Homogeneous Enzyme Immunoassay Research Articles

Comparative Studies between Dry Reagent System (Dry Chemistry) and Homogenous Enzyme Immunoassay for Measurement of Blood Theophylline Level in Bronchinl Asthma in Childhood

Comparative Studies between Dry Reagent System (Dry Chemistry) and Homogenous Enzyme Immunoassay for Measurement of Blood Theophylline Level in Bronchinl Asthma in Childhood

4560648 Homogeneous enzyme immunoassay for ferritin: Richar Armenta assigned to Syntex (U S A) Inc

4560648 Homogeneous enzyme immunoassay for ferritin: Richar Armenta assigned to Syntex (U S A) Inc

Laboratory Detection of Marijuana Use

To the Editor.— Schwartz and Hawks have provided an excellent discussion of the laboratory's role in the detection of marijuana use. 1 However, their statement that false-positive results of the homogeneous enzyme immunoassay technique (EMIT) and radioimmunoassay are rare when the tests are performed under optimal conditions requires clarification. Frederick et al 2 were unable to confirm by gas chromatography— mass spectrometry (GC-MS) the positive results obtained using the EMIT-d.a.u. (drugs of abuse in urine) on three of 42 urine specimens. Another study, using high-performance liquid chromatography and high-efficiency thin-layer chromatography, confirmed only 72% of the EMIT results that were positive at a lower cutoff of 20 ng/mL of 11-nor-Δ 9 -tetrahydrocannabinol-9-carboxylic acid (9-carboxy-THC). 3 The confirmation rate rose to 91% when 75 ng/mL was used as the lower detection limit. In a multicenter study, it was found that 2% of the results obtained from the EMIT single-test and EMIT-d.a.u.

Homogeneous colorimetric enzyme inhibition immunoassay for cortisol in human serum with Fab anti-glucose 6-phosphate dehydrogenase as a label modulator

The Fab fragment of rabbit IgG antibody to bacterial glucose 6-phosphate dehydrogenase covalently linked to the cortisol retained the capacity to inhibit the enzyme completely. In optimal conditions the antibody to cortisol effectively bound the cortisol residues of the cortisol-Fab conjugate, making it incapable of inhibiting the enzyme. The enzyme modulatory properties of the cortisol-Fab conjugate were exploited to set up a direct competitive homogeneous enzyme immunoassay for cortisol in human serum. The procedure involved the use of the auxiliary enzyme diaphorase, specific for NADH, which converts the nitro blue tetrazolium salt to a colored formazan. The procedure detects modulated glucose 6-phosphate dehydrogenase activity by a single-point measurement without serum interference. The assay working range was between 20 and 640 μg/1 of cortisol and used 50 μ1 of sample.

Laboratory Detection of Marijuana Use

Frequent smoking of cannabis (marijuana) has been shown to be associated with a decline in social, mental, and perceptual skills and, during adolescence, with maladaptive emotional development. Urinalysis for the detection of such use can be a useful tool for the physician responsible for treatment and counseling of adolescents who develop habitual use of marijuana. Primary methods for urinalysis detection of cannabis use include the homogeneous enzyme immunoassay (EMIT) and the radioimmunoassay. These and other methods are discussed along with the issues of "false" results (both positive and negative) and the "limits of interpretation" that can be placed on a positive urine result. The pharmacokinetics and metabolism of the active constituents of cannabis are described as well as the interpretation of urinalysis results as they relate to use patterns. Guidelines are presented for the primary care physician for selecting candidates for such testing and for the use of such tests in the treatment or counseling of adolescents for whom marijuana abuse has become a psychological and social problem.

Instability of succinyl ester linkages in O2′-monosuccinyl cyclic AMP-protein conjugates at neutral pH

Chromatographic and immunological evidence is presented regarding the hydrolysis of the ester linkage of O 2′-monosuccinyl cyclic AMP in neutral solutions. Such hydrolysis occurs whether the nucleotide derivative is present in free form in solution or conjugated through its succinyl carboxyl group via an amide bond to proteins. The latter process apparently occurs when succinyl cyclic AMP is conjugated to human serum albumin for use as an immunogen in the production of anti-cyclic AMP antibodies and when the derivative is coupled to the enzyme glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49). The enzyme conjugate has been used in developing a homogenous enzyme immunoassay for cyclic AMP. Inhibition of the catalytic activity of enzyme-cyclic AMP conjugates by anti-cyclic AMP antibody decreases with time, apparently due to the loss of cyclic AMP from enzyme-cyclic AMP conjugates stored in neutral solutions. In addition, the ability of free cyclic AMP to completely reverse the inhibition process decreases with time because of the presence of antibodies in the anti-cyclic AMP sera that apparently inhibit enzyme activity because of their binding specificity for the residual succinate-protein determinant sites of the enzyme conjugates. Lyophilization of the conjugates immediately after preparation helps to overcome the problem; however, in vivo hydrolysis of immunogens prepared with the succinyl cyclic AMP derivative may always occur. The consequence of this hydrolysis reaction and the subsequent formation of anti-succinyl-protein antibodies will be discussed with regard to existing RIAs for cyclic AMP and a new homogeneous enzyme immunoassay for the nucleotide.

Improved sensitivity in homogeneous enzyme immunoassays using a fluorogenic macromolecular substrate: An assay for serum ferritin

A new highly sensitive nonseparation enzyme immunoassay for human serum ferritin is described. Reagents include a β-galactosidase-ferritin conjugate, sheep anti-ferritin, anti-sheep IgG, and dextran-linked β-galactosylumbelliferone as enzyme substrate. The method is based on inhibition of enzyme activity when anti-ferritin binds to the enzyme-ferritin conjugate. Ferritin in the sample and enzyme-labeled ferritin compete for a limited quantity of antiferritin. The enzyme activity of the reaction mixture is directly related to the ferritin content of the sample. Some patients' samples caused strong interference in the assay due to the presence of antibody to β-galactosidase. Several ways of eliminating the interference are presented. When measures were adopted to suppress sample interference, the assay results correlated well with those of other immunoassay methods.

Extraction of Interfering Substance from the Sera of Uremic Subjects Prior to Homogeneous Enzyme Immunoassay

Extraction of Interfering Substance from the Sera of Uremic Subjects Prior to Homogeneous Enzyme Immunoassay

エミット法による血清中の低濃度フェニトインの測定

Determination of lower level of serum phenytoin (PHT) was made by homogeneous enzyme immunoassay (Emit) in the following methods:(A) Wide range calibration method, (B) Extrapolated calibration method, and (C) Mixed sample method. In the method A and B, calibration curves of serum PHT levels were generated using a nonlinear least-squares regression program (EMITFIT) with a microcomputer. It was concluded that method A was most reliable of the three. In method A, the PHT recoveries in spiked serum samples in the concentrations of 0.25, 0.5, 1.0 μg/ml were 104.6, 102.6, 100.1%, and coefficients of variation were 11.4, 6.8, 2.3%, respectively.

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.

4510240 Homogeneous enzyme immunoassay with heating step after incubation, theresia: Jurgen Schrenk, Wilzhofen, Federal Republic of Germany assigned to Boehringer Mannheim GmbH

4510240 Homogeneous enzyme immunoassay with heating step after incubation, theresia: Jurgen Schrenk, Wilzhofen, Federal Republic of Germany assigned to Boehringer Mannheim GmbH

4510240 Homogeneous enzyme immunoassay with heating step after incubation, theresia: Jurgen Schrenk, Wilzhofen, Federal Republic of Germany assigned to Boehringer Mannheim GmbH

4510240 Homogeneous enzyme immunoassay with heating step after incubation, theresia: Jurgen Schrenk, Wilzhofen, Federal Republic of Germany assigned to Boehringer Mannheim GmbH

血しょう中カルバマゼピン濃度測定におけるＳＬＦＩＡ法の評価 ＥＭＩＴ法およびＨＰＬＣ法との比較

Concentrations of carbamazepine (CBZ) in plasma were measured by AMES TDATM carbamazepine kit, a newly developed substrate-labeled fluorescent immunoassay (SLFIA). The study produced results which showed good correlation with those of homogeneous enzyme immunoassay (EMIT) and high-performance liquid chromatography (HPLC). There was a significant correlation in the plasma concentrations between SLFIA and EMIT methods (n= 92, r= 0.994, p<0.01), whereas a high correlation between SLFIA and HPLC was also found (n=92, r= 0.990, p<0.01).For the determination of plasma concentrations of antiepileptics in the clinical field, SLFIA procedure seems to have considerable advantages over hitherto available method: This method requires only 50μl of plasma for a single determination and is quite a simple method for monitoring plasma concentrations of antiepileptics.Concentrations of CBZ in plasma after administration of CBZ alone were compared with those after concurrent administration of antiepileptics, including CBZ, phenobarbital (PB) and phenytoin (PHT). From these results, it was found that plasma concentrations of CBZ administered concurrently with PB and PHT were significantly lower than those after administration of CBZ alone.

Time‐Dependent Absorption of Theophylline in Man

Sixteen healthy volunteers were given either oral or intravenous doses of aminophylline (125 mg) at 9:00 A.M. and 9:00 P.M. under controlled food conditions. Measured at regular time intervals by homogeneous enzyme immunoassay, the plasma theophylline concentrations 1.5 and 2 hours after oral aminophylline were significantly higher in the morning than in the evening (P less than 0.05). Also, the mean peak plasma concentration was significantly higher (P less than 0.05) and the time to peak concentration was faster (P = 0.02) after the morning dose. Neither the morning mean elimination half-life nor the morning mean area under the plasma concentration-time curve differed significantly from those after the evening dose. After intravenous aminophylline, no significant differences were found in the plasma theophylline concentrations and in the elimination half-life between morning and evening. Therefore, the small but statistically significant time-dependent differences in theophylline kinetics must be due to changes in absorption from the gastrointestinal tract and not to changes in distribution or elimination of the drug.

Homogeneous enzyme immunoassay for cortisol with a centrifugal analyzer.

We determine serum cortisol by a homogeneous enzyme immunoassay in the Cobas Bio centrifugal analyzer. To unbind cortisol from its protein complex, serum is treated for 15 min with an acidic solution. The reaction then proceeds automatically in the analyzer at 37 degrees C. To 50 microL of sample mixture is added 125 microL of reagent (cortisol antibodies, glucose 6-phosphate, and NAD+). This mixture is incubated for 60 s, after which 25 microL of a cortisol derivative labeled with glucose 6-phosphate is added; the increase in absorbance is monitored at 340 nm. The standard curve was linear from 10 to 500 micrograms of cortisol per liter. Within-assay precision (CV) varied from 0.2 to 0.6%, between-assay precision from 6.2 to 10.6%. Analytical recovery ranged from 100 to 103%. Results for control samples deviated from target values by 1.4 to 7.8%. Results compared well with those by radioimmunoassay. The method is reliable and practicable and will usefully replace previous routine methods for serum cortisol.

Controlled coupling of aminoglycoside antibiotics to proteins for use in homogeneous enzyme immunoassays

Selective N-acylation of aminoglycoside antibiotics with the N-hydroxysuccinimide ester of methyldithioacetic acid, followed by reaction with methanethiol or dithioerythritol, gives sulfhydryl labeled antibiotics. Alternatively, the nucleophilic sulfhydryl group is incorporated into an antibiotic by treatment with N-acetyl-d,l-homocysteine thiolactone. These derivatives couple readily with proteins that have previously been modified with bromoacetylglycyl groups to provide conjugates for use in the development of homogeneous enzyme immunoassays.

Homogeneous potentiometric enzyme immunoassay for human immunoglobulin G

Homogeneous potentiometric enzyme immunoassay for human immunoglobulin G

Micro-Scale Method for Determination of Tobramycin in Serum Using High-Performance Liquid Chromatography

Abstract A rapid, simple, accurate, and micro-scale method for the determination of tobramycin, sisomicin and netilmicin in serum using high-performance liquid chromatography has been developed. The method is sensitive to 0.3 pg/ml using only 20 μl of serum. The serum is deproteinized with methanol containing an internal standard: sisomicin for the tobramycin, netilmicin for the sisomicin, and sisomicin for the netilmicin. After centrifugation, a counter-ion reagent is added to the supernatant, then an aliquot of the solution is injected into the chromatograph. Tobramycin, sisomicin and netilmicin are separated by reversed-phase, ion-pair chromatography and detected by fluorescence using continuous-flow, post-column derivatization with o-phthalaldehyde. For the tobramycin, within-run and day-to-day variation was below 2.5%. Correlation of this method with microbiological assay and homogeneous enzyme immunoassay was good.

Homogeneous enzyme-linked assays mediated by enzyme antibodies; a new approach to electrode-based immunoassays

A new homogeneous enzyme—immunoassay system is described. The assay employs an ammonia-liberating enzyme covalenty coupled to protein antigens along with two antibodies. An anti-enzyme antibody inhibits the enzyme. However, an antibody selective for the antigen reverses the inhibition process. When samples containing free antigen are present in the assay mixture, there is competition for anti-antigen antibody sites and protection against the anti-enzyme antibodies is diminished. The extent of the enzymatic reaction is monitored with an ammonium ion-selective electrode. Preliminary data demonstrating the feasibility of this approach for human serum albumin are presented.

Analytical specificity of commercially available methods for serum phenytoin determination.

Phenytoin concentrations in serum obtained from uremic patients and patients with normal renal function were measured by fluorescence polarization immunoassay (FPI), rate nephelometric inhibition immunoassay ( RNII ), and homogeneous enzyme immunoassay (EIA) and compared to corresponding values determined by high performance liquid chromatography (HPLC). In serum specimens from patients with normal renal function, phenytoin concentrations measured by FPI, RNII , and EIA averaged within 12% of the corresponding concentrations determined by HPLC. Phenytoin concentrations in uremic serum were 20% (FPI), 60% (EIA), and 83% ( RNII ) higher than corresponding values determined by HPLC. Accumulation of phenytoin metabolites in patients with end-stage renal disease was associated with artifactually elevated measured serum concentrations of phenytoin. FPI was the most specific of the three commercial methods evaluated.