One-step Enzyme-linked Immunosorbent Assay Research Articles

Development and application of one-step ELISA for the detection of neomycin in milk

A sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin (NEO) in milk was developed. Two conjugates were synthesized as the immunogen and coating antigen. Four BALB/c female mice were immunised and the antisera were screened by indirect competitive ELISA (icELISA). The icELISA was further optimised using different coating methods, pH values, ionic strengths of assay buffer and reaction times. After optimised, the indirect competitive ELISA (icELISA) could be finished in 85min with an IC50 value of 0.74±0.05 ng/mL. One-step indirect competitive ELISA (icELISA) exhibited similar sensitivity with an IC50 value of 0.73±0.03 ng/mL, which was sensitive enough for NEO detection and could be finished in 55 min. No cross-reactivity of the antibody was observed with other aminoglycosides based on icELISA, indicating that the antibody is highly specific for neomycin. Milk samples were further analyzed with recovery ranging from 85 to 110% at spiked level of 0.25–10 ng/mL, and the detection limit was 0.08 ng/mL.

One-Step Monoclonal Antibody Based Enzyme-Linked Immunosorbent Assay for Direct Determination of Cortisol in Serum

Monoclonal antibodies to cortisol have obvious potential advantages as starting materials for assay systems to detect their levels in body fluids. This is very important for monitoring pituitary gland and adrenal functions. To develop a one-step competitive heterogeneous enzyme-linked immunosorbent assay (ELISA), a monoclonal anti-cortisol antibody was generated using a reasonably designed haptenic derivative. Cortisol-3-O-carboxymethyloxime was coupled to carrier protein bovine serum albumin (BSA) to enhance its immunogenicity. Spleen cells were prepared from a BALB/c mouse, which had repeatedly been immunized with a conjugate of cortisol-3-O-carboxymethyloxime-bovine serum albumin (cortisol-3-O-CMO-BSA), to be fused with SP2/0 myeloma cells. After one fusion experiment, four hybridoma clones secreting a practical antibody were established. One of the resulting monoclonal antibodies, 2C9D11B5, showed an affinity constant (Ka) of 1.4 × 10(10) M(-1) for cortisol and provided a practical calibration curve (limit of detection [LOD], 0.26 ng per assay) in this ELISA system employing cortisol-21-hemisuccinate-horseradish peroxidase (cortisol-21-HS-HRP) as a tracer. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (3.5%), cortisone (0.47%), corticosterone (<0.01%), progesterone (<0.01%), 17-hydroxyprogesterone (1.2%), 6-hydroxycortisol (7.6%), and tetrahydrocortisol (<0.01%). The intra-assay and inter-assay coefficient of variations (CVs) ranged from 4.3% to 9.2% and 3.8% to 10.4 %, respectively. The analytical recoveries were 92.3% to 116.3%. Serum cortisol levels of healthy volunteers were determined after chilled acetone, stripped to be 292.76 ± 201.38 ng/mL (n=5), which are in the reference range.

A Novel Accurate Rapid ELISA for Detection of Urinary Connective Tissue Growth Factor, a Biomarker of Chronic Allograft Nephropathy

Background Chronic allograft nephropathy (CAN) is a leading cause of kidney graft failure. The latest evidence suggests that connective tissue growth factor (CTGF) may be a biomarker of CAN. Detection of urinary CTGF levels is a potential noninvasive strategy to predict the early onset of CAN. Compared to the traditional “sandwich” enzyme-linked immunosorbent assay (ELISA), we established a novel, accurate, faster, one-step competitive indirect ELISA (Ci-ELISA) to estimate the urinary CTGF concentrations in humans, rats, and mice. Materials and Methods We used recombinant human CTGF, which is an 11.2 kDa protein of 98 amino acid residues containing the C-terminal portion of the full-length CTGF protein having 96% and 94% homology with rats and mice, as the coating antigen and standard competitor. We then applied polyclonal antibody against the CTGF C-terminal portion, secondary antibody conjugated with horseradish peroxidase (HRP), and urine samples to establish one-step Ci-ELISA. Rat kidney allograft recipients were sacrificed at 4, 8, or 12 weeks posttransplantation. We harvested serum, urine, and transplanted kidneys. Hematoxylin-eosin (H&E), periodic acid-Schiff (PAS), and Masson trichrome stains were used to estimate the histopathologic changes according to the Banff schema. Serum creatinine (SCr) levels were measured to represent renal function. CTGF in kidney was detected by immunohistochemistry. Urinary CTGF concentrations were measured using the one-step Ci-ELISA. Results The whole one-step Ci-ELISA test can be accomplished within 2 hours. The detection limit of the assay was 0.16 ng/mL CTGF. The time-dependent elevated recipient concentration of urinary CTGF was positively correlated with SCr, histologic changes, and CTGF in the kidney after transplantation. The 95% confidence intervals of urinary CTGF concentrations in control rats were 24.4 to 35.2 ng/mL; at 4, 8, and 12 weeks posttransplantation they were 139.4 to 395.6, 826.7 to 1429.5, and 3206.0 to 4448.2 ng/mL, respectively. Conclusions A rapid one-step Ci-ELISA was successfully established to detect the CTGF levels in urine. These results further indicated that detection of urinary CTGF is a potential noninvasive strategy to predict the early onset of CAN.

Evaluation of Two New Commercial Tests for the Diagnosis of Acute Dengue Virus Infection Using NS1 Antigen Detection in Human Serum

BackgroundWe compared the performance of two new commercial tests for the detection of dengue NS1 protein during the clinical phase of dengue virus (DENV) infection—an immunochromatographic test allowing rapid detection of the NS1 antigen, Dengue NS1 Ag STRIP (Bio-Rad Laboratories - Marnes La Coquette, France), and a two-step sandwich-format microplate enzyme-linked immunosorbent assay (ELISA), pan-E Dengue Early ELISA (Panbio - Brisbane, Australia)—with a one-step sandwich-format microplate ELISA, the Platelia Dengue NS1 Ag test (Bio-Rad).MethodsWe tested 272 serum samples from patients with dengue disease. Of these, 222 were from patients with acute infection of one of the four dengue serotypes, detected by RT-PCR and/or virus isolation. Forty-eight acute-phase serum samples from patients not infected with dengue virus were also included.ResultsThe sensitivity of the Platelia Dengue NS1 Ag test on acute serum samples (n = 222) was 87.4% (95% confidence interval: 82.3% to 91.5%); that of Dengue NS1 Ag STRIP was 81.5% (95% CI: 75.8% to 86.4%) after 15 minutes and 82.4% (95% CI: 76.8% to 87.2%) after 30 minutes. Both tests had a specificity of 100% (97.5% CI, one-sided test: 92.6% to 100.0%). The pan-E Dengue Early ELISA had a sensitivity of 60.4% (95% CI: 53.4% to 66.8%) and a specificity of 97.9% (95% CI: 88.9% to 99.9%).ConclusionOur findings support the use of diagnostic tools based on the NS1 antigen detection for the diagnosis of acute DENV infection. The immunochromatographic test, Dengue NS1 Ag STRIP—the first rapid diagnostic test for DENV infection—was highly sensitive and specific, and would therefore be a suitable first-line test in the field. The pan-E Dengue Early ELISA was less sensitive than the Platelia test; this two-step ELISA should be combined with DENV IgM antibody detection for the diagnosis of DENV infection.

One-step immunochromatographic separation and ELISA quantification of glycyrrhizin from traditional Chinese medicines

The bioactive constituent, glycyrrhizin or glycyrrhizic acid (GA), was purified from two traditional Chinese medicines (TCM), Shaoyao gancao tang and Dahuang gancao tang, and from crude extracts from licorice roots by means of immunoaffinity chromatography using anti-GA monoclonal antibody (MAb) and was quantified with an enzyme-linked immunosorbent assay (ELISA). Laboratory preparations included the synthesis of conjugate GA-human serum albumin (GA-HSA), the production of anti-GA-MAb, the optimization of the immunoaffinity column packed with the anti-GA-MAb coupled to hydrazide gel and the determination of the GA content in TCM and crude drugs from five different sources by ELISA and high performance liquid chromatography (HPLC). The experimental results reveal that the anti-GA-MAb coupled to Affi-Gel Hz gel results in a coupling efficiency of 95.2%, and the immunoaffinity chromatography gives a mean recovery of 97.6% of GA with a capacity of 33.5 ± 2.40 μg/mL of immunoaffinity gel under the given conditions. The GA content of the crude extracts (ranging 74.8–114.6 μg/mg) from different sources by the ELISA method is much greater than that of the TCM (16.4–25.1 μg/mg) which is, in good agreement with the results of the HPLC method. Our report provides a rapid, reliable and sensitive approach for one-step separation and quantification of GA.

Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface

Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which (10)Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.

“Melanoma inhibitory activity” (MIA): a promising serological tumour marker in metastatic uveal melanoma

To assess the role of "melanoma inhibitory activity" (MIA) as a potential serum marker for screening and detection of metastatic uveal melanoma. Prospective, clinical study. Serum samples of 305 patients with uveal melanoma were collected. Serum samples were analysed by a one-step enzyme-linked immunosorbent assay (ELISA) to quantify the MIA serum levels. All patients underwent a standardized echography of the globe to evaluate maximum tumour height and were checked for systemic metastasis of the tumour by liver enzyme tests and ultrasonography of the liver. Twenty patients (6.6%) had proven metastatic disease; eight of them developed it during follow-up. The mean serum concentration of MIA in the 285 patients without metastasis was 6.72 ng/ml, whereas the mean serum concentration of MIA in the 20 patients with metastasis was 13.03 ng/ml (P<0.001). The eight patients who developed metastatic disease during follow-up showed an MIA of 5.92 ng/ml before detection of metastasis and 12.21 ng/ml afterwards (P<0.001). MIA serum levels did neither correlate with the tumour height or to whether local therapy had been applied. The elevation of MIA serum levels in patients with metastatic disease from melanoma supports its promising role as a serum marker for monitoring patients with uveal melanoma.

Comparative analytical evaluation of thyroid hormone levels in pregnancy and in women taking oral contraceptives: a study from an iodine deficient area

Increase of serum thyroxine binding globulin (TBG) resulting from estrogen action may lead to problems in thyroid diagnostics. The aim of the present study was to define the most diagnostically reliable thyroid parameters in women exposed to differentially elevated estrogens. Sera of three groups of healthy women were analyzed: women taking no medicine (controls), those taking oral contraceptives and pregnant women (in weeks 16 or 32 of gestation). All women involved in the study lived in a moderately iodine-deficient geographical area. Thyroid stimulating hormone (TSH), TBG, total thyroxine (T4), total triiodothyronine (T3) and free T3 were determined and free T4 indices (total T4 × T3 uptake; total T4/thyroxine binding capacity (TBC); total T4/TBG) were calculated. Free T4 was measured simultaneously with a one-step T4-analog enzyme-linked immunosorbent assay (ELISA), a labeled T4 antibody radioimmunoassay (RIA), and a two-step microparticle enzyme immunoassay (MEIA). Estrogen-dependent differences were found in all investigated parameters; however, they remained in the reference interval for TSH, total T4 × T3 uptake, total T4/TBC, free T3 and free T4 MEIA. It was concluded that simultaneous estimations of free T4 and free T3 should follow a primary TSH measurement. The necessity of a distinct reference range has emerged for free thyroid hormones in midterm and late pregnancy as well as in the use of oral contraceptives, especially in iodine-deficient areas.

Melanoma inhibitory activity: a novel serum marker for uveal melanoma.

The aim of this study was to evaluate the tumour-associated antigen melanoma inhibitory activity (MIA) as a potential novel serological tumour marker in primary and metastatic uveal melanoma in both the laboratory and the clinical setting. In the laboratory setting, immunohistochemical staining with MIA antibody was performed in paraffin-embedded tissues from six amelanotic uveal melanomas and eight metastatic lesions of uveal melanomas. In the clinical setting, serum samples of 139 patients with uveal melanoma were examined; eight of these patients had overt metastatic disease. Sixty-one initially metastatic disease-free patients were followed over time (median follow-up 240 days, 95% confidence interval 60-883 days) and MIA levels were assessed repeatedly. A one-step enzyme-linked immunosorbent assay was used to quantify the MIA serum levels. In the laboratory setting, five of the six primary uveal melanomas and seven of the eight metastatic lesions stained immunohistologically positive for MIA. In the clinical setting, the 131 patients without overt metastatic disease demonstrated a median serum concentration of MIA of 6.6 ng/ml. In the eight patients with overt metastatic disease, the median serum concentration of MIA was 26.28 ng/ml. This difference was highly statistically significant (P < 0.001, analysis of variance). During follow-up, three initially metastatic disease-free patients developed overt metastatic disease, and the MIA level increased from a median of 6.6 ng/ml before to 29.2 ng/ml after clinical detection of metastatic disease. In the 58 other patients, the serum level remained stable during the entire follow-up period. In conclusion, MIA is expressed in primary and metastatic lesions of uveal melanomas, and a statistically significant elevation in MIA serum levels in patients who develop metastatic disease due to uveal melanoma indicates its promising role as a serum marker for monitoring uveal melanoma patients for metastasis.

Melanoma inhibitory activity

The aim of this study was to evaluate the tumour-associated antigen melanoma inhibitory activity (MIA) as a potential novel serological tumour marker in primary and metastatic uveal melanoma in both the laboratory and the clinical setting. In the laboratory setting, immunohistochemical staining with MIA antibody was performed in paraffin-embedded tissues from six amelanotic uveal melanomas and eight metastatic lesions of uveal melanomas. In the clinical setting, serum samples of 139 patients with uveal melanoma were examined; eight of these patients had overt metastatic disease. Sixty-one initially metastatic disease-free patients were followed over time (median follow-up 240 days, 95% confidence interval 60–883 days) and MIA levels were assessed repeatedly. A one-step enzyme-linked immunosorbent assay was used to quantify the MIA serum levels. In the laboratory setting, five of the six primary uveal melanomas and seven of the eight metastatic lesions stained immunohistologically positive for MIA. In the clinical setting, the 131 patients without overt metastatic disease demonstrated a median serum concentration of MIA of 6.6 ng/ml. In the eight patients with overt metastatic disease, the median serum concentration of MIA was 26.28 ng/ml. This difference was highly statistically significant (P < 0.001, analysis of variance). During follow-up, three initially metastatic disease-free patients developed overt metastatic disease, and the MIA level increased from a median of 6.6 ng/ml before to 29.2 ng/ml after clinical detection of metastatic disease. In the 58 other patients, the serum level remained stable during the entire follow-up period. In conclusion, MIA is expressed in primary and metastatic lesions of uveal melanomas, and a statistically significant elevation in MIA serum levels in patients who develop metastatic disease due to uveal melanoma indicates its promising role as a serum marker for monitoring uveal melanoma patients for metastasis.

One-step enzyme-linked immunosorbent assay (ELISA) for measurement of serum free leptin

In man, circulating leptin levels are increased with obesity and are regulated by a complex of hormonal, feeding and body-weight changes. Accurate and precise methods to quantitate circulating serum free leptin (f-leptin) concentrations are needed for physiological and clinical studies. We developed a one-step enzyme immunoassay to measure human f-leptin in serum. The detection limit was 0.40 ng/ml. The recovery of leptin added to serum was 90.8–102.8%. The within-run and between-day coefficients of variation (C.V.) ranged from 2.8 to 7.7 and 5.7 to 9.7%, respectively, and the immunoassay had an overall recovery rate for serial dilution in the range of 94.0–109.9%. Measured serum f-leptin concentrations in 201 adults correlated ( r=0.449, P<0.001) directly with body mass index (BMI kg/m 2), particularly when results were separated by gender ( r=0.709 for male, P<0.001; r=0.643 for female, P<0.001). We conclude that this one-step enzyme immunoassay is accurate for measuring f-leptin in human serum.

A New Principle for Rapid Immunoassay of Proteins Based on In Situ Precipitate-Enhanced Ellipsometry

A new technique is presented that allows measurement of protein concentrations in the picomolar range with an assay time of only 10–20 min. The method is an enzyme-linked immunosorbent assay (ELISA), but uses in-situ ellipsometric measurement of a precipitating enzyme product instead of the usual colorimetric detection of accumulating enzyme product in solution. Quantitative validation was obtained by use of annexin V, a protein with high binding affinity for phosphatidylserine-containing phospholipid membranes, resulting in a transport-limited adsorption rate. This property was exploited to obtain a range of low surface concentrations of annexin V by timed exposures of phospholipid bilayers to known concentrations of annexin V. Using polyvinylchloride (PVC)-coated and silanized silicon slides, various versions of this technique were used for the rapid assay of fatty acid-binding protein (FABP), a recently introduced early marker for acute myocardial infarction with a normal plasma concentration below 1 nmol/l, interleukin 6 (IL-6), a cytokine with normal plasma concentrations below 1 pmol/l, and again, annexin V. A possible future application of the method in the development of a one-step ELISA is discussed.

Development of a monoclonal antibody to urinary degradation products from the C-terminal telopeptide α1 chain of type I collagen. Application in an enzyme immunoassay and comparison to CrossLaps™ ELISA

A monoclonal antibody MAbA7 was raised against a synthetic peptide having a sequence (EKAHDGGR) specific for a part of the C-telopeptide α1 chain of type I collagen. MAbA7 was labelled with horseradish peroxide and used in a competitive one-step enzyme-linked immunosorbent assay (ELISA) for measurement of urinary type I collagen degradation products. The assay was technically evaluated and preliminary clinical data are presented. The measuring range was 200–7000 µg 1−1 with a detection limit of 25 µg 1−1. Within-run and total CVs were 5.5 and 8.0%, respectively. Analytical recovery averaged 96.6%±M5.3 (mean±1SD). Values obtained in the ELISA were highly correlated (r=0.93) to values obtained by a commercially available assay (CrossLaps™ ELISA) known to measure urinary degradation products derived from the C-telopeptide of type I collagen reflecting the rate of bone resorption. Investigation of the urinary fragments responsible for the immunological response in the two assays revealed, however, that they are not identical. Values obtained in urine samples from postmenopausal women (n=108) and patients with Paget's disease (n=6) increased 43% (p<0.01) and 28-fold (p<0.001), respectively, when compared to a premenopausal level (n=50). A decrease in the urinary concentrations of 67% (p<0.01) was seen after 6 months in urine samples from postmenopausal women (n = 13) receiving hormone replacement therapy (HRT) compared to a group receiving placebo (n=9). Likewise, the urinary concentrations decreased 88% (p<0.001) in early postmenopausal women receiving bisphosphonate therapy (n = 11) for a period of 9 months compared to a group receiving placebo (n = 12). These results suggest that the monoclonal antibody and the new assay may be useful for further investigations of the physiological and clinical importance of type I collagen degradation.

Extraction and Enzyme Immunoassay of Sulfadimethoxine Residues in Channel Catfish (Ictalurus punctatus) Muscle

Four commercially available enzyme immunoassays were evaluated for the detection of sulfadimethoxine (SDM) residues in channel catfish muscle. Matrix solid-phase dispersion (MSPD) extracts of samples (n = 60, 0.5 g) fortified with SDM at 0, 25, 50, 100, and 250 ng/g were used in all assays. Intra-assay variations for 2 MICROTITER well-based quantitative assays, SIGNAL sulfamethazine test and IDS SDM One-Step ELISA (enzyme-linked immunosorbent assay), were 5.6 and 7.7%. Interassay variations for these tests were 7.9 and 16.6%. The agreements between evaluators for 2 membrane-based, visually determined assays, CITE Sulfa Trio and EZ-SCREEN: SDM, were 77 and 95%. Performance values, as indicated by sensitivity, specificity, efficiency, and positive and negative predictive values, were 100, 92, 95, 89, and 100%, respectively for the SIGNAL test; 100, 94, 97, 92, and 100% for the IDS test; 98, 71, 82, 69, and 98% for the CITE test; and 98, 94, 96, 92, and 99% for the EZ-SCREEN assay. Eight sulfonamides cross-reacted in the SIGNAL test; EC-50 values (concentrations causing 50% inhibition of color development compared with blanks) varied from < 0.1 to 45 micrograms/mL. The EC-50 value for SDM was 0.25 microgram/mL. The CITE test cross-reacted with sulfachloropyridazine at 10 micrograms/mL. The IDS and EZ-SCREEN tests had no significant cross-reactivity with other sulfonamides. N-Acetyl SDM reacted like the parent SDM in all assays. Performance results indicated that MSPD extracts of catfish muscle may be used in these immunoassays to screen catfish muscle samples for violative levels of SDM residue.

Rapid serotyping of infectious pancreatic necrosis virus by one-step enzyme-linked immunosorbent assay using monoclonal antibodies

A one-step ELISA has been developed for detection and serotyping of infectious pancreatic necrosis virus (IPNV) in infected cell cultures using monoclonal antibodies (mAb) raised against strains representing the Sp, Ab and VR 299 serotypes of IPNV. This assay uses a serotype-specific mAb as capture and a mAb directed against a common epitope as detector antibody. Avidin-peroxidase was employed for amplification. The assay was performed in 90 min by simultaneous incubation of the samples, the biotin labelled mAb and the avidin-peroxidase, and detected 37 ng/ml of purified virus. Serotyping of 34 isolates from different areas of Europe, Asia and America showed a total concordance with the results obtained by the neutralization assay.

A one-step enzyme-linked immunosorbent assay to detect anti-CMV antibodies; development and clinical validation

A simple one-step ELISA to detect anti-CMV antibodies was developed. The test was based on the inhibition principle, and used anti-CMV coated microtitre plates, CMV nuclear antigen and anti-CMV Fab'-HRP conjugate; total assay time was 1.5 h. The use of Fab'-HRP conjugates improved discrimination between positive and negative sera as compared with IgG-HRP conjugates. In an in-house clinical study, the one-step ELISA showed a 98.4% correlation with the latex agglutination test. The test could be demonstrated to be suitable for both serum and citrate- or EDTA-plasma specimens; heparin-plasma gave somewhat higher absorbance values. In an external clinical validation, the one-step ELISA showed a 99.5% correlation with the latex agglutination test, and was found to be highly suitable for screening of blood donor specimens in a blood bank setting.