Comparative Evaluation of LAMP, qPCR, Conventional PCR, and ELISA to Detect Ralstonia solanacearum in Kenyan Potato Fields.

Bacterial wilt caused by Ralstonia solanacearum is considered among the most damaging diseases of potato in Sub-Saharan Africa and the most significant biotic constraint of potato production alongside late blight. Unlike late blight, which can be managed by chemical means, R. solanacearum can only be managed through cultural methods and clean seed. Laboratory testing to certify seed before planting is required to confirm the absence of the pathogen in Kenya. A loop-mediated isothermal amplification (LAMP) assay was developed using the UDP-(3-O-acyl)-N-acetylglucosamine deacetylase gene (IpxC) to screen seed potato for R. solanacearum strains. The assay was assessed using DNA extracted from R. solanacearum and other soil and potato pathogens to demonstrate specificity and sensitivity. The LAMP assay was validated using field samples from different potato growing regions of Kenya collected over two growing seasons and compared with established nucleic acid and protein-based assays. The IpxC LAMP assay was found to be specific and sensitive to R. solanacearum, detecting as low as 2.5 pg/µl of R. solanacearum DNA. Of the 47 potentially infected field samples collected, both IpxC LAMP and quantitative polymerase chain reaction (PCR) detected R. solanacearum DNA in 90% of the samples, followed by conventional PCR (86%) and ELISA (75%). This IpxC LAMP assay is a promising diagnostic tool to rapidly screen for R. solanacearum in seed potato with high sensitivity in Kenya. Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Comparison of Methods Study between a Photonic Crystal Biosensor and Certified ELISA to Measure Biomarkers of Iron Deficiency in Chronic Kidney Disease Patients.

The total analytical error of a photonic crystal (PC) biosensor in the determination of ferritin and soluble transferrin receptor (sTfR) as biomarkers of iron deficiency anemia in chronic kidney disease (CKD) patients was evaluated against certified ELISAs. Antigens were extracted from sera of CKD patients using functionalized iron-oxide nanoparticles (fAb-IONs) followed by magnetic separation. Immuno-complexes were recognized by complementary detection Ab affixed to the PC biosensor surface, and their signals were followed using the BIND instrument. Quantification was conducted against actual protein standards. Total calculated error (TEcalc) was estimated based on systematic (SE) and random error (RE) and compared against total allowed error (TEa) based on established quality specifications. Both detection platforms showed adequate linearity, specificity, and sensitivity for biomarkers. Means, SD, and CV were similar between biomarkers for both detection platforms. Compared to ELISA, inherent imprecision was higher on the PC biosensor for ferritin, but not for sTfR. High SE or RE in the PC biosensor when measuring either biomarker resulted in TEcalc higher than the TEa. This did not influence the diagnostic ability of the PC biosensor to discriminate CKD patients with low iron stores. The performance of the PC biosensor is similar to certified ELISAs; however, optimization is required to reduce TEcalc.

A Direct Assay for Measuring Free 25-Hydroxyvitamin D.

Recent studies suggest that the concentration and genotype of vitamin D binding protein (VDBP) are important factors that determine the bioavailability of 25-hydroxyvitamin D [25(OH)D] in blood. Accumulating data indicate that, e.g., in pregnant women, hemodialysis patients, chronic kidney disease, liver failure, and bladder and pancreatic cancers, the measurement of free 25(OH)D in serum provides more relevant diagnostic information than measurement of total 25(OH)D. The aim of this study was to develop and validate an ELISA for direct measurement of free 25(OH)D in serum. A simple and direct ELISA was developed, based on a two-step immunoassay procedure performed in a microtiter plate. The assay has been characterized in terms of precision (4-10% CV, according to concentration), sensitivity (limits of blank = 0.5-1.0 pg/mL and LODs = 1.3-1.8 pg/mL), accuracy (correlation to dialysis, ELISA = 0.99xdialysis-0.5 pg/mL, r2 = 0.74), cross-reactivity of the antibody for the D2 form (77%), and addition of both VDBP and albumin (35-38% recovery upon addition of VDBP, 53-58% upon addition of albumin). The assay has already been used in multiple studies, including its comparison with calculation methods and in studies of patients with liver failure, different ethnic groups, supplemented mice, respiratory diseases, and obesity. The free 25(OH)D ELISA can be used in studies as a valuable tool to establish the clinical relevance of free 25(OH)D.

Sex-specific and inter-individual differences in biomarkers of selenium status identified by a calibrated ELISA for selenoprotein P.

Selenoprotein P (SELENOP) is a liver-derived transporter of selenium (Se) in blood, and a meaningful biomarker of Se status. Se is an essential trace element for the biosynthesis of enzymatically-active selenoproteins, protecting the organism from oxidative damage. The usage of uncalibrated assays hinders the comparability of SELENOP concentrations and their pathophysiological interpretation across different clinical studies. On this account, we established a new sandwich SELENOP-ELISA and calibrated against a standard reference material (SRM1950). The ELISA displays a wide working range (11.6-538.4µg/L), high accuracy (2.9%) and good precision (9.3%). To verify whether SELENOP correlates to total Se and to SELENOP-bound Se, serum samples from healthy subjects and age-selected participants from the Berlin Aging Study II were analyzed by SELENOP-ELISA and Se quantification. SELENOP was affinity-purified and its Se content was determined from a subset of samples. There was a high correlation of total Se and SELENOP concentrations in young and elderly men, and in elderly women, but not in young women, indicating a specific sexual dimorphism in these biomarkers of Se status in young subjects. The Se content of isolated SELENOP was independent of sex and age (mean±SD: 5.4±0.5). By using this calibrated SELENOP-ELISA, prior reports on pathological SELENOP concentrations in diabetes and obesity are challenged as the reported values are outside reasonable limits. Biomarkers of Se status in clinical research need to be measured by validated assays in order to avoid erroneous data and incorrect interpretations, especially when analyzing young women. The Se content of circulating SELENOP differs between individuals and may provide some important diagnostic information on Se metabolism and status.

Development, validation, and application of ELISA for detection of anti-HD105 antibodies in pre-clinical safety evaluation using monkeys.

Unwanted immunogenicity of protein therapeutics can result in severe side effects and should be assessed in animals before applying the treatment to humans. Monkeys are the most relevant choice for pre-clinical toxicity testing of antibody-based therapeutics. To assess the immunogenicity of HD105, a novel antibody therapeutic that targets both vascular endothelial growth factor and Delta-like-ligand 4, a bridging enzyme-linked immunosorbent assay was developed as an anti-drug antibody (ADA) assay and validated for use in pre-clinical studies using non-human primates. This method was found to have suitable assay sensitivity, intra- and inter-assay precision, confirmation, drug tolerance, recovery, and sample stability for measuring ADA in monkey serum samples. The results showed that ADA elevation occurred following repeated doses of HD105, and that ADA production was negatively associated with serum HD105 concentration. These results suggest that intravenous administration of HD105 induces production of ADA in monkeys and that the detection of ADA may be negatively influenced by free HD105 in serum.

Comparison of coprological, immunological and molecular methods for the detection of dogs infected with Angiostrongylus vasorum before and after anthelmintic treatment.

Timely diagnosis of the nematode Angiostrongylus vasorum in dogs is important in view of severe and permanent lung and cardiovascular lesions that may occur. The performance of the classical Baermann coprological method was compared with ELISAs for the serological detection of circulating antigen and specific antibodies and with Polymerase chain reaction (PCR) performed on EDTA blood, feces and tracheal swabs of serial samples from experimentally inoculated dogs over 13 weeks post inoculation (wpi) (n = 16) and following anthelmintic treatment (n = 6). Patency was observed from 6.7 to 7.6 wpi in all dogs, Baermann results were then mostly positive (116/119, 97%) during the patent period, with wide variations in the numbers of first stage larvae numbers. Blood PCR was tested positive on 1-2 occasions in 11/16 dogs in the pre-patent period, while all tested positive by antibody-detection ELISA by 6 wpi. The proportion of dogs testing positive by fecal PCR and antigen-detection ELISA rose early in the patent period. Tracheal swabs were occasionally DNA-positive in 3/16 dogs starting from 10 wpi. Following treatment, larval excretion stopped within 3 weeks and blood PCR results became negative within 1 week (5/6 dogs), while 4/6 dogs were positive for parasite DNA in tracheal swabs. Parasite antigen and specific antibodies both persisted in the blood for 3-9 weeks after treatment, with average optical densities and the proportion of positive dogs falling gradually, while results using other tests were much more variable. Results indicate that the earliest and most consistent results are obtained by the ELISAs, which can also be used for monitoring dogs after anthelmintic treatment.

Enzyme-linked immunosorbent assay by enhanced chemiluminescence detection for the standardization of estrogenic miroestrol in Pueraria candollei Graham ex Benth.

Miroestrol (ME) is a potent phytoestrogen from the P. candollei tuberous root. It has been approved for use in clinical trials due to its beneficial effect on disorders associated with estrogen deficiency. To ensure medical efficacy and safety, high performance analytical methods for ME analysis are required to standardize products from the P. candollei root. An enhanced chemiluminescence enzyme-linked immunosorbent assay (ECL-ELISA) was developed and validated using a polyclonal antibody against ME and a chemiluminescent system of luminol-H2 O2 -horseradish peroxidase-4-(1-imidazolyl) phenol. The ECL-ELISA system exhibited linearity over a concentration range of 0.31-10.00 ng mL(-1) , for which the relative standard variation (%RSD) was less than 10% for both intra- and interplate determinations. The ECL-ELISA is reliable for the determination of ME as reflected by the high recovery percentage (101.22-103.06%). As a comparative analysis, the ME content in each sample determined by ECL-ELISA was correlated with high coefficients of determination with colorimetric ELISA (R(2) = 0.998) and high performance liquid chromatography (HPLC) (R(2) = 0.998) methods. The ECL-ELISA method could be applied to all of the commercial products containing P. candollei root, when the products contain between 0.706 ± 0.046 and 13.123 ± 0.794 µg g(-1) dry wt. of ME. This method is useful as a high performance analytical method for the quantity control of ME in raw materials and end products at both the research and industrial levels.

High performance enzyme-linked immunosorbent assay for determination of miroestrol, a potent phytoestrogen from Pueraria candollei.

Pueraria candollei associated preparation is widely applied in folk Thai medicine for rejuvenating purpose in aged people, which correlated with its pharmacological activities reported by pre-clinical and clinical trials. Therefore, standardized products of this plant are needed by consumers and health care personnel. Miroestrol, a potent and stable phytoestrogen in P. candollei, exhibited potential to be biomarker for quality control of P. candollei samples in research or industrial levels. Indirect competitive enzyme-linked immunosorbant assay (ELISA) for miroestrol determination was developed and validated by using polyclonal antibody from rabbit immunization. The polyclonal antibody recognized specifically to miroestrol, which exhibited cross-reactivity to deoxymiroestrol and isomiroestrol with 6.68% and 1.05%, respectively. The linearity range of measurement was 0.73-3000 ng mL(-1), which coefficient of variation (CV) of both intra- and inter-plate determination was less than 5%. With spiked samples of known amount miroestrol, the percentages of recovery were 98.80-104.37% and 98.31-106.69% in P. candollei and its involved product samples, respectively. Validated ELISA was comparable with published HPLC method (R(2)=0.9996) (Yusakul et al.) in samples with various miroestrol contents. For application, the P. candollei involved preparations contained miroestrol 0.695±0.037-12.108±0.285 µg g(-1) dry wt. The developed ELISA was high performance for miroestrol determination, which could be applied for P. candollei quality control in research fields and industrial productions.

Development of an indirect competitive enzyme-linked immunosorbent assay (icELISA) using highly specific monoclonal antibody against paclitaxel.

Paclitaxel, the major active component of the yew tree, is used as an important anti-cancer agent. To obtain the monoclonal antibody (MAb) against paclitaxel for paclitaxel determination using immunoassay, 7-xylosyltaxol was conjugated to the carrier protein bovine serum albumin (BSA) to construct the immunogen, and the ratio of hapten in XylTax-BSA conjugate was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. After immunization of mice with this conjugate, hybridomas secreting MAbs against paclitaxel were obtained by fusing the splenocytes with the mouse myeloma cell line SP2/0. After hybridoma screening, the anti-paclitaxel MAb 3A3 was obtained, which showed a relatively high specificity to paclitaxel (cross-reactivities against other naturally occurred taxanes: 7-xylosyltaxol, 31.8%; cephalomannine, 6.17%; baccatin III, 10-deacetyl-baccatin III, 1-hydroxybaccatin I, 13-acetyl-9-dihydrobaccatin III and 1-acetoxyl-5-deacetyl-baccatin I, <0.11%). Using the MAb 3A3, we established an indirect competitive enzyme-linked immunosorbent assay (icELISA) for paclitaxel determination with a detection range of 0.098-312.5 µg ml(-1). Determination of paclitaxel contents in various yew tree samples with this icELISA resulted in recovery rates ranging from 92 to 94.8%, and intra- and inter-assay variations of 3.6 and 4.7%, respectively. This icELISA provides a valuable method of paclitaxel determination for various purposes.

Utilizing design of experiments to characterize assay robustness.

BACKGROUND: Design of experiments (DOE) is a systematic approach to assess the effects of many factors on a response of an assay. This paper provides a case study whereby DOE was successfully utilized to evaluate robustness parameters for a ligand-binding assay (LBA). METHODOLOGY: A 24-run Plackett-Burman design was developed to investigate factors that may have caused a lack of robustness in this particular LBA. We modeled five main effects and their ten two-way interactions, using the standard curve signal as the response. RESULTS: By utilizing DOE, we were able to quickly identify the factors that affected our assay's performance. The lack of robustness was attributed to the handling of the coat reagent. Factors that had an adverse effect on the coat material were vortexing and freeze-thaw cycles. CONCLUSION: We recommend that a robustness DOE be conducted prior to the validation of an assay for early identification of critical factors that may impact assay performance.

[Postcolostral immunoglobulin G (IgG) levels in newborn piglets using three different ELISA detection systems]. / Untersuchungen zur postkolostralen Immun-globulin-G-Versorgung neugeborener Saugferkel mittels unterschiedlicher ELISA-Nachweissysteme.

UNLABELLED: Objective of the study was to evaluate whether the Colostrum Quality Counter (CQC), a new test method for immunoglobulin G (IgG) levels in newborn piglets, is easy to handle and provides comparable results to established testing regimes. MATERIAL AND METHODS: Blood samples from 219 piglets from four different farms were tested for their IgG-concentrations using three different ELISA tests. Furthermore, double samples from 30 piglets were taken from both the anterior vena cava and from the tail to determine whether the collection site affects the results. The three tests used were the Colostrum Quality Counter (CQC; FarmulaONE, NL-Best), the internal IgG-ELISA from our laboratory (MUC) and a commercially-available IgG-ELISA (NAT; NatuTec, Frankfurt/Main, Germany). RESULTS: MUC and NAT showed a higher correlation to each other than to the CQC when referring to the individual results per single piglet. The results from the CQC were higher and the standard deviation was significantly greater. The sampling site had no significant effect on the IgG concentrations measured. CLINICAL RELEVANCE: The CQC is a straightforward and simple test, being very convenient for sampling a large number of piglets. CQC results were inhomogeneous with some unusually high IgG-concentrations. MUC and NAT provided comparable results to one another and the IgG-concentrations showed a good correlation.

Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.

Development of heterologous enzyme linked immunosorbent assay for dehydroepiandrosterone in serum.

Homologous and heterologous combinations of enzyme conjugate with immunogen in steroid enzyme immunoassay (EIA) influence labeled steroid recognition by an antibody that affects the sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) enzyme linked immunosorbent assay (ELISA), antibodies were generated against dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) and dehydroepiandrosterone-7-carboxymethyloxime- bovine serum albumin (DHEA-7-CMO-BSA). Two dehydroepiandrosterone (DHEA) horse radish peroxidise (HRP) enzyme conjugates were prepared using two dehydroepiandrosterone derivatives (DHEA-17-CMO and DHEA-7-CMO). Four combinations of homologous and heterologous assays were evaluated. The use of heterologous combination improved the sensitivity of the assay.

Development of a sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the antimalaria active ingredient artemisinin in the Chinese herb Artemisia annua L.

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the Chinese medicinal plant Artemisia annua L. It has been widely used in South-East Asia and Africa as an effective drug against sensitive and multidrug-resistant Plasmodium falciparum malaria. A monoclonal antibody (mAb), designated as 3H2, was generated with artesunate-bovine serum albumin conjugate as the immunogen. mAb 3H2 was used to develop a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) for artemisinin. The concentration of analyte producing 50% of inhibition (IC(50)) and the working range of the icELISA were 1.3 and 0.2-5.8 ng/mL, respectively. The mAb 3H2 recognized the artemisinin analogs artesunate, dihydroartemisinin, and artemether with cross-reactivity of 650%, 57%, and 3%, respectively, but negligibly recognized deoxyartemisinin and the artemisinin precursors arteannuin B and artemisinic acid. The average recoveries of artemisinin fortified in A. annua samples at concentrations from 156 to 5,000 microg/g determined by icELISA ranged from 91% to 98%. The icELISA was applied for the determination of artemisinin in different wild A. annua samples and the results were confirmed by high-performance liquid chromatography (HPLC) analysis. The correlation coefficient (R(2)) between the two assays was larger than 0.99, demonstrating a good agreement between the icELISA and HPLC results. This ELISA is suitable for quality assurance of A. annua L. materials.

Biomarker method validation in anticancer drug development.

Over recent years the role of biomarkers in anticancer drug development has expanded across a spectrum of applications ranging from research tool during early discovery to surrogate endpoint in the clinic. However, in Europe when biomarker measurements are performed on samples collected from subjects entered into clinical trials of new investigational agents, laboratories conducting these analyses become subject to the Clinical Trials Regulations. While these regulations are not specific in their requirements of research laboratories, quality assurance and in particular assay validation are essential. This review, therefore, focuses on a discussion of current thinking in biomarker assay validation. Five categories define the majority of biomarker assays from 'absolute quantitation' to 'categorical'. Validation must therefore take account of both the position of the biomarker in the spectrum towards clinical end point and the level of quantitation inherent in the methodology. Biomarker assay validation should be performed ideally in stages on 'a fit for purpose' basis avoiding unnecessarily dogmatic adherence to rigid guidelines but with careful monitoring of progress at the end of each stage. These principles are illustrated with two specific examples: (a) absolute quantitation of protein biomarkers by mass spectrometry and (b) the M30 and M65 ELISA assays as surrogate end points of cell death.

Detection and quantification of soy allergens in food: study of two commercial enzyme-linked immunosorbent assays.

The objective of this study was to evaluate the efficacy of 2 commercially available soy enzyme-linked immunosorbent assays (ELISA) and use them in detecting soy proteins in selected food commodities. Both ELISA kits exhibited high sensitivity. The determined limits of detection (LOD) (approximately 2 and <1 microg/mL for Tepnel Biosystems and Elisa Systems kits, respectively) were lower than those claimed by the manufacturer. Quantification range for both kits was, however, narrower and in a lower concentration range than defined by the kit providers. Our examination revealed a positive cross-reactivity with chickpea proteins and matrix interferences for both kits. The immunoreactivity of soy proteins, when tested by the Tepnel Biosystems kit, was partially reduced by papain and bromelain hydrolysis; it was significantly decreased by protein glycation (>47%). Nondenatured and nonheated soy protein isolate (SPI) samples were also significantly less antigenic than the treated ones.

Validation of an ELISA test kit for the detection of ochratoxin A in several food commodities by comparison with HPLC.

An ELISA Microtiter Plate, Ochratoxin Test called AgraQuant was validated to measure ochratoxin A in a range from 2 to 40 ppb in corn, milo, barley, wheat, soybeans and green coffee. The test is performed as a solid phase direct competitive ELISA using a horseradish peroxidase conjugate as the competing, measurable entity. For the test method, ochratoxin A is extracted from ground samples with 70% methanol and sample extracts plus conjugate are mixed and then added to the antibody-coated microwells. After 10 min incubation at room temperature, the plate is washed and enzyme substrate is added and allowed to incubate for an additional 5 min. Stop solution is then added and the intensity of the resulting yellow color is measured optically with a microplate reader at 450 nm. Results obtained from internal validation studies assessing accelerated stability indicate a 1 year shelf life; accuracy and precision are comparable to HPLC from 0 to 80 ppb and limit of detection in corn is 1.9 ppb and other food commodities is up to 3.8 ppb. Comparison of the method to HPLC, ability to detect individual ochratoxins, and ruggedness of the test kits determined this test to be rugged from 18 to 30 degrees C, sensitive, accurate, precise and effective comparable to HPLC for measuring ochratoxin A ranging from 2 to 40 ppb in several commodities.

Development and validation of a sensitive, specific, and rapid hybridization-ELISA assay for determination of concentrations of a ribozyme in biological matrices.

Ribozymes are RNA or modified RNA polymers capable of catalyzing cleavage reactions in target strands RNA, and are under development as human therapeutics. Previous methods used for quantitation of nucleic acid polymers in serum or plasma required extraction of the polymer followed by capillary electrophoresis, HPLC, or gel electrophoresis. These methods are time consuming and lack sensitivity. A bioanalytical method has been developed that does not require extraction of the ribozyme analyte from serum. This technique relies on hybridization of the ribozyme molecule to two complementary biotin and digoxigenin labeled oligonucleotide probes. Serum containing the ribozyme is mixed with the labeled probes, and the mixture is heated at 75 degrees C for 5 min to disrupt the ribozyme secondary structure. Samples are then cooled to permit probe annealing and are added to a streptavidin-coated 96-well plate. The bound complex is detected with an anti-digoxigenin alkaline phosphatase (AP) conjugate using PNPP (p-nitrophenyl phosphate) as a substrate. The amount of colored product is measured on a microtiter plate reader at a wavelength of 405 nm. Concentrations of unknown ribozyme samples are estimated based on a standard curve (0.37-270 ng/ml) prepared in serum. The validated lower and upper limits of quantification are 5.0 and 120 ng/ml, respectively. The assay can be completed in approximately 5h and does not require extraction procedures or electrophoretic/chromatographic separation. It is therefore a simple, sensitive and rapid technique. This assay has been validated and has been used for quantitation of serum levels of the HEPTAZYME ribozyme in mouse, monkey, and human pharmacokinetic studies.

[Inter-laboratory evaluation studies for establishment of notified ELISA methods for allergic substances (buckwheat)].

Inter-laboratory evaluation studies were conducted for the ELISA methods for allergic substances (buckwheat). Extracts of snack, bun and udon spiked with buckwheat standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate at 10 laboratories. Coefficients of variation (CVs) of the ELISA methods using a Buckwheat Protein ELISA Kit (Buckwheat kit) and a FASTKIT Buckwheat ELISA kit (Buckwheat ELISA kit) were mostly below 10%. Mean recoveries of the buckwheat standard protein from the food extracts were over 40% in the two ELISA methods. Repeatability relative standard deviations of buckwheat standard protein in three food extracts were in the ranges of 6.8-78.5% and 5.0-33.9% for the Buckwheat kit and the Buckwheat ELISA kit, respectively. Reproducibility relative standard deviations of buckwheat standard protein in three food extracts were 11.9-69.5% and 16.5-34.1% for the Buckwheat kit and the Buckwheat ELISA kit, respectively. The detection limits of both ELISA methods were 1 ng/mL in sample solutions. These results suggest that the notified ELISA methods are reliable and reproducible for the inspection of buckwheat protein levels in extracts of snack, bun and udon.