated, growth-based method for detection and semi-quantitative determination of Escherichia coli in foods. The method can be used in dilute-to-specification (threshold) or presence/absence modes. OBJECTIVE: The objective of the study was to validate four modifications to the method: (1) a change in the vial detection window plug composition from agar to agarose to improve plug consistency and robustness, (2) a change in pre-enrichment incubation time for presence/absence testing from 6 h to 18-24 h, (3) a change in vial incubation temperature from 44.5 to 43.5°C, and (4) incorporation of a simple direct-from-vial confirmation test as an alternative to traditional procedures. METHODS: Elements of the study included inclusivity/exclusivity testing, matrix testing in comparison to the ISO 7251:2005 reference method, reagent stability/lot-to-lot consistency testing, and an independent laboratory study. RESULTS: In inclusivity testing, all 55 Escherichia coli strains tested produced positive results. In exclusivity testing, 30 of 31 strains of other bacterial species produced negative results, the sole exception being a strain of Enterobacter cloacae. In internal and independent laboratory matrix testing of mozzarella cheese, condensed milk, pasteurized liquid egg, and frozen green beans, results showed no significant differences in performance of the Soleris and reference methods with two exceptions, one in which the Soleris method produced more positive results, and one in which the reference method produced more positive results. CONCLUSION: Performance characteristics of the modified Soleris E. coli method are consistent with those of the original validated method. HIGHLIGHTS: The Soleris E. coli method offers improvements in ease of use and reagent robustness.

Validation of the One Broth One Plate for Salmonella Method for Detection of Salmonella Spp. in Select Food and Environmental Samples: AOAC Performance Tested MethodSM 102002.

BACKGROUND: One Broth One Plate for Salmonella (OBOP Salmonella) is a rapid and simple method for detection of Salmonella spp. in food and environmental samples using traditional culture methodology. The method utilizes single-step enrichment followed by plating to a selective/differential, chromogenic agar. OBJECTIVE: The purpose of the validation study was to measure the effectiveness of the OBOP Salmonella method in comparison to reference culture procedures. METHOD: Performance of the OBOP Salmonella method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5 reference method for queso fresco, smoked salmon, cantaloupe, chocolate, black pepper, chili powder, dry pet food, and sponge samples from a stainless steel surface, or to that of the U.S. Department of Agriculture Microbiology Laboratory Guidebook Chapter 4.10 method for raw ground turkey, chicken carcass rinse, and pasteurized liquid egg. Inclusivity/exclusivity, robustness, and stability/lot-to-lot consistency testing was also performed. RESULTS: In the matrix study, there were no statistically significant differences in performance between the OBOP Salmonella and reference methods, as determined by probability of detection analysis (P < 0.05), for any of the matrixes examined. All 104 Salmonella spp. strains produced positive results in inclusivity testing, and all 33 non-salmonellae exclusivity strains tested negative with the OBOP Salmonella method. CONCLUSIONS: Results of the validation study show that the OBOP Salmonella method is a reliable procedure for detection of Salmonella spp. in select matrixes. The method is simple to perform, requires no specialized equipment, and produces results in as little as 37 h. HIGHLIGHTS: The OBOP Salmonella method was awarded AOAC PTMSM (#102002) for detection of Salmonella in queso fresco, smoked salmon, cantaloupe, chocolate, black pepper, chili powder, dry pet food, sponge samples on a stainless steel surface, raw ground turkey, chicken carcass rinse, and pasteurized liquid egg. The method is also approved by MicroVal® for a broad range of foods under certification number 2019LR88.

Soleris®Enterobacteriaceae for the Detection of Enterobacteriaceae in Select Foods: AOAC Performance Tested MethodSM 121901.

BACKGROUND: Soleris®Enterobacteriaceae is a growth-based, automated method for detection of Enterobacteriaceae in food. OBJECTIVE: A study was conducted to validate the Soleris method for detection of Enterobacteriaceae in select foods (pasteurized milk, yogurt, mozzarella cheese, ice cream, dried milk, pasteurized liquid egg, frozen cooked chicken, deli ham, lettuce, and dry dog food) at a threshold of ≥ 10 CFU/g of product. METHODS: Inclusivity and exclusivity of the Soleris method were assessed by testing 55 and 38 target and non-target bacterial strains, respectively. Matrix testing was performed with one naturally contaminated and nine inoculated foods. Efficacy of the Soleris method was compared to that of the ISO 21528-2:2017 direct plating reference method using probability of detection analysis. Independent laboratory testing was conducted to verify method performance in two matrixes (yogurt and deli ham). Method robustness, stability, and lot-to-lot consistency of the Soleris reagents were also assessed. RESULTS: Inclusivity of the Soleris test was 91% and exclusivity was 100%. In matrix testing, there were no significant differences in the number of positive results obtained with the Soleris and reference methods for any of the matrixes examined. Overall, of 370 test portions, there were 176 positive results by the Soleris method and 177 positive results by the reference procedure. CONCLUSIONS: Soleris Enterobacteriaceae is an effective method for detection of Enterobacteriaceae in the foods evaluated, with performance equivalent to that of the ISO 21528-2:2017 reference method. HIGHLIGHTS: The Soleris method offers the advantages of labor savings and results within 18 h.

Validation of the Reveal® 3-D for Peanut Lateral Flow Test: AOAC Performance Tested MethodSM 111901.

BACKGROUND: Reveal® 3-D for Peanut is an immunochromatographic, lateral flow test for qualitative detection of peanut residue in food manufacturing and food preparation settings. The test can detect low ppm levels of peanut in clean-in-place (CIP) rinses and in swabs from environmental surfaces and can serve as a tool in managing allergen risk. OBJECTIVE: The objective of the study was to validate the lateral flow method for detection of peanut in CIP rinses, specifically water, peroxyacetic acid/hydrogen peroxide, and quaternary ammonium compound rinses, and in swabs taken from stainless steel and plastic surfaces. METHODS: CIP rinses spiked with low levels of peanut were tested, as were surfaces inoculated with peanut. Specificity and assay interference were assessed in testing of food commodities with and without added peanut. Assay robustness and test kit stability and consistency testing were also performed. RESULTS: Results demonstrated that the lateral flow test can detect peanut in CIP rinses in the range of 2-4 ppm and in environmental surface swabs in the range of 3-4 µg/100 cm2. Results of specificity testing with 29 common food items showed lack of cross-reactivity, and potential assay interference only from walnut. Data from stability trials supports expiration dating for the kit of up to 23 months post-manufacture. CONCLUSIONS AND HIGHLIGHTS: The lateral flow test is a sensitive, specific, and rapid method for detection of low levels of peanut residue in CIP rinses and environmental samples and can be an important component in a comprehensive allergen risk management program.

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.

NeoSeekTM STEC: A Multiplex Molecular Method for Detection and Identification of Select Shiga Toxin-Producing Escherichia coli in Beef.

BACKGROUND: NeoSeekTM STEC is a single-source, service-based method for detection and identification of select Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7 and STEC of somatic groups O26, O45, O103, O111, O121, and O145. The method is a multiplex molecular method utilizing more than 80 genetic targets to identify STEC in complex matrices such as food enrichment cultures. OBJECTIVE: A study was conducted to validate the NeoSeek method for detection of select STEC in raw beef trim. METHODS: Performance of the NeoSeek STEC method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service reference methods for E. coli O157:H7 and non-O157 STEC for detection of E. coli O157:H7 and E. coli O26:H11 in raw beef trim. Additionally, inclusivity/exclusivity testing and method robustness testing were performed. RESULTS: Results of raw beef trim testing showed no statistically significant differences in performance between the NeoSeek and reference methods in the ability to detect either E. coli O157:H7 or E.coli O26:H11, as determined by probability of detection analysis. Results of inclusivity and exclusivity testing showed 100% expected results with target and nontarget bacteria, with the exception of a single strain of E. coli O157:H7, which was subsequently verified to be stx-negative by PCR. Conclusions and Highlights: NeoSeek STEC is an accurate, reliable method for rapid detection and identification of select STEC in complex populations such as beef trim enrichment cultures.

Matrix Extension Study: Listeria Right Now™ Test for Detection of Listeria spp. from Selected Environmental Surfaces Without Enrichment: AOAC Performance Tested MethodSM 081802.

BACKGROUND: Listeria Right Now™ is a novel, enrichment-free test for the detection of Listeria spp. in swab samples taken from environmental surfaces. Results are available in less than 1 h. In a previous Performance Tested MethodSM (PTM) study, the test was validated for swab samples from stainless-steel and sealed concrete surfaces. OBJECTIVE: A PTM matrix extension study was conducted to validate the method for the detection of Listeria spp. in swab samples from ceramic tile, plastic, and rubber surfaces. METHODS: Performance of the Listeria Right Now method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedure for the detection of Listeria spp. in swab samples taken from inoculated ceramic tile, plastic, and rubber surfaces. Data were analyzed using a probability of detection model. RESULTS: There were no significant differences in performance between the Listeria Right Now and reference culture methods for any of the three surfaces tested, as determined by probability of detection analysis. CONCLUSIONS: The Listeria Right Now method is an effective procedure for the detection of Listeria spp. from a variety of environmental surfaces. HIGHLIGHTS: Listeria Right Now provides accurate results, without enrichment, in real time. This enables food industry personnel to react swiftly to suspected Listeria contamination incidents.

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 Listeria Right NowTM Test for Detection of Listeria spp. from Selected Environmental Surfaces Without Enrichment.

Background: Listeria Right NowTM is a novel, enrichment-free molecular method for detection of Listeria spp. in swab samples from environmental surfaces. The test provides results in real time, indicating the current or recent presence of Listeria spp. in the environment. After sampling, the entire contents of the swab are subject to sample processing, releasing large quantities of target ribosomal RNA molecules into the lysate. A portion of the lysate is then tested using the ANSR® for Listeria isothermal nucleic acid amplification assay. Objective: A Performance Tested MethodSM study was conducted to validate the method for detection of Listeria spp. in swab samples from stainless steel and sealed concrete surfaces. Methods and Results: In inclusivity testing, 60 of 60 Listeria spp. strains tested positive. In exclusivity testing, 31 of 31 nontarget bacterial strains tested negative. In LOD testing, the test was able to detect as few as 2 CFU of L. monocytogenes applied to a stainless steel surface. In matrix testing of inoculated stainless steel and sealed concrete surfaces, there were no statistically significant differences in method performance comparing the Listeria Right Now and U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedures as determined by probability of detection analysis. In robustness testing, modest changes to three assay operating parameters simultaneously did not significantly affect performance of the test. Conclusions and Highlights: Results can be obtained in less than 1 h using the Listeria Right Now test, allowing food industry personnel to take immediate corrective action in the case of Listeria contamination incidents.

Validation of a Modified ANSR® for E. coli O157:H7 Method for Detection of E. coli O157:H7 in Select Foods.

Background: The ANSR method is based on isothermal nucleic acid amplification technology. The modifications to assay components improve sensitivity of the assay and robustness of the internal positive control. Objective: A Performance Tested MethodSM validation study was conducted to assess performance of a modified version of the ANSR® for Escherichia coli O157:H7 method. Methods: The validation study included inclusivity/exclusivity, matrix, robustness, accelerated stability, and independent laboratory testing. Results: In inclusivity testing of 55 strains of E. coli O157:H7 and E. coli O157:NM variants, all strains produced positive results. In exclusivity testing of 41 strains including E. coli of other serotypes and bacteria of closely related genera, all strains produced negative results. In matrix testing of beef trim, raw ground beef, spinach, and sprout-irrigation water, ANSR method performance was compared with that of the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook or the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedures. Conclusions: all trials, ANSR method performance was not statistically different from that of the reference methods. Results of independent laboratory testing of ground beef corroborated those of internal testing. Introducing modest changes to three assay operating parameters did not materially affect ANSR method performance. Finally, accelerated stability testing results of three independently manufactured lots of ANSR reagents support a shelf-life of 1 year when stored at 2-8°C.

Age, calorie restriction, and age of calorie restriction onset reduce maturation of natural killer cells in C57Bl/6 mice.

Calorie restriction (CR), also known as energy restriction, has been shown to have a deleterious impact on both adult and aged mouse survival during influenza virus infection. Natural killer (NK) cell phenotypic differences contribute to increased susceptibility of adult CR mice. We hypothesized NK cell phenotype from adult and aged C57Bl/6 mice fed NIH-31 diet ad libitum (AL) would be different from NK cell phenotype from adult and aged mice fed NIH-31/NIA fortified diet at 40% CR. We hypothesized NK cell phenotype from mice consuming 40% CR initiated at 20 months of age would not be different from 40% CR initiated at 3 months of age. We initiated the 40% restriction either at the standard 12 weeks of age or at 78 weeks of age. NK cells were isolated and quantified from various tissues using flow cytometry. Aged CR mice had significantly reduced levels of terminally mature (CD27-CD11b+) NK cells, increased expression of the immature marker CD127, and decreased expression of the mature marker DX5. Total number of NK cells among cells was significantly lower in the lung and spleen of old-onset aged CR mice compared to aged AL mice, while there was no significant difference between young-onset aged CR and aged AL mice. Old-onset aged CR mice had significantly less early mature (DX5+ and CD27+CD11b+) NK cells compared to young-onset aged CR and aged AL fed mice. Overall, we found that CR in aged mice is detrimental to maturation of NK cells, which is exacerbated when CR is initiated in old age.

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.

Short-term supplementation with active hexose correlated compound improves the antibody response to influenza B vaccine.

Administration of bioactive nutritional supplements near or at the time of immunization has been a recent approach to stimulate human immune response to vaccination. Active hexose correlated compound (AHCC), a mushroom extract, has been shown to protect mice against lethal primary influenza infection. Moreover, when AHCC was administered pre-vaccination in mice, they showed improved protection from lethal avian flu infection when compared to mice vaccinated alone. In this study, we hypothesized that AHCC will also improve the immune responses of healthy individuals to influenza vaccine. A randomized controlled study was performed with 30 healthy adults to evaluate the effects of AHCC supplementation on the immune response to the 2009-2010 seasonal influenza vaccine. Blood was drawn pre-vaccination and 3 wk post-vaccination. Immediately post-vaccination, the AHCC group began supplementation with AHCC (3 g/d). Flow cytometric analysis of lymphocyte subpopulations revealed that AHCC supplementation increased NKT cells (P < .1), and CD8 T cells (P < .05) post-vaccination compared to controls. Analysis of antibody production 3 weeks post-vaccination revealed that AHCC supplementation significantly improved protective antibody titers to influenza B, while the improvement was not significant in the control group. Overall, our study showed that AHCC supplementation improved some lymphocyte percentages and influenza B antibody titers over the control. Future studies are required to determine the kinetics of AHCC supplementation to improve the overall response to influenza vaccination.