Validation Of Enzyme-linked Immunosorbent Assays Research Articles

Development and validation of enzyme-linked immunosorbent assays for the measurement of infliximab and anti-drug antibody levels

Infliximab and its anti-drug antibody (ADA) serum concentrations exhibit a strong correlation with clinical response and loss of response. The use of therapeutic drug monitoring to measure the concentration of infliximab and ADA can facilitate clinical decision-making, helping patients attain optimal therapeutic effects. However, there are still limitations to the existing infliximab and its ADA detection methods. Therefore, this study aimed to develop and validate enzyme-linked immunosorbent assay (ELISA)-based methods for measuring infliximab and its ADA levels in human plasma according to the general recommendations for immunoassays. Free infliximab is bound by recombinant TNF-α and detected using HRP-labeled anti-human antibody. The ADA is captured by on-plate-coated infliximab and recognized by biotin-labeled infliximab. Two bridging ELISA assays were developed and after assay optimization and validation, these assays have been applied in ten patients with inflammatory bowel disease (IBD). In infliximab detection assay, a standard curve ranging from 0.10 μg/mL to 8.0 μg/mL with great precision and accuracy has been established. Drug tolerance of the ADA assay was that 100 ng/mL ADA could tolerate at least 5.0 μg/mL infliximab in the plasma using a commercially available monoclonal anti-infliximab antibody as the positive control. The ADA screening and confirmatory assays achieved a sensitivity of 36.74 ng/mL and 37.15 ng/mL, respectively. All other assay characteristics met the requirements. The mean concentration of infliximab in eight patients with IBD was 7.88 (1.87–21.1) μg/mL, and the ADA levels were all negative. Moreover, the concentrations of infliximab in the remaining two patients were below the LLOQ and the ADAs were positive. Thus, accurate and sensitive ELISA methods have been developed and validated for the detection of infliximab and its ADA concentrations and have been successfully applied to clinical therapeutic drug monitoring.

Development and validation of a specific sandwich ELISA for determination of soybean allergens and its application in processed foods

Soybean allergens are commonly quantified using enzyme-linked immunosorbent assays (ELISAs) with antibodies produced by the single allergen. To enhance the specificity and avoid the false-negative results, the sandwich ELISA method was developed using a rabbit anti-bulk soybean proteins polyclonal antibody and a goat anti- bulk soybean proteins polyclonal antibody as the detection and capture antibody, respectively. The proposed method has been optimized and validated according to the requirements of the AOAC Allergen Community. The method has a wide quantification range from 0.0625 μg/mL to 6.0 μg/mL and can quantify soybean residues at a low level down to 0.25 mg/kg with satisfactory accuracy (recovery range from 89.5% to 118%) and precision (RSDs < 16.67%). The new approach performs high specificity to soybean residues without obvious cross-reactivity to common allergic foods and legumes, and has been successfully applied to detect soybean proteins in commercial foods. Our data demonstrated that the established sandwich ELISA method has a higher specificity, accuracy, and robustness that could meet the quantification requirements for soybean allergens.

Development and validation of ELISAs for the quantitation of interleukin (IL)-1β, IL-6, IL-8 and IL-10 in ovine plasma

There is growing evidence that inflammation underpins many common diseases. Inflammatory/immunomodulatory/immune mediators, such as cytokines, are key modulators of inflammation and mediate both immune cell recruitment and complex intracellular signalling pathways. Ovine models of disease are increasingly utilized in pre-clinical research, however existing methods for measuring cytokine levels are limited. We established and validated enzyme-linked immunosorbent assays (ELISAs) targeting interleukin (IL)-1β, IL-6, IL-8 and IL-10 in sheep plasma. These ELISAs showed high sensitivity and specificity with intra- and inter-assay CV's below 10%, and recovery rates between 82 and 123%. Sensitivity for IL-1β, IL-6, IL-8 and IL-10 were 117.6 pg/mL, 443.1 pg/mL, 30.9 pg/mL, and 64.3 pg/mL, respectively. ELISA test result reproducibility decreased significantly after 12 weeks of plasma storage at −80 °C. Therefore, for accurate cytokine measurements, plasma samples need to be tested within three months of sample collection to account for cytokine protein degradation. These ELISAs offer a reliable and convenient method to identify inflammatory cytokine changes in sheep, allowing key insights into the disease pathogenesis of these ruminants.

Development and validation of a competitive ELISA based on bacterium-original virus-like particles of serotype O foot-and-mouth disease virus for detecting serum antibodies.

Foot-and-mouth disease (FMD) is a highly contagious disease that affects all susceptible cloven-hoofed animals, resulting in considerable economic losses to animal industries worldwide. Numerous categories of enzyme-linked immunosorbent assays (ELISA) have been developed and widely used to evaluate herd immunity. Manufacturing inactivated FMD virus (FMDV) as a diagnostic antigen requires a facility with a high level of biosafety, but this requirement raises concern on viral leakage. In our previous study, bacterium-original FMD virus-like particles (VLPs) resemble the authentic FMDV and induce protective immunity against homologous viral challenges, thereby demonstrating that they are sufficiently safe without limitations on biosafety facilities and easily prepared. Herein, we developed a competitive ELISA (cELISA) based on FMDV-VLPs as a diagnostic antigen to evaluate herd immunity. The criterion of this cELISA was determined by detecting panels of positive sera with different antibody titers and negative sera. The working parameter of cELISA was optimized, and samples with a percentage inhibition of ≥ 50% were considered positive. The specificity of cELISA to test 277 serum samples with various antibody titers was 100%, and the sensitivity reached 96%. The coincidence rates of cELISA with a VDPro® FMDV and a PrioCHECK® FMDV type O antibody ELISA kit were 97.8% and 98.2%, respectively. Repeatability tests demonstrated that the coefficients of variation within and between runs were less than 7% and 14%, respectively. Our data demonstrated that cELISA based on bacterium-original VLPs had high specificity, sensitivity, and reproducibility. The cELISA could also be used for evaluating vaccination herd immunity effects, especially in developing countries.

Validation of commercial ELISAs for quantifying anabolic growth factors and cytokines in canine ACD-A anticoagulated plasma.

Platelet-rich plasma has been studied extensively in dogs, but validation of enzyme-linked immunosorbent assays (ELISAs) for quantifying anabolic growth factors and inflammatory cytokines in canine plasma prepared with citrate-based anticoagulants is not available. We performed a validation of commercial ELISAs for transforming growth factor-beta 1 (TGF-β1), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β) for use with canine plasma prepared with acid-citrate-dextrose, solution A (ACD-A). Platelet-poor plasma (PPP) anticoagulated with ACD-A as well as PPP anticoagulated with ACD-A and spiked with the relevant canine recombinant proteins were evaluated with each ELISA to calculate the efficiency of spike recovery. Replicates of the spiked PPP were also assessed in 2 additional assays to quantify intra-assay and interassay precision. The efficiency of spike recovery was within 75-125% of the expected concentration for the TGF-β1, PDGF-BB, and VEGF ELISAs. The intra- and interassay variability were <25% for the TGF-β1, PDGF-BB, VEGF, and TNF-α ELISAs. The TGF-β1, PDGF-BB, and VEGF ELISAs demonstrate acceptable efficiency of spike recovery and intra- and interassay variability, whereas the TNF-α and IL-1β ELISAs did not meet industry standards of performance with ACD-A anticoagulated canine plasma.

Validation Parameters for Quantitating Specific Proteins Using ELISA or LC-MS/MS: Survey Results

Genetically modified organisms (GMOs) express one or more exogenous genes inserted through genetic engineering resulting in the production of novel protein(s). Agencies responsible for deregulating GMOs require that levels of the newly expressed protein(s) be characterized. Ideally, the quantitation methods used will be validated according to Good Laboratory Practices (GLPs) that are accepted by these global regulatory agencies. Targeted protein quantitation may be performed using enzyme-linked immunosorbent assays (ELISAs) with commercial kits or specially produced and validated antibodies. Proteins may also be quantitated by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Although some scientific guidance exists for validation of ELISA, the need remains for a harmonized set of protocols for protein quantitation in testing of GMOs, particularly for LC-MS/MS. We surveyed various industry experts to determine current practices for assay linearity, accuracy, and precision; assay specificity; detection limits; and methods to assess analyte stability for both ELISA and LC-MS/MS. We compared the survey results with assay criteria suggested in the literature and governmental guidance documents.

A Review of Cry Protein Detection with Enzyme-Linked Immunosorbent Assays.

The widespread use of Cry proteins in insecticide formulations and transgenic crops for insect control has led to an increased interest in the environmental fate of these proteins. Although several detection methods are available to monitor the fate of Cry proteins in the environment, enzyme-linked immunosorbent assays (ELISAs) have emerged as the preferred detection method, due to their cost-effectiveness, ease of use, and rapid results. Validation of ELISAs is necessary to ensure accurate measurements of Cry protein concentrations in the environment. Validation methodology has been extensively researched and published for the areas of sensitivity, specificity, accuracy, and precision; however, cross validation of ELISA results has been studied to a lesser extent. This review discusses the use of ELISAs for detection of Cry proteins in environmental samples and validation of ELISAs and introduces cross validation. The state of Cry protein environmental fate research is considered through a critical review of published literature to identify areas where the use of validation protocols can be improved.

Validation of an ELISA for the concurrent detection of total antibodies (IgM and IgG) to Rift Valley fever virus.

Rift Valley fever virus (RVFV) infects humans and livestock, causing haemorrhaging and abortions in animals. Three major RVF epizootics have occurred in South Africa since the 1950s and the outbreak in 2010 had a mortality rate of 10.7% in humans. Accurate and early detection is therefore essential for management of this zoonotic disease. Enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of either IgM or IgG antibodies to RVFV in animal sera. In this study, data are presented on the validation of a double-antigen ELISA for the simultaneous detection of both classes of antibodies to RVFV ina single test. ELISA plates were coated with a recombinant nucleoprotein. The nucleoprotein,conjugated to horseradish peroxidase, was used as the detecting reagent. A total of 534 sera from sheep and cattle were used in the validation. The sheep sera were collected during a RVF pathogenesis study at the Agricultural Research Council (ARC) - Onderstepoort Veterinary Institute and the cattle sera were collected during an outbreak of RVF in 2008 at the ARC -Animal Production Institute in Irene, Pretoria. The ELISA had a diagnostic sensitivity of 98.4% and a specificity of 100% when compared to a commercial cELISA. This convenient and fast assay is suitable for use in serological surveys or monitoring immune responses in vaccinated animals.

Validation and Preclinical Correlation of a New Sandwich ELISA for Measuring Murine Hepcidin.

AbstractAbstract 2100Although enzyme-linked immunosorbent assays (ELISA) exist for measuring human hepcidin, no commercially available antibodies to mouse hepcidin are validated for this purpose. Thus, we developed an ELISA to measure mouse hepcidin using low plasma volumes to allow for repeat testing of individual mice over time. To this end, human hepcidin knock-in mice (Hep2 mice) were used to generate a panel of anti mouse hepcidin antibodies. A sandwich ELISA using these antibodies was validated. Pooled plasma from iron-deficient (combining diet and phlebotomy), iron-replete, and inflamed (injecting sub-clinical amounts of lipopolysaccharide) FVB mice was used to prepare frozen aliquots of low, medium, and high reagent control plasma. These samples were tested on 5 different days (n=8 wells/sample/day). Mean hepcidin levels for the high, medium, and low reagent were 791, 504, and 117 ng/mL, respectively. The intra-assay coefficient of variation (CV) was 10, 4.5, and 15.6% for the high, medium, and low samples, respectively. Inter-assay CV was 4.7, 4.0, and 11.1% for the high, medium, and low samples, respectively. Analytical sensitivity was 0.7 ng/mL and the assay was linear (Pearson r=0.99) to 500 ng/mL at a 1:100 dilution of sample to blocking buffer (i.e. only 1 uL of sample needed per test). With 20 plasma samples spanning the range of expected hepcidin levels, there was high correlation between hepcidin determined by ELISA and the sum of hepcidin-1 and hepcidin-2 by time-of-flight mass spectrometry (Pearson r = 0.99; P<0.0001). Stability was analyzed using samples from repeated freeze-thaw cycles at −80°C or from incubation at 4°C. Each freeze-thaw cycle induced up to a 15% loss in hepcidin. Overnight refrigeration led to a 31%, 21%, and 4% decrease in hepcidin for the high, medium, and low samples, respectively. Severe hemolysis (i.e. 8 g/dL hemoglobin) caused interference, decreasing apparent hepcidin levels up to 52%; mild hemolysis (0.1 g/dL hemoglobin) decreased hepcidin levels up to 15%. To provide clinical correlation in mouse models, we used cohorts (n=5/group) of C57BL/6 females with differing iron status: 1.Iron deficient: weanling mice on a low iron diet (0–5 ppm FeSO4; AIN diet) for 8 weeks2.Iron-replete: weanling mice on a normal iron diet (45 ppm FeSO4; AIN diet) for 8 weeks3.Iron-overload: mice received weekly iron-dextran injections (5 mg for 7 weeks)4.Transfusional iron-overload: mice received weekly syngeneic RBC transfusions (1 unit for 7 weeks)Hepcidin mirrored ferritin levels and total liver iron measured at necropsy (see Table). To test whether this ELISA can track hepcidin during infection, and whether iron status affects this response, iron-deficient or -replete mice (n=10/group) were inoculated with 1000 colony forming units of Salmonella typhimurium and blood samples obtained pre-infection and 1–7 days post-infection to measure hepcidin and interleukin (IL)-6. Pre-infection, iron-deficient mice had significantly lower hepcidin levels than iron-replete mice (<0.7 vs. 136 ± 56 ng/mL, mean ± sd). On Day 7 post-infection, IL-6 was much higher in surviving iron-deficient mice (up to >2000 pg/mL) than in iron-replete mice (207 ± 70 pg/mL). Despite dramatic increases in IL-6, hepcidin increased less in iron-deficient mice (260 ± 132 ng/mL) than in iron-replete mice (636 ± 273 ng/mL). Thus, despite a stronger inflammatory stimulus, hepcidin levels are muted in iron-deficient animals. In conclusion, this validated ELISA is a new tool that can be used to study iron regulation in mice.Liver iron (μg)Ferritin (ng/mL)Hepcidin (ng/mL)Iron replete62 ± 10849 ± 100204 ± 32Iron deficient31 ± 5387 ± 1142.4 ± 1Iron overload (Fe-Dextran)11531 ± 1163109912 ± 11872633 ± 54Iron overload (Transfusion)352 ± 13302 ± 872323 ± 64Data presented as mean ± sd. Disclosures:Winters:Amgen, Inc.: Employment. Cooke:Amgen, Inc.: Employment. Sasu:Amgen, Inc.: Employment.

Validation and quality control of ELISAs for the use with human saliva samples

Enzyme-linked immunosorbent assays (ELISAs) have proven to be a powerful tool for fast and reliable sample analysis, in both clinical diagnostics and in research. Most assays are now available for use with a range of different analytical fluids, including serum, plasma or urine. In recent years, saliva has drawn attention as a potentially valuable diagnostic fluid; however few ELISAs have been validated for use with saliva, or their validation is often incomplete. Saliva has a number of different physical characteristics than, for example, cell culture medium or serum and assuming an ELISA which works well with serum samples will also do so with saliva potentially could lead to erroneous data and conclusions.In this report, we provide a detailed protocol to validate any ELISA for use with saliva samples and show the results of validation procedures for 13 ELISAs for using saliva. Our findings suggest that the majority of ELISAs work reliably with saliva, even if the assay was not specifically designed for this biological fluid. However, we also report a few cases where recovery or intra-and inter-assay variations were unexpectedly high, emphasising the importance of performing a validation procedure for each assay before using it with saliva to ensure accurate and reliable data.

Development, validation and application of ELISAs for pharmacokinetic and HACA assessment of a chimeric anti-CD40 monoclonal antibody in human serum

As part of a Phase I chimeric anti-CD40 monoclonal antibody clinical trial, two enzyme-linked immunosorbent assays (ELISAs) were developed for secondary endpoints: 1) for the pharmacokinetic (PK) monitoring of serum antibody levels and 2) for immunogenic screening of human anti-chimeric antibody (HACA) responses. The ELISA is a well established immunoassay, with clear guidelines for validation when used as a quantitative assay. However, these parameters may not always be relevant for a semi-quantitative assay used to assess whether a sample is positive or negative for a novel marker such as an antibody developed against a therapeutic antibody. We report here the development of a quantitative PK ELISA and a semi-quantitative HACA ELISA, and the different approaches of validation to prove each assay are ‘fit for purpose.’ The parameters of linearity (R 2 > 0.99), accuracy (±30%), lowest level of detection (4 μg/ml), intra-assay (coefficient of variation (CV) < 20%) and inter-assay (CV < 20%) variability were assessed for the quantitative PK assay. For the semi-quantitative HACA assay, parameters of linearity (R 2 > 0.99), lowest level of detection, intra (CV < 10%) and inter-assay (CV < 30%) variability were assessed using a surrogate positive control. The validation outcome showed that each assay was robust, reliable and accurate to meet the requirements of the intended analytical application, that being to 1) quantitatively determine the concentration of antibody in the serum and 2) determine whether a sample is positive or negative for human anti-chimeric antibodies. Each assay has been successfully translated for use in a clinical trial with adequate quality controls and acceptance criteria set for monitoring consistency and performance.

Quantitative ontogeny of murine insulin-like growth factor (IGF)-I, IGF-binding protein-3 and the IGF-related acid-labile subunit

Objective The mouse serves as an important model for insulin-like (IGF)-related research. However, lack of homologous mouse assays has prevented studies of the normal ontology of the murine IGF system. Therefore, we developed and validated immunoaassays for murine IGF-I, IGFBP-3 and ALS and studied levels of these analytes in mice. Methods Using commercially available reagents, we developed and validated specific enzyme-linked immunosorbent assays (ELISAs) for murine IGF-I, IGFBP-3, and ALS. Levels of these analytes were measured in sera from CD-1 mice, male and female, sampled at 1, 2, 4, 8, 20 and 32 weeks of age. In addition, sera from pregnant and postpartum CD-1 mice were also studied. Results Validation of specific ELISAs for murine IGF-I, IGFBP-3 and ALS are described; all 3 assays were highly sensitive, precise and accurate. As measured by our homologous ELISA, IGF-I levels (ng/mL, mean ± SD) in male mice were relatively low at 1 week (53 ± 8), rising sharply to peak at 8 weeks of age (636 ± 48), and gradually declining thereafter, reaching 395 ± 64 at 32 weeks. IGF-I levels in non-pregnant female mice peaked at 4 weeks of age (548 ± 77) declined at 8 weeks (417 ± 61), then recovered to plateau at 539 ± 78 and 535 ± 88 at 20 and 32 weeks, respectively. In male mice, trends in IGFBP-3 were similar to the patterns of IGF-I. However, in non-pregnant female mice, the IGFBP-3 levels declined relatively slowly after peaking at 4-weeks of age, unlike IGF-I levels during this period. ALS levels followed the same pattern as IGF-I in both sexes. IGF-I to IGFBP-3 molar ratios (percent) were similar between sexes, rising continuously with age: ∼30% at 1 week, 80% at 4 weeks, 135% at 32 weeks. IGF-I was reduced in 8 week old mice in mid-pregnancy (354 ± 75 vs 417 ± 61 in non-pregnant 8 week females), reaching a nadir in late-term (146 ± 40), and only partially recovering in the postpartum period (239 ± 23). IGFBP-3 was also lower in late-pregnancy (1245 ± 100 vs 1925 ± 439) and remained depressed postpartum. In contrast to IGF-I and IGFBP-3, ALS increased more than threefold in mid-pregnancy (12180 ± 1641 vs 3741 ± 910), followed by a 4-fold decrease in late-pregnancy (2964 ± 489), recovering postpartum (6104 ± 1178). Conclusions We report the first ontological studies of IGF-I, IGFBP-3 and ALS in mice using newly-characterized sensitive, homologous immunoassays. Our results indicate that mice have a generally similar pattern in IGF-related axis components, with low levels early in life, increasing to peak during sexual maturation and declining thereafter. Significant gender differences in non-pregnant levels and dramatic changes during pregnancy were also found. Knowledge of the normal developmental changes in the murine IGF system and accurate tools for investigations of this system are a necessary foundation for research in this field.

Diagnosis of Paratuberculosis by Fecal Culture and ELISA on Milk and Serum Samples in Two Types of Chilean Dairy Goat Herds

Fecal culture has been the primary method used to diagnose paratuberculosis in goats. It is laborious, slow, and expensive. Validation of enzyme-linked immunosorbent assays (ELISAs) on milk samples could make paratuberculosis testing more widely available for goat farmers. The aim of this study was to determine the accuracy of serum and milk ELISAs for paratuberculosis, relative to fecal culture, in Chilean dairy goats. Eight dairy goat herds were selected. Feces, blood, and milk samples were collected from all female goats >2 years old. Fecal samples were cultured using Herrold egg yolk medium with mycobactin J and antibiotics. Serum and milk samples were tested using a commercial ELISA kit for Mycobacterium avium subsp. paratuberculosis antibody detection. A total of 383 goats were tested by ELISA and fecal culture. The sensitivity of ELISA on serum and milk relative to fecal culture was 74.3% (95% CI: 59.8-88.8) and 60% (95% CI: 43.8-76.2), respectively. The corresponding values for ELISA specificity based on the percentage of non- M. avium subsp. paratuberculosis-infected goats testing ELISA-negative were 98.6% (95% CI: 96.6-100) and 99.3% (95% CI: 97.9-100) on serum and milk, respectively. Proportions of positive results for serum and fecal samples were significantly different, whereas the proportions of positive results for milk and fecal samples were not significantly different. The milk ELISA had a moderate level of agreement with fecal culture results (Kappa = 0.57). The paratuberculosis ELISA on goat milk samples may be a cost-effective, accurate alternative to fecal culture.

Validation of enzyme-linked immunosorbent assays for the diagnosis of bovine brucellosis

The purpose of this study was to evaluate the performance of the indirect enzyme immunoassay (IELISA) and the competitive enzyme immunoassay (CELISA) for the diagnosis of bovine brucellosis in comparison to conventional serological tests routinely used in Argentina. Serum samples ( n = 3500), from Brucella-free herds, from vaccinated cattle and from naturally infected cattle, were tested by the following tests: buffered antigen agglutination test (BPAT), rose bengal test (RBT), 2-mercaptoethanol test (2-ME), complement fixation test (CFT), IELISA and CELISA. Sensitivity and specificity of the BPAT, RBT, IELISA and CELISA were determined relative to the 2-ME and the CFT. The CELISA was considered suitable for eliminating most serological reactions of vaccinated animals and was more specific than the other tests. The results indicate the potential use of the CELISA as a complementary assay in the brucellosis control and eradication program in Argentina and other countries, where Brucella abortusstrain 19 vaccination is mandatory.