A convenient homogeneous enzyme immunoassay for estradiol detection

A convenient homogeneous enzyme immunoassay for estradiol is described. Unlike heterogeneous immunoassays, which require time-consuming separation steps or expensive automated systems, homogeneous immunoassays, wherein all reagents are freely suspended in bulk solution, can be simple and fast without costly instrumentation. The key component of this assay system, an estradiol-reporter enzyme conjugate, was prepared by covalently binding β-estradiol-6-(O-carboxymethyl)oxime to glucose-6-phosphate dehydrogenase (G6PDH) by an N-hydroxysuccinimide-enhanced, carbodiimide-mediated coupling reaction. The estradiol-G6PDH activity can be repressed up to 46% upon anti-estradiol antibody binding. The lower detection limit of the assay is 1 nM estradiol in aqueous solution, and the standard curve is linear on logit-log scale-up to 6.7 μM estradiol. A detection limit of 11.5 nM in estradiol-spiked human serum samples suggests the feasibility of applying this assay to monitor estradiol levels for the prediction and prevention of ovarian hyperstimulation syndrome.     

Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A.

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.     

Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A.

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.     

Capillary electrophoretic enzyme immunoassay with electrochemical detection for thyroxine

A capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) has been developed. In this method, antigen (Ag) competes with horseradish peroxidase (HRP)-labeled antigen (HRP-Ag) for a limited number of antibody (Ab) binding sites. The free HRP-Ag and the bound HRP-Ag-Ab complex are separated by capillary electrophoresis in a separation capillary. Then they catalyze the oxidation of their enzyme substrate 3,3',5,5'-tetramethylbenzide (TMB (reduced form)) with H(2)O(2) in a reaction capillary, which follows the separation capillary. The reaction product (TMB (oxidized form)) is amperometrically determined using a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Due to the amplification of the enzyme, the concentration of TMB(Ox) is much higher than those of free HRP-Ag and the bound HRP-Ag-Ab complex. Therefore, the limit of detection (LOD) of CE-EIA-ED is very low. The method has been used to determine thyroxine in human serum. A concentration of LOD of 3.8 x 10(-9)mol/L, which corresponds to a mass LOD of 23.2 amol, was achieved.     

Heterogeneous enzyme immunoassay.

During the past 10 to 15 years immunoassays have gained acceptance as the methods of choice in the diagnosis of a number of disease states. At present the immunodiagnostic techniques employed range from radioimmunoassay for haptens through immunofluorescence for autoimmune diseases to complement fixation for viral infections. All of these assays have their own individual limitations such as: safety, short shelf life and sensitivity. The development of enzyme immunoassays, in particular enzyme linked immunosorbent assay (ELISA), has led to a substantial literature which offers the view that enzyme immunoassays provide a safe, sensitive and specific alternative to standard methods for the detection of antibodies or antigens. The application of heterogeneous enzyme linked immunosorbent assays for the quantitation of haptens, macromolecular antigens and antibodies is reviewed.     

An enzyme immunoassay for detection of Japanese encephalitis virus-induced chemotactic cytokine

Japanese encephalitis virus (JEV) induces human peripheral blood monocytes to secrete a chemotactic cytokine [human macrophage-derived factor (hMDF)] which causes chemotaxis of neutrophils. The only known assay for hMDF cannot quantify its level in samples, so an enzyme immunoassay has been standardized for detection of hMDF and hMDF-specific antibodies in test samples. The reported enzyme linked immunosorbent assay (ELISA) was found to be sensitive (89%), specific (91%), accurate (92 2%) and reproducible and was able to detect a minimum concentration of 23 ng hMDF/ml in test samples. The chemotactic factor could be detected in JEV inoculated mouse sera and JEV infected culture fluids. Significant finding of the test was the detection of hMDF in sera of human cases of JE.     

A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles–chemiluminescence enzyme immunoassay (MPs–CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol–H₂O₂ chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs–CLEIA method had a linear range from 0.31 to 100 ng/ml with a detection limit of 0.24 ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA.     

Subfemtomole enzyme immunoassay for human growth hormone using affinity chromatography and enzyme amplified detection

An immunometric assay is described which allows fast detection of attomole amounts of an antigen. The sensitivity is 100 to 1000 times better than that of classical sandwich immunometric assays. Our system allowed the measurement of human growth hormone in the range of 0.1 amol to 100 fmol in a 4-h time period overall. A chromatography column is sequentially filled with two immunoaffinity resins: SP-M1--E1-Ab1 in the upper half and SP-M2--E2-Ab2 in the lower half, where Ab1 and Ab2 represent complementary antibodies reacting with the antigen to be assayed, E1 and E2 represent enzymes, M1 and M2 represent substances reacting reversibly with E1 and E2, respectively, and SP represents the chromatographic solid phase; the sign - represents covalent linkages and the sign--reversible linkages. The sample solution is passed through the column, resulting in binding of the antigen to the first encountered antibody, yielding the immobilized complex SP-M1--E1-Ab1--Ag. The M1 bound is then destabilized by washing with solution of agonist to M1. The freed complex is immediately trapped by the second antibody in the lower part of the column, resulting in the entity SP-M2--E2-Ab2--Ag--Ab1-E1. After a washing step, and amplified detection allows the measurement of the antigen through the activity of the enzyme E1. The antigen-antibody reactions occur in the presence of a very large excess of antibody. The continuous equilibrium displacement due to the chromatographic procedure enhances the yield of complex formation. These factors explain the extremely low levels (subattomole) capable of being detected with this original technique.     

Rapid detection of Staphylococcus aureus using bioluminescent enzyme immunoassay

A bioluminescent enzyme immunoassay (BLEIA) method for detecting protein A-bearing Staphylococcus aureus was developed using biotinylated firefly luciferase. The BLEIA was able to detect protein A at one pg ml-1 and 103 cfu ml-1 level of Staph. aureus. The BLEIA showed significant signals with overnight cultures of all 24 Staph. aureus strains, and the BLEIA did not show any significant signals with overnight cultures of all 44 strains of coagulase-negative staphylococci and the other genus bacteria. After 5 h cultivation beginning at approximately 50 cfu ml-1, the BLEIA was able to detect all 35 Staph. aureus strains isolated from healthy humans.     

Deaminating enzyme labels for potentiometric enzyme immunoassay

Adenosine deaminase, asparaginase, and urease are examined as possible enzyme labels for immunoassays using potentiometric detection with the ammonia gas-sensing membrane electrode. Considerations of binding ability, retained activity, and stability reveal asparaginase to be the most effective enzyme label for immunoassay purposes. The utility of the potentiometric approach with this enzyme label is demonstrated via model hapten assays for dinitrophenyl groups and for cortisol.     

A modified rapid enzyme immunoassay for the detection of rabies and rabies-related viruses: RREID-lyssa.

This paper presents a modification of the previously described Rapid Rabies Enzyme Immuno-Diagnosis test (RREID) by using biotinylated antibodies, streptavidin conjugate and a mixture of monospecific polyclonal antibodies against several lyssaviruses. In the modified technique (RREID-lyssa), microplates were sensitized with a mixture of purified antibodies against ribonucleoprotein (RNP) from Pasteur virus (Lyssavirus serotype 1), European Bat Lyssavirus (EBL, unclassified) and Mokola virus (Lyssavirus serotype 3). Bound RNP was detected by the same antibodies labelled with biotin and peroxidase-strepavidin conjugate. These techniques were used for the detection of RNP of different Lyssavirus serotypes (rabies and rabies-related viruses). For lyssavirus specimens of serotype 1, the threshold of detection of RREID and RREID-lyssa were similar. However, a smaller amount of labelled antibodies was needed when biotinylated antibodies were used. For specimens infected by rabies-related strains (serotypes 2, 3, 4 and EBL), the threshold of detection of the RREID-lyssa was between two and 512 times lower than with the RREID. The sensitivity and the specificity of the RREID-lyssa for rabies virus (serotype 1) when tested on a small field trial (53 specimens) were found to be identical to the RREID. Consequently, RREID-lyssa can be a useful tool for diagnostic laboratories that receive specimens infected by rabies-related viruses.     

An enzyme immunoassay for secretin

A sensitive and specific enzyme immunoassay for secretin was developed with the use of enzyme-labeled antigens. Synthetic porcine secretin and its carboxy-terminal fragments (residues 11-27 and 18-27) were conjugated with beta-D-galactosidase for use in the immunoassay, and the assay method with the latter fragment (residues 18-27) linked to beta-D-galactosidase was found to be the most sensitive. The minimum amount of secretin detectable by this method was 1-2.5 pg/assay. Serum levels of secretin after intravenous injection of the peptide in rats were determined by both the enzyme immunoassay and a commercial radioimmunoassay kit. The correlation coefficient between the levels measured by the two methods was 0.984. The enzyme immunoassay could detect immunoreactive secretin levels in normal human sera, giving a value of 16.9 +/- 2.2 pg/ml (mean +/- SE of six human subjects).     

Type-specific enzyme immunoassay for detection of pneumococcal capsular polysaccharide antigens in nasopharyngeal specimens

We developed a new competitive EIA method for the demonstration of pneumococcal capsular polysaccharides from respiratory samples. The pediatric types 4, 6B, 9V, 14, 18C, 19F and 23F were selected for this study, because these capsular polysaccharides were included in the first heptavalent pneumococcal conjugate vaccines, which were used in the Finnish Otitis Media Vaccine Trial. Sensitivity of the EIA tests for purified polysaccharide antigens varied between 5 and 100 ng/ml, depending on the type. The assays performed well in 100 nasopharyngeal samples (NPS) samples processed through an enrichment culture, with an almost 100% sensitivity compared with routine culture. The method appeared type-specific, except that EIA for 6B capsule also detected 6A. The method is applicable for type-specific identification of pneumococcus in carriage studies.     

An indirect enzyme immunoassay for the mycotoxin citrinin.

An indirect competitive enzyme immunoassay using rabbit antisera could detect citrinin in buffer solutions at 1 to 13 ng/ml (0.05 to 0.65 ng per assay). Cross-reactivity with austdiol, alternariol, ochratoxin A, and deoxynivalenol was < 0.1% relative to citrinin. Recovery of citrinin added to wheat flour at 200 to 2,000 ng/g was 89 to 104%, with a coefficient of variation of 6.9 to 13%.     

Simultaneous competitive enzyme immunoassay for human chorionic gonadotropin.

A simultaneous competitive enzyme immunoassay (SICEIA) for hCG was developed using beta-D-galactosidase (beta-Gal) as a labelled enzyme and anti-hCG antibody coated sheep red blood cells (SRBC) as a solid phase. In this report, a new coupling agent, MCAE, was used to couple beta-Gal with hCG. The sensitivity was improved to the degree of 2.5 mIU/ml, equal to that of RIA. The present procedure was safer and rapider than RIA. The value of hCG in urine by our procedure had good correlation with that by RIA.     

Electrochemical cell-based chip for the detection of toxic effects of bisphenol-A on neuroblastoma cells

A cell-based chip was fabricated for the electrochemical detection of the dose-dependent effects of bisphenol-A (BPA) on neuroblastoma cells (SH-SY5Y), which showed dual-mode correlation as a standard curve. Toxicity assessment of BPA became very important in environmental toxicants detection since BPA can be reached out easily from various common plastic-based product and give negative cellular effects on living organism. Cell chip was fabricated by immobilizing cells on C(RGD)(4) peptide coated electrode to detect the cytotoxicity of BPA electrochemically. Redox properties in living cells were determined by cyclic voltammetry using a home-made three-electrode system, and the cathodic peak current (I(pc)) was used as a parameter for measurement of the effect of BPA on cell viability. The peak current, I(pc) value increased with the concentration of BPA up to 300 nM and then decreased because of the stimulation of cancer cell activity at the concentration of BPA below 300nM and cytotoxicity at the concentration of BPA above 300 nM, respectively. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and optical microscopy-based morphological analysis confirmed the results of electrochemical study. This dual-mode correlation between the concentration of BPA and voltammetric signal intensity should be firstly considered to analyze its dose-dependent stimulus and cytotoxic effects on neuroblastoma cells by cell chip.     

Fluorophotometric enzyme immunoassay of thyroid-stimulating hormone.

A method for enzyme immunoassay of thyroid-stimulating hormone (TSH) is described, TSH was conjugated with horseradish peroxidase according to periodate oxidation method. Separation of the bound and free was obtained by double-antibody solid-phase technique using Sepharose 4B-anti-rabbit immunogiobulin G (IgG)-geat IgG. The fluorescence reaction using tyramine and hydrogen peroxide as substrates was used for the determination of enzyme activity in order to increase the sensitivity of enzyme immunoassay. The standard curve for serum TSH was satisfactory to recognize TSH concentrations as 0.06 μU/tube. TSH values obtained by this method correlated well with those obtained by radioimmunoassay (r, 0.96). The coefficients of variation were 1.8 to 5.3% (within assay) and 5.1 to 10.5% (between assay). The method is about equal to radioimmunoassay with respect to sensitivity. Since it requires minimal equipment and is less expensive than radioimmunoassay, it is possible to perform routine assays even in laboratories with limited facilities.