Enzyme-linked Immunosorbent Assay Procedure Research Articles

Development of a High Sensitivity Rapid Sandwich ELISA Procedure and Its Comparison with the Conventional Approach

A highly sensitive and rapid sandwich enzyme-linked immunosorbent assay (ELISA) procedure was developed for the detection of human fetuin A/AHSG (alpha2-HS-glycoprotein), a specific biomarker for hepatocellular carcinoma and atherosclerosis. Anti-human fetuin A antibody was immobilized on aminopropyltriethoxysilane-mediated amine-functionalized microtiter plates using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysulfosuccinimide-based heterobifunctional cross-linking. The analytical sensitivity of the developed assay was 39 pg/mL, compared to 625 pg/mL for the conventional assay. The generic nature of the developed procedure was demonstrated by performing human fetuin A assays on different polymeric matrixes, i.e., polystyrene, poly(methyl methacrylate), and polycyclo-olefin (Zeonex), in a modified microtiter plate format. Thus, the newly developed procedure has considerable advantages over the existing method.

Single-step ELISA capillary sensor based on surface-bonded glucose oxidase, antibody, and physically-adsorbed PEG membrane containing peroxidase-labeled antibody

A single-step enzyme-linked immunosorbent assay (ELISA) capillary immunosensor that exploits the combination of surface-bonded glucose oxidase (GOD), antibody and physically-adsorbed poly(ethyleneglycol) (PEG) membrane containing peroxidase (POD)-labeled antibody in a single capillary, is presented. The present simple detection technique involves a proximity-based two-enzyme system where the product of the first enzyme reaction is used as a substrate into the second enzyme reaction. Since the degree of antigen–antibody reaction controls the second enzymatic reaction process, amount of final fluorescent product varies with antigen concentration that leads to the making ELISA procedure single step. Sample solution that contains antigen, glucose, ascorbic acid and a fluorescent substrate (amplex red) is simply introduced into the capillary immunosensor by capillary action. In the presence of an antigen in the nanoliter sample volume, the released POD-labeled antibody forms a sandwich immunocomplex on the surface bearing the antibody. This leads to a proximity of the POD and GOD. We confirmed that the rate of conversion of a non-fluorescent probe, amplex red, to a red fluorescent dye, resorufin, is dependent on the two-enzyme proximity in a certain experimental condition. Preliminary results yielded an approximate detection limit of around subnanogram per milliliter concentration for human IgG. The relative standard deviation of the fluorescence response was ca. 4.1% for the capillary pieces prepared by cutting a long immunosensor capillary. This approach could be an enabling technology applicable to microfluidic device integration for multiple analyte sensing. Realization of such an endeavor could be very promising for drug screening and clinical diagnostic applications.

IgG anti-gliadin determination with an immunological microfluidic system applied to the automated diagnostic of the celiac disease

In the present article, a novel microfluidic immunosensor coupled with electrochemical detection for anti-gliadin IgG antibody quantification is proposed. This device represents an important tool for a fast, simple, sensitive, and automated diagnostic for celiac disease, which is carried out through detection of anti-gliadin IgG antibodies present in human serum samples. Celiac disease (CD) is an autoimmune disease generated by gluten protein fractions called prolamins. This pathology affects about one in 250 people around the world, produces intestinal inflammation, villous atrophy, and crypt hyperplasia, which causes a range of symptoms including altered bowel habits, malnutrition and weight loss. Our immunosensor consists of a Plexiglas device coupled to a gold electrode, with a central channel containing 3-aminopropyl-modified controlled pore glass (AP-CPG). The quantification of anti-gliadin IgG antibodies was carried out using a heterogeneous, non-competitive enzyme-linked immunosorbent assay (ELISA) in which IgG antibodies bound to gliadin protein, immobilized on AP-CPG, were determined by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP, and the electroactive product was quantified on a gold electrode at 0.250 V. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.52 and 2.72 UR mL(-1), respectively, and the within- and between-assay coefficients of variation were below 5.8%. The optimized procedure was applied to the determination of anti-gliadin IgG antibodies in human serum samples.

Enhanced Gold Nanoparticle Based ELISA for a Breast Cancer Biomarker

In this work, we developed an optical enzyme-linked immunosorbent assay (ELISA) immunoassay for the analysis of CA15-3 antigen, an important biomarker present in blood samples and useful for the follow-up of the medical treatment of breast cancer. Gold nanoparticles (AuNPs) were used as carriers of the signaling antibody anti-CA15-3-HRP (horseradish peroxidase) in order to achieve an amplification of the optical signal. In the range between 0 and 60 U/mL, the assay adopting AuNPs as an enhancer resulted in higher sensitivity and shorter assay time when compared to classical ELISA procedures. The application of AuNPs to the commercially available ELISA test can be useful to improve important immunoanalysis procedures where a more confident result is needed.

Variations of brain endothelial nitric oxide synthase concentration in rat and mouse cortex

No information exists on the differences of eNOS concentration in brain tissue, [eNOS] br, between animals during normal and hypotensive blood pressure and both between and within animals during moderate hypotension. To address these questions, we modified a commercially available enzyme-linked immunosorbent assay (ELISA) kit for determining murine [eNOS] br since no method exists to measure [eNOS] br. Optimization of the kit ELISA procedure using brain cortex homogenates from 3 normotensive rats and 1 wild-type and 1 eNOS −/− (ko) mouse included recovery evaluation for each sample and the use of an “eNOS-free” homogenate calibrator diluent obtained from a mutant eNOS-ko mouse. Initial spike-and-recovery values of 12.5–27% suggesting a substantial sample matrix effect were improved with lipid removal treatment to 37.3% and to 70% with 1:20 dilution of the sample. Calibration standards prepared using eNOS-free buffer increased recovery values to 78% in micro-punch samples. The optimized ELISA was used in micro-punch (<1 mg) brain cortex samples from 6 hypotensive rats. Whole brain [eNOS] br varied considerably from 5–11 fmol/mg wet weight and was different between normo- and hypotensive animals ( p = 0.023). The variability of [eNOS] br due to moderate hypotension in micro-punch rat brain cortex samples was composed of both between (24%) and within (76%) animal components. The differences and variability of [eNOS] br between normo- and hypotensive animals, and between and within hypotensive animals suggests the potential utility of its measurement for investigations of cerebrovascular physiology and that [eNOS] br itself could be an important factor in cerebrovascular regulation.

APPLICATION OF STEPWISE AMMONIUM SULFATE PRECIPITATION AS CLEANUP TOOL FOR AN ENZYME-LINKED IMMUNOSORBENT ASSAY OF GLYPHOSATE OXIDOREDUCTASE IN GENETICALLY MODIFIED RAPE OF GT73

ABSTRACT The method of enzyme-linked immunosorbent assay after stepwise ammonium sulfate (AS) purification (AS-ELISA) was developed and used to detect genetically modified (GM) rape of GT73 containing glyphosate oxidoreductase (Gox). Gox protein encoded by the Gox gene from Achromobacter sp. was highly expressed as inclusion bodies in Escherichia coli BL21 (DE3) and purified to homogeneity by Ni2+affinity chromatography. A simple and efficient extraction and purification procedure of Gox protein from the seeds and leaves of GM rape was developed by means of stepwise AS precipitation. Purified polyclonal antibodies against Gox was produced and enzyme-linked immunosorbent assay (ELISA) procedures were established further on to measure the Gox protein. AS-ELISA allowed 5% GMOs to be detected in the seeds of GT73 and 0.5% GMOs to be detected in the leaves of GT73 rape, which makes this method an acceptable method to access Gox protein in GM rape of GT73. PRACTICAL APPLICATIONS Many GMOs containing Gox gene have been approved worldwide such as GT73 rape, 1,445 cotton and Mon832 maize. Protein based methods were more important than DNA based methods, because protein performs a specific and concrete function and is closely interconnected with crop traits. AS-ELISA method can be used in the screening of GM plant, Gox protein expression assay and quantitative detection for GMO labeling. AS-ELISA Gox detecting method was established in this paper and was being evaluated of Inter-laboratory Comparison in some of Chinese GMO detection and assessment centers. With the knowledge of ELISA, ELISA method will be the national standards and international and will be a beneficial supplement for the DNA based GMO detecting methods.

Rapid identification of grouper and wreck fish meals by ELISA: a field study in restaurants

SummaryGrouper (Epinephelus marginatus) and wreck fish (Polyprion americanus) are two costly and demanding fish species which are subjected to substitution in the restaurant industry. In our study, 44 purported grouper and wreck fish meals (24 from school and university lunch rooms and 20 from restaurants) were analysed using an indirect enzyme‐linked immunosorbent assay (ELISA) (microtiter plates and immunostick format) and a MAb specific to these fish species. The results showed that 29 out of 44 meal samples were misdescribed in the restaurant menus. This ELISA procedure, which has proved to be specific, sensitive, cheap and very simple to develop, could be very useful for accurate authentication of meals in the restaurant industry.

Aflatoxin M 1 determination and stability study in milk samples using a screen-printed 96-well electrochemical microplate

A highly sensitive method for the determination of aflatoxin M 1 (AFM 1) is described that involves the use of a disposable multichannel microplate coupled to intermittent pulse amperometry (IPA) for immunosensor development based on a direct competitive assay. Immunoassay parameters were evaluated and optimized to establish an electrochemical enzyme-linked immunosorbent assay (ELISA) procedure for AFM 1 in milk samples. The suitability of the immunosensors for the direct analysis of the toxin in milk was assessed. AFM 1 was measured with a working range of 5–250 pg mL −1 and a detection limit of 1 pg mL −1. Good recovery values (90–105%) were obtained. The method was used to study the recovery of this toxin in frozen (−30 °C) or lyophilized (4 °C) milk samples stored for up to 3 months. A significant, but variable, decrease (>50%) of the measured AFM 1 concentration with respect to the initial toxin contamination levels was found.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma

Trastuzumab, a humanized monoclonal antibody, is used for the treatment of breast cancer patients who overexpress the HER2 receptor. To optimize therapy, pharmacokinetic studies are necessary. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for trastuzumab to support these pharmacokinetic studies. For this immunoassay, we raised anti-idiotype antibodies in rabbits. After purification of the rabbit material, the anti-idiotype antibodies are used as capturing antibodies on the ELISA plate. After trastuzumab has bound to the catcher antibody, a sandwich ELISA procedure is followed whereby biotinylated anti-idiotype antibodies can bind to trastuzumab. Detection is performed by streptavidin–polyHRP (poly-horseradish peroxidase) conjugate and (3,5,3′,5′)-tetramethylbenzidine (TMB) substrate. The reaction is stopped using sulfuric acid, and the absorbance is measured at 450 nm. The calibration range of the assay is 0.039 to 5 ng/ml in well. Because samples are analyzed in multiple dilutions, the validated range corresponds to 1.6 to 1600 ng/ml in undiluted serum. Samples above the upper limit of quantification (ULOQ) can be diluted before transfer to the assay plates. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum and plasma. The assay is now used to support pharmacokinetic studies with trastuzumab in human serum and plasma.

Pressure: A Novel Tool for Enzyme-Linked Immunosorbent Assay Procedure

The enzyme-linked immunosorbent assay (ELISA) technique is the backbone of the diagnostic assay for detection of infectious and allergic diseases. Here, we demonstrate a unique ELISA method for the detection of an antigen or antibody, in which ELISA steps are carried out under pressure instead of conventional thermal incubation. Pressure-mediated ELISA (PELISA), carried out in 1 h shows more than a 2-fold increase in absorbance value than the control experiment carried out at the same time and temperature without applying pressure. Estimation of total IgE by the 1-h PELISA method gives similar absorbance value (1.081+/-0.031, 823.12 IU) to that obtained by 3-h heat-mediated ELISA on an activated surface (HELISA) (1.165+/-0.037, 810.96 IU). Since PELISA is sensitive, specific, and reproducible (intra- and interassay CVs were 6.47% and 9.65%, respectively), it could be an excellent alternative to HELISA or conventional ELISA procedures.

Urinary calreticulin in the diagnosis of bladder urothelial carcinoma

To evaluate the potential suitability of calreticulin (CRT) as a urinary marker for bladder cancer. Urine specimens were collected from patients with histologically confirmed bladder urothelial carcinoma (Group 1; n = 109), urological patients without urothelial carcinoma (Group 2; n = 60), and non-urological patients (Group 3; n = 40). We developed an enzyme-linked immunosorbent assay (ELISA) procedure using commercially available anti-CRT mono/polyclonal antibodies, and then measured the concentration of urinary CRT. Urinary CRT concentration of group 1 was significantly higher than group 2 and 3 (Mann-Whitney U-test, P < 0.001). Groups 2 and 3 were joined together and considered as a non-bladder cancer group (n = 100), and a cutoff value (2.85 ng/mL) was determined using receiver operating characteristic (ROC) analysis. The sensitivity, specificity, and the area under the curve were 67.9%, 80.0%, and 0.742, respectively. The overall sensitivity of voided urine cytology (VUC) was 39.0% (n = 105), and the sensitivity of urinary CRT was significantly superior to VUC (McNemar test, P < 0.001). Higher sensitivity was observed especially in Ta, G1-2, and <or=3 cm tumors. Urinary CRT may be useful for diagnosis of bladder urothelial cancer. However, given that its specificity is relatively low, further evaluation in larger series is needed to define its clinical usefulness.

Problem solved: a modified enzyme-linked immunosorbent assay for detection of human antibodies to pertussis toxin eliminates false-positive results occurring at analysis of heat-treated sera

Enzyme-linked immunosorbent assay (ELISA) for measurement of antibodies to pertussis toxin (PT) is a widely used method for diagnosis of whooping cough and is also frequently used for seroprevalence studies and for assessment of antibodies after vaccinations against whooping cough. The recommended ELISA procedure for assessment of PT antibodies in human serum does, however, have a serious problem with false-positive results when heat-treated sera are analyzed. Historic sera might have been exposed to routine heat treatment, and diagnostic sera from warm geographic regions are at risk of unintentional exposure to heat. A modified version of the PT ELISA, incorporating a blocking step with 1% milk and addition of 0.1% milk to the dilution buffer, eliminates the false-positive phenomenon occurring with heat-treated sera. Results for serum antibody concentrations correlate well with the currently recommended method. This ELISA modification is straightforward and cheap, and it should be recommended at all analyses incorporating sera with unknown history of heat exposure.

Comparison of the Anti‐inflammatory Activities of Imidazole Antimycotics in Relation to Molecular Structure

The objective of this study was to compare the anti-inflammatory potencies of the imidazole antimycotics, fluconazole, itraconazole, ketoconazole and voriconazole (0.5 and 5 microM) in relation to their molecular structures. Anti-inflammatory activity was determined according to the magnitude of inhibition of production of leukotriene B4 and influx of Ca2+ following activation of the cells with the chemo-attractant platelet-activating factor (200 nM), using enzyme-linked immunosorbent assay and spectrofluorometric procedures, respectively. Treatment of platelet-activating factor-activated neutrophils with the imidazole antimycotics resulted in inhibition of production of leukotriene B4 and attenuation of Ca2+ influx, the order of potency being itraconazole > ketoconazole > fluconazole = voriconazole. These observations demonstrate the requirement for both the diazole/triazole moiety (all four agents), and the substituted phenylpiperazinyl ether side chain (itraconazole and ketoconazole only) for maximal anti-inflammatory activity of this class of pharmacological agents.

In-house validation of an ELISA method for screening of semicarbazide in eggs

An enzyme-linked immunosorbent assay (ELISA) method is described for the semi-quantitative determination of semicarbazide (SEM), the marker residue for the banned nitrofuran drug, nitrofurazone, in chicken eggs. The sample homogenate is subjected to acid hydrolysis and derivatisation with o-nitrobenzaldehyde, followed by ethyl acetate/hexane extraction and detection by ELISA. The ELISA procedure has been validated using 0.3, 1.0 and 3 µg kg−1 of SEM in fortified samples. Detection capability (CCß) was based on the acceptance of 5% false compliant results for a given concentration level according to Commission Decision 2002/657/EC and was determined to be 0.3 µg kg−1 with a respective limit of detection of 0.13 µg kg−1. A validated LC–MS/MS method was used for the analysis of incurred egg samples and the results compared with ELISA. A good correlation between the results obtained from ELISA and LC–MS/MS within the concentration range 0.12–20.3 µg kg−1 was observed in samples collected from chickens fed with a medicated ration of nitrofurazone (r = 0.992, n = 14). Validated ELISA enabled reliable monitoring of SEM levels in eggs collected from incurred chickens over a 90-day period.

Enzyme-Linked Immunosorbent Assay (ELISA) based on superparamagnetic nanoparticles for aflatoxin M 1 detection

Five different clones of antibodies developed against the aflatoxin M 1 were investigated by using the classical indirect and direct competitive Enzyme-Linked Immunosorbent Assay (ELISA) formats, and also the direct competitive ELISA based on the use of the superparamagnetic nanoparticles. The purpose of this study was to assess if not so friendly time classical ELISA procedures can be further improved, by reducing the coating, blocking and competition time. Here we showed that a complete dc-ELISA (coating, blocking and competition step) based on the use of superparamagnetic nanoparticles can be performed in basically 40 min, if coating step (20 min) should be taken into account. Moreover, the standard analytical characteristics of the proposed method fulfil the requirements for detecting AFM 1 in milk, in a wide linear working range (4–250 ng/L). The IC 50 value is 15 ng/L. The matrix effect and the recovery rate were assessed, using the European Reference Material (BD282, zero level of AFM 1), showing an excellent percentage of recovery, close to 100%.

Soluble Triggering Receptor Expressed on Myeloid Cells in Patients With Suspected Meningitis, Peritonitis, or Pleuritis

The recently identified Triggering Receptor Expressed on Myeloid cells 1 (TREM-1) is up-regulated on the surfaces of inflammatory cells in the presence of extracellular bacteria and fungi, and the soluble form, sTREM, has been used to improve the diagnostic accuracy of ventilator-associated pneumonia and septicemia in critically ill patients. The current study, which included 265 patients, was undertaken to evaluate the potential role of sTREM in the diagnosis of meningitis, peritonitis, and pleuritis by comparing sTREM concentrations, measured by means of an enzyme-linked immunosorbent assay procedure, in samples of cerebrospinal fluid, peritoneal fluid, and pleural fluid, with the results of laboratory investigations used to detect the presence of microbial pathogens in these fluid samples. Significantly higher concentrations of sTREM were associated with the presence of diverse microbial pathogens and may be useful in predicting the presence of microbial pathogens in peritoneal and pleural fluid samples.

Bayesian semiparametric ROC curve estimation and disease diagnosis

We develop a novel semiparametric modeling framework involving mixtures of Polya trees for screening data with the dual purpose of diagnosing infection or disease status and of assessing the accuracy of continuous diagnostic measures. In this framework, we obtain (i) predictive probabilities of 'disease' based on continuous diagnostic test outcomes in conjunction with other information, including relevant covariates and results from one or more independent binary diagnostic tests. An example would be the modeling of a serum enzyme-linked immunosorbent assay (ELISA) procedure for detecting antibodies to an infectious agent when used in conjunction with culture for antigen detection. Our second goal is to (ii) characterize measures of diagnostic performance of continuous tests by estimating receiver-operating characteristic curves and area under the curve, primarily when such extra information is available. When true disease status is unknown, parametric and nonparametric analyses require sufficient separation between the distributions of outcome values for the diseased and nondiseased populations. However, this overlap becomes less problematic when additional information in the form of either an informative 'prior' that is based on real (preferably data-based) scientific input, or when additional information, or both, are available. The additional information can be used to distinguish 'diseased' from 'nondiseased' individuals. We present an example using simulated data that illustrates this point. We also present an example involving data from an animal-health survey for Johne's disease, where the performance of a serum ELISA is evaluated using additional information obtained from fecal culture. Issues related to identifiability and partial identifiability are also discussed.

Detection of Group D Salmonellae Including Salmonella Enteritidis in Eggs by Polymyxin-Based Enzyme-Linked Immunosorbent Assay

A high-throughput, rapid method was devised for the detection of Salmonella Enteritidis in egg products. For each target organism, preenrichment in nutrient broth was followed by selective enrichment in Rappaport-Vassiliadis soya peptone and tetrathionate brilliant green broths or by plating on modified semisolid Rappaport Vassiliadis (MSRV) agar medium. The presence of Salmonella Enteritidis was determined by subjecting portions of the selective broth cultures or swarming growth on MSRV medium to an enzyme-linked immunosorbent assay (ELISA) procedure using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for lipopolysaccharide (LPS) antigens. Sample extracts were reacted with polymyxin-coated microwells, and captured LPS antigens were detected immunoenzymatically with a commercially available Salmonella factor O9–specific antibody. The polymyxin-ELISA was 100% sensitive and 100% specific for Salmonella strains bearing the O9 antigen. When the ELISA was combined with enrichment using either the selective broths or plating on MSRV medium, the system was an effective means for detection of Salmonella Enteritidis in artificially inoculated egg products. The polymyxin-ELISA is a simple and inexpensive assay for group D salmonellae (including Salmonella Enteritidis) in a convenient 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.

Acute Pulmonary Effects of Combined Exposure to Carbon Nanotubes and Ozone in Mice

Ozone (O3) is a well-investigated gaseous air pollutant known to produce acute and chronic toxicity in the respiratory system. Whether prior exposure to nanoparticles influences the toxicity of O3 has not been well investigated. To determine if there are toxicological interactions between particulate and gas exposures, we examined acute pulmonary effects of a 3-h ozone exposure (0.5 ppm) in female C57Bl mice that had been preexposed to a single dose of 20 μ g multiwall carbon nanotubes (CNT) by pharyngeal aspiration 12 h earlier. A total of four groups were compared: (1) PBS/air-control, (2) PBS/O3, (3) CNT/air, and (4) CNT/O3. Analyses of the bronchoalveolar lavage fluid (BALF) and lung tissue samples collected at 5 and 24 h post O3 exposure were performed for various markers of cytotoxicity and inflammation using standard enzyme-linked immunosorbent assay (ELISA) and immunoblot procedures. The results showed a pronounced cellular response and increase in various cytotoxicity/inflammatory markers in the lungs of CNT-exposed mice. Ozone by itself produced minimal effects, but in CNT-exposed animals there was a significant increase in total brochoalveolar lavage (BAL) cells and polymorphonuclear leukocytes. Additionally, protein, lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and mucin levels in BALF at 5 and 24 h were higher in CNT-exposed animals than in corresponding air-exposed controls or animals exposed to O3 alone. A comparable increase over the controls was also observed in the CNT/O3 group, but neither an additive nor a synergistic interaction was observed in mice that received sequential exposure to CNT and ozone. In fact, some CNT-induced cytotoxic/inflammatory responses were attenuated in mice following exposure to both CNT and low levels of ozone. These results are contrary to enhanced responses that were anticipated and may represent the development of “cross-tolerance” reported by others for some sequentially administered pollutants.

Identification and Elimination of False-Positives in an ELISA-Based System for Qualitative Assessment of Glycoconjugate Binding using a Selection of Plant Lectins

Systematic optimization of a lectin-based enzyme-linked immunosorbent assay (ELISA) procedure using a panel of 21 biotinylated plant lectins and a glycoprotein with defined glycosylation (i) identified blocking as a limiting step in solid phase sugar binding analysis and (ii) found Synblock to be a better alternative to bovine serum albumin (BSA) as blocking agent.