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.     

Modeling of homogeneous cloned enzyme donor immunoassay

One of the most widely used analytical techniques for sensitive detection of biologically and clinically significant analytes is the immunoassay. In recent years direct immunoprobes allowing label-free detection of the interaction between the antibody and the target analyte have proved their capabilities as fast, simple, and nevertheless highly sensitive methods. Cloned enzyme donor immunoassay (CEDIA) homogeneous assay is based on the bacterial enzyme beta-galactosidase, which has been genetically engineered into two inactive fragments, enzyme donor and enzyme acceptor. Reassociation of the fragments in the assay forms active enzyme, which acts on substrate to generate a colored product. A comprehensive kinetic model of CEDIA is developed to aid in understanding this method and to facilitate development of a truly homogeneous version, potentially applicable to a dipstick-type multianalyte point of care analytical device (ChemChip). Although the standard assay involves a two-step process, we also chose to model a single-combined process, which would be simpler to apply in a ChemChip device. From the modeling simulation, we obtain the time courses of the amounts of product and active enzyme, from which the dynamic ranges can be obtained as 10(-6)-10(-7) and 10(-5)-10(-7)M analyte concentration for two-step and single-combined processes under the conditions of the assumed parameters, respectively. A simple one-step immunoassay has the merit of reducing time and cost and has an improved dynamic range.     

Enzyme channeling immunoassay: A new homogeneous enzyme immunoassay technique

A homogeneous enzyme immunoassay has been developed in which an antigen and glucose-6-phosphate dehydrogenase are coimmobilized on agarose beads. Binding of hexokinase-labeled antibody to the bead-bound antigen results in an accelerated conversion of glucose, ATP, and NAD⁺ to 6-phosphogluconolactone, ADP, and NADH. Critical parameters affecting assay response are discussed.     

A homogeneous enzyme immunoassay with avidin-ligand conjugate as the enzyme-modulator

A homogeneous enzyme immunoassay (EIA) based on the immunomodulation of an avidin-ligand conjugate and the inhibition of pyruvate carboxylase is described. The conjugation of the ligand, 5,5-diphenylhydantoin (DPH), to avidin does not affect avidin's capacity to bind biotin or inhibit pyruvate carboxylase. The DPH-avidin conjugate and free DPH were shown to compete for a limited number of antibody sites. The interaction of anti-DPH with the DPH-avidin conjugate sterically inhibited enzyme inactivation. Enzyme activity was correlated with DPH concentrations in the therapeutic range found in serum.     

Development of enzyme immunoassay for the herbicide chlorsulfuron

Antibodies against the herbicide chlorsulfuron have been raised and characterized. Enzyme immunoassays (EIAs) for chlorsulfuron, involving labeled antigen or labeled antibodies, have been developed. The kinetics of antigen-antibody interactions in the EIA systems developed has been studied. Both systems exhibit equal sensitivity (1 ng/ml). The values of the coefficient of variation (CV), determined within the range of chlorsulfuron concentrations 1-100 ng/ml to be measured by the systems, are not in excess of 8%. The possibility of using glucose oxidase as a label in EIAs for chlorsulfuron has been demonstrated. Lack of cross-reactivity with a series of sulfonyl- and arylurea derivatives and triazines makes it possible to recommend the EIA systems developed for chlorsulfuron determination in the environment.     

Development of an enzyme immunoassay for cryptococcal antibody

An enzyme immunoassay (ELISA) for measurement of cryptococcal Ig G antibody in human serum is described. Clinical studies indicate that the assay is a useful addition to the currently available techniques for measuring antibodies in cryptococcosis. IgG-specific antibody (titers 4 to 1,024) was detected in the serum of 78 % of the cryptococcosis patients tested and in 61 % of the serum from healthy individuals with positive delayed skin hypersensitivity to cryptococcin. The micro-ELISA for cryptococcal antibody is of potential value in patient management, and in epidemiological studies.     

Mutagenic activity of the organophosphorus insecticide chloracetophon

Chloracetophone (0,0-dimethyl-2,2,2-trichloro-1-(1-chloroacetoxy)phosphonate) is a new organophosphorus insecticide. The LD50 for male rats is 420 mg/kg b.w. The mutagenic activity was evaluated with the method of cytogenetic analysis of rat bone marrow after acute and short-term oral exposure. In the acute study groups of male and female animals were treated with chloracetophone at a dose of 1/5 LD50 and then examined at periods of 6, 12, 24, 48 and 72 h following administration. Separate groups of animals were examined 24 h after single administration of the doses 1/5, 1/10 and 1/20 LD50. In the short-term study groups of male animals were treated for 5 successive days. The slides were prepared 6 or 24 h after the last administration. Concurrent negative and positive (cyclophosphamide 20 mg/kg b.w.) control groups were used. 50 cells at metaphase per animal were scored for chromosomal aberrations. The results did not show significant increase in the frequency of chromosomal breaks in the groups with chloracetophone.     

Hapten synthesis for enzyme-linked immunoassay of the insecticide triazophos

Two haptens of the insecticide triazophos (O,O-diethyl O-[1-phenyl-1H-1,2,4-triazol-3-yl] phosphorothioate) were synthesized by introducing appropriate spacers in the O-ethyl site of the analyte molecular structure. First, thiophosphoryl chloride (PSCl(3)) reacts with methanol at low temperature to give O-ethyl dichlorothiophosphate. After reacting with 1-phenyl-3H-1,2,4-triazol, the O-ethyl dichlorothiophosphate was transformed into the intermediate O-ethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) chlorothiophosphate. Then the intermediate reacts with 4-aminobutyric acid and 6-aminobutyric acid to produce hapten I and hapten II, respectively. The molecule structures of the two haptens were identified by (1)H nuclear magnetic resonance spectrum and mass spectrum. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was also developed to evaluate the two haptens. Results showed that the monoclonal antibodies with high titers were obtained after immunizing with protein conjugates of these haptens and that the immunoassay has high affinity and specificity to triazophos. These results suggested that the haptens were synthesized successfully and could be used for immunoassay for the rapid screening and sensitive determination of this insecticide.     

Biodetoxification of coumaphos insecticide using immobilizedEscherichia coli expressing organophosphorus hydrolase enzyme on cell surface

Recently, we reported an improved technology for the degradation of organophosphate nerve agents using whole cells of genetically engineeredEscherichia coli that anchored and displayed the enzyme organophosphorus hydrolase on the cell surface. In this paper we report the immobilization of these cells on highly porous sintered glass beads and the subsequent application of the immobilized cell in a continuous-flow packed bed bioreactor for the biodetoxification of a widely used insecticide, coumaphos.     

Genotoxicity studies on the organophosphorus insecticide chloracetophone

Chloracetophone (O,O-dimethyl-2,2,2-trichloro-1-(chloroacetoxy)phosphonate), a new insecticide of the organophosphorus group of pesticides, was tested for genotoxicity in a variety of systems with different genetic end-points and varying parameters. The test systems included 2 microbial systems, Salmonella and Aspergillus for point mutations and mitotic segregation, respectively, and human lymphocyte cultures and mammalian bone marrow cells (from rats and hamsters treated acutely and subacutely) for chromosomal aberrations and micronuclei. Chloracetophone was negative in Aspergillus at concentrations of 1-500 micrograms/ml, in human lymphocyte cultures at concentrations of 2.5-40 micrograms/ml, in rats at doses of 420-21 mg/kg b.w. and in hamsters at doses of 210-42 mg/kg b.w. for chromosomal aberrations. It did not cause any increase of micronuclei in human lymphocytes and rat bone marrow cells but did cause a significant increase in hamster bone marrow cells. Chloracetophone induced base-pair substitutions in strain TA100 of Salmonella with and without metabolic activation at a concentration range of 2000-6000 micrograms/plate.     

A new homogeneous enzyme immunoassay. Its application to measurement of alpha-fetoprotein

A rapid and sensitive homogeneous enzyme immunoassay (homogeneous EIA) was developed for determination of serum proteins such as alpha-fetoprotein (AFP). There are two assay systems, one is a competitive system including horseradish peroxidase (HRP)-labeled antigen, antibody and substrate, and the other is a non-competitive system including HRP-labeled antibody and substrate. When the aggregate was formed through the binding of HRP-labeled AFP and anti-AFP antibody or through the binding of HRP-labeled anti-AFP antibody and AFP, HRP of the aggregates, as compared with HRP of free conjugates, exhibited marked activity in the presence of 35 mM H2O2. The extent of stimulation of HRP activity depended on the amount of AFP. This new assay method is very simple and sensitive, and can be used for the determination of any kind of protein, hormone, or drug.     

A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion

A consortium comprised of two engineered microorganisms was assembled for biodegradation of the organophosphate insecticide parathion. Escherichia coli SD2 harbored two plasmids, one encoding a gene for parathion hydrolase and a second carrying a green fluorescent protein marker. Pseudomonas putida KT2440 pSB337 contained a p-nitrophenol-inducible plasmid-borne operon encoding the genes for p-nitrophenol mineralization. The co-culture effectively hydrolyzed 500 microM parathion (146 mg l(-1)) and prevented the accumulation of p-nitrophenol in suspended culture. Kinetic analyses were conducted to characterize the growth and substrate utilization of the consortium members. Parathion hydrolysis by E. coli SD2 followed Michaelis-Menten kinetics. p-Nitrophenol mineralization by P. putida KT2440 pSB337 exhibited substrate-inhibition kinetics. The growth of both strains was inhibited by increasing concentrations of p-nitrophenol, with E. coli SD2 completely inhibited by 600 microM p-nitrophenol (83 mg l(-1)) and P. putida KT2440 pSB337 inhibited by 1,000 microM p-nitrophenol (139 mg l(-1)). Cultivation of the consortium as a biofilm indicated that the two species could cohabit as a population of attached cells. Analysis by confocal microscopy showed that the biofilm was predominantly comprised of P. putida KT2440 pSB337 and that the distribution of E. coli SD2 within the biofilm was heterogeneous. The use of biofilms for the construction of degradative consortia may prove beneficial.     

Development of a competitive enzyme immunoassay for factor VIII-related antigen

An enzyme immunoassay system basing on a competitive method has been developed to measure factor VIII related antigen (F. VIII R:Ag). A sufficient discrimination at low F. VIII R:Ag concentrations was gained. This method appears to be sensitive to 7,8 X 10(-3) U/ml F. VIII R:Ag showing an intraassay coefficient of variation (CV) of 0,11. In comparison to the commonly used Laurell electroimmunodiffusion assay for factor VIII significant less antisera per sample for the enzyme immunoassay technique is necessary.     

Development of a fast solid-phase enzyme immunoassay for C-reactive protein

A fast sandwich enzyme immunoassay has been developed for C-reactive protein (CRP). This method can be used for screening CRP concentration in large numbers of samples providing a non precipitation, non agglutination and non radioactive alternative for assessment of human CRP. Advantages over previously reported CRP sandwich assays include: assay time was reduced from 4 1/2 h to 45 min, incubations were made at room temperature instead of 37 degrees C and serum dilutions required were 100-400 fold instead of 10000-20000 fold. Correlations were good with both nephelometry and phosphorylethanolamine binding assay. The 45% false positives found with the slide-latex anti C-reactive protein method were reduced to 0% by the use of the described method.     

Effects of methanol and temperature on enzyme immunoassay with monoclonal antibodies specific to the insecticide etofenprox

We examined the effects of methanol and temperature on the reactivity of monoclonal antibodies specific to the insecticide etofenprox. When the antigen-antibody reaction was done at 4 degrees C in 10% methanol, the sensitivity in the enzyme immunoassay with each antibody was more than 10-fold higher than that measured at 37 degrees C. Although in 10% methanol one of the antibodies reacted equally with both etofenprox and the carbonate-derivative of etofenprox, in 50% methanol the antibody reacted with etofenprox, but not with the derivative.     

Direct and homogeneous immunoassay for IgG analyses

A concept involving the use of antibody conjugated with reporter molecules for direct sensing of subtle changes in the local electrostatic environment of an antigen-antibody complex due to the antigen binding is presented. The proposed direct sensing mechanism is studied using the protein A-immunoglobulin G (IgG) complex as the model system. The change in the local electrostatic potential and pH around the protein A-IgG complex is observed by measuring the fluorescence intensity of fluorescein molecules conjugated with protein A before and after the introduction of IgG. Factors affecting the efficiency of our direct sensing mechanism including the conjugation ratio of fluorescein per protein A molecule, Solution conditions, and the amount of protein A-fluorescein conjugate used in the experiment are investigated. The IgG(2A) concentrations in the cell supernatants are analyzed by using our direct sensing method and then compared with the results obtained from enzyme-linked immunosorbent assay (ELISA). The comparisons between our homogeneous assay and heterogeneous ELISA in the analysis time, analysis procedures, cost, and sensitivity are reported and discussed. (c) 1992 John Wiley & Sons, Inc.     

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.     

Indication for weak mutagenicity of the organophosphorus insecticide dimethoate in Drosophila melanogaster

The organophosphorus insecticide dimethoate was tested for induction of genetic damage in male germ cells of Drosophila melanogaster. Sex-linked recessive lethals, sex-chromosome loss and non-disjunction induction were studied following different routes of administration: adult feeding, injection and larval feeding. Our results show that, after injection, dimethoate induces a slight but significant increase in the frequency of point mutations.     

Development and clinical application of a bioluminescence enzyme immunoassay for oxytocin

The development of a highly sensitive analytical method for oxytocin could be useful in the diagnosis and treatment of autistic spectrum disorder. We previously developed a colorimetric enzyme immunoassay (EIA) for plasma oxytocin measurement. In this study, we developed a method to measure oxytocin concentrations using a higher sensitivity bioluminescent EIA. Biotinylated oxytocin bridged with five lysine residues was used in a competitive format. The standard curve range for oxytocin was 1.0 to 1000 pg/assay. In addition, there was good correlation between the colorimetric and bioluminescent immunoassays in terms of measured oxytocin concentration (r = 0.9665, n = 48). The bioluminescent EIA for plasma oxytocin was more rapid and provided higher sensitivity than the colorimetric immunoassay, making it suitable for clinical application.