Classical Enzyme-linked Immunosorbent Assay Research Articles

Comparison of Electrochemical and Spectro-Photometric Detection in Hrp-Based Elisa for Tobacco Mosaic Virus

ABSTRACT An electrochemical method was compared with a spectrophotometric method using a horseradish peroxidase (HRP)-based indirect enzyme-linked immunosorbent assay (ELISA) with direct antigen coating (DAC) technique for the determination of tobacco mosaic virus (TMV). In substrates for a spectrophotometric HRP-based ELISA method, was selected as the substrate for the electrochemical method as well as the spectrophotometric method. 2, 3-Diaminophenazine is the product of the oxidation of OPD with H2O2 catalyzed by HRP in the selected conditions. It is electroactive and has a sensitive voltammetric response at -0.93 V (vs. Ag/AgCl) in alkaline solution. The detection limit of free HRP, by second-order derivative linear-sweep voltammetry, is 1.1 mU/L. This method can be further used to detect TMV antigen. The peak current is linear with TMV concentration in the range of 0.25-5.0 ng/mL. The detection limit for TMV is 0.25 ng/mL and the highest dilution ratio detected for the infected leaf sap is 1:102400. Compared with the classical OPD ELISA spectrophotometric method, the detection limits of the electrochemical method for the clarified TMV and the infected leaf sap detection are about ten and four times lower, respectively.

Failure of soluble fibrin polymers in the diagnosis of clinically suspected deep venous thrombosis.

A new diagnostic test has recently become available which is highly specific for plasma soluble fibrin polymers, the thrombus precursor protein (TpP) enzyme immunoassay. In order to evaluate the diagnostic accuracy of this test and that of a new rapid and automated test for the determination of D-dimers, the BC D-dimer test, in patients with clinically suspected deep vein thrombosis (DVT), 70 consecutive symptomatic patients underwent laboratory analysis with both tests and with the classic enzyme-linked immunosorbent assay (ELISA) D-dimer test, followed by the execution of a compression ultrasound (CUS) test of the affected limb. Patients with a positive CUS test were considered to have DVT (20 of 70), whereas those with a serially negative test and an uneventful 3-month follow-up test were regarded as not having DVT (50 of 70). The sensitivity of the TpP test (45.0%) was significantly lower than that of both the BC D-dimer test (80.0%; P = 0.02) and the classic ELISA test (90.0%; P = 0.002). The specificity of the TpP test (66.0%) did not differ from that of either D-dimer test (60.0 and 64.0%, respectively). The negative predictive value of the TpP test (75.0%) was significantly lower than that of the classic ELISA D-dimer test (94.1%; P = 0.02), which in turn did not differ from that of the BC D-dimer test (88.2%). The positive predictive value was similarly low for each investigated test (34.6, 44.4, and 50.0%, respectively). In conclusion, the TpP test can neither be used to detect a DVT nor to exclude its development in patients with the clinical suspicion of this disease. By contrast, the BC D-dimer might safely replace the classic ELISA test for excluding DVT in symptomatic patients.

Prognostic value of urokinase plasminogen activator in primary breast carcinoma: comparison of two immunoassay methods

Urokinase-type plasminogen activator (uPA) is a potentially important prognostic factor in breast cancer for identifying patients at high risk of recurrence. This retrospective study assessed two enzyme-linked immunosorbent assay (ELISA) methods measuring uPA antigen levels in 499 primary breast cancer cytosols. Both uPA methods were applied to cytosols used routinely for oestrogen (ER) and progesterone (PgR) receptor assays. uPA was determined using a classical ELISA method (Imubind; American Diagnostica) and a novel automatic immunoluminometric assay (Lia; Sangtec Medical). The uPA Imubind method revealed about twice as much uPA antigen (median 0.75 ng mg(-1) protein) as the uPA Lia method (median 0.38 ng mg(-1) protein). The correlation coefficient between the two methods was acceptable (r = 0.81), but the two techniques are not interchangeable. Univariate analyses confirmed the poor outcome of patients whose tumours contained large amounts of uPA, regardless of the technique used. Multivariate analyses showed that uPA Imubind and uPA Lia values were both strong independent prognostic factors.

Reliability of Five Rapid D-Dimer Assays Compared to ELISA in the Exclusion of Deep Venous Thrombosis

Studies measuring the fibrin degradation product D-Dimer (DD) using enzyme-linked immunosorbent assays (ELISA) in patients with venographically proven deep venous thrombosis (DVT) suggest that it is possible to exclude DVT when DD level is below a certain cut-off level. However, ELISA methods are time-consuming and not available in all laboratories. Different rapid latex-agglutination assays have been investigated, but their sensitivity is considerably lower. In the present study we compared the value of four novel latex DD tests (Tinaquant, Minutex, Ortho and SimpliRed) and one rapid ELISA (VIDAS) to a classical ELISA DD assay (Organon Mab Y18) in 132 patients suspected of DVT. The VIDAS, a new quantitative automated ELISA, had a sensitivity of 100% and a negative predictive value of 100% for both proximal and distal DVT at a cut-off level of 500 ng/ml. The Tinaquant assay, a new quantitative latex method, had a sensitivity of 99% and a negative predictive value of 93% for both proximal and distal DVT at a cut-off level of 500 ng/ml. For proximal DVT only, both assays had a sensitivity and negative predictive value of 100%. VIDAS and Tinaquant correlated well with ELISA (correlation of r = 0.96 and r = 0.98 respectively). Sensitivities of the semi-quantitative latex assays Minutex, Ortho and SimpliRed were considerably lower (77%, 51% and 61% respectively). These results suggest that VIDAS and Tinaquant may be used instead of ELISA DD in the exclusion of DVT. Tinaquant can be performed within 20 min and VIDAS within 35 min. Both assays might be used as a routine screening test and should be evaluated in large clinical management studies.

A new, rapid, noninvasive screening test for celiac disease

Serum levels of IgA and IgG antigliadin antibodies were evaluated in 267 subjects by using both the classic enzyme-linked immunosorbent assay and a rapid test (strip-AGA test) performed on a drop of whole blood. This new test was easy to perform and gave results comparable to those of the enzyme-linked immunosorbent assay; it could be useful in screening for celiac disease in both at-risk and normal populations.

Dual HIV-1 and HIV-2 infection in West Africa supported by synthetic peptide analysis.

Double Western blot human immunodeficiency virus (HIV)-1 and HIV-2 antibody-positive patients have been reported from West Africa suggesting the phenomenon of double infection. To evaluate this possibility synthetic peptide analysis was performed on 58 sera from a West African population. Site-directed serologic studies based on the use of synthetic peptides located downstream of HIV-1 gp 41 and HIV-2 gp 36 have been shown to be type specific and well suited for discrimination between HIV-1 and HIV-2 antibody responses. When tested with radioimmunoprecipitation assay or Western blot 10 of these 58 serum samples were reactive with both HIV-1 gp 41 and HIV-2 gp 36; the remainder reacted with other combinations of HIV-1 and HIV-2 envelope glycoproteins. When submitted to an indirect enzyme-linked immunosorbent assay (ELISA) test in peptide 39 or 41.2.1 coated microtiterplate wells 3 reacted only with peptide 39 25 reacted only with peptide 41.2.1 and 30 reacted with both these peptides. To further investigate the double reactivity observed in these 30 sera 14 such samples were submitted to a classic competition ELISA test. The binding capacity of antibodies to peptide 39 was only inhibited by peptide 39; similarly the binding capacity of antibodies to peptide 41.2.1 was only inhibited by this peptide. No significant cross-reactions could be observed between the 2 peptides in any of the samples analyzed indicating the simultaneous presence of the 2 antibody species.

Detection of Salmonella infections by polyvalent enzyme-linked immunosorbent assay.

Serum immunoglobulin G (IgG) and IgM were measured for individual Salmonella species by the enzyme-linked immunosorbent assay (ELISA). F344 rats were experimentally infected with Salmonella typhimurium (serogroup B), S. enteritidis (serogroup D), and S. rubislaw (serogroup G.) Endotoxin extracted from each serogroup served as the antigen in a classical indirect ELISA. Antibody specific for each Salmonella serogroup was detected by ELISA. Normal gut flora from control animals appeared not to cause cross-reactions in the ELISA. Specificity and sensitivity of the IgG ELISA were determined by statistically evaluating false-positives and false-negatives. Ideal values of 90% or better were achieved in nearly all instances. Each antigen was also tested with heterologous antisera in an effort to develop a polyvalent assay for Salmonella species. No single antigen detected all positive heterologous antisera. Therefore, a polyvalent antigen composed of the three serogroup antigens was tested. The results suggested that Salmonella infections can be detected by measuring serum IgG levels with a polyvalent ELISA 6 to 9 days postinfection.