Polarization Fluoroimmunoassay Research Articles

Development of a polarization fluoroimmunoassay for sulfamethazine using an automated analyser

A rapid, non-isotopic, polarization fluoroimmunoassay for the antibiotic sulfamethazine has been developed. The influence of the structure of fluorescein-labelled tracers on the sensitivity of the assay was investigated. Reagents were adapted for use with the Abbott TDx Analyzer and existing software was adapted for automated analyses. Total time for the assay of 1 and 20 samples was 14 and 22 min, respectively. All analytical criteria for the assay were satisfied. The detection limit for sulfamethazine in 20 µl of aqueous sample was 10 ng ml–1(200 pg), which compares well with the sensitivity of enzyme linked immunosorbent assay methods.

Detection of ephedrine and phenylpropanolamine in urine using a polarization fluoroimmunoassay

A polarization fluoroimmunoassay, with a high specificity for ephedrine and phenylpropanolamine, has been adapted for screening urine samples on the Abbott TDx analyser. The antiserum was produced from an ephedrine derivative conjugated to keyhole limpet haemocyanin, and, as a tracer, a fluorescein-labelled derivative of phenylpropanolamine was used. The antiserum showed very low cross-reactivity for amphetamine and other structurally related amines.

Urinary cotinine fluoroimmunoassay for smoking status screening adapted to an automated analyser.

A polarization fluoroimmunoassay for cotinine, a major metabolite of nicotine, has been adapted for fully automated screening of urine samples on the Abbott TDx analyser. The method has sensitivity and specificity suitable for the discrimination of active smokers from non-smokers (including passive smokers) by application of a cut-off at 0.5 mg l-1 of total urinary cotinine. Most active smokers' urine gave results over 1 mg l-1, whereas apparent levels in non-smokers were 0.08 mg l-1 or lower. A result for one sample can be obtained in about 5 min and a throughput of 80 samples h-1 can be maintained for large-scale screening applications.

Design and development of a single-reagent polarization fluoroimmunoassay for methamphetamine.

The design and development of reagents suitable for use in single-reagent polarization fluoroimmunoassay of methamphetamine is described. The single-reagent method requires that a substantial fraction of antibodies exhibit fast dissociation of bound tracer. Antiserum obtained early in the course of immunization of a sheep with a methamphetamine immunogen had suitable dissociation characteristics. Tracers with bridges of different length between methamphetamine and the fluorescein label group were prepared and evaluated. A single-reagent assay with sensitivity and specificity suitable for urine screening application was demonstrated.

Single-reagent polarization fluoroimmunoassay of methamphetamine in urine.

This simple polarization fluoroimmunoassay for detection of amphetamine in urine involves the use of a single reagent: suitably diluted antiserum plus fluorescein-labeled amphetamine as tracer. To this, one adds 10 microL of urine, incubates for a few minutes at room temperature, and measures the fluorescence polarization. By using an antiserum obtained against an immunogen conjugated via the para position of the drug's phenyl ring, we have attained greater specificity than that of most existing immunoassays. As little as 1 mg of amphetamine per liter was easily detected. Results for 266 patients' urines agreed completely with those by gas-liquid chromatography. When a nonseparation enzymoimmunoassay (EMIT-d.a.u., Syva) was applied to 62 specimens chosen without conscious bias, three results were falsely negative, three falsely positive.

Multiple drugs of abuse in urine detected with a single reagent and fluorescence polarization.

We describe a simple polarization fluoroimmunoassay for the detection of several commonly abused drugs in urine. The single reagent used, prepared by mixing four different antisera and three fluorescein-labeled derivatives, produces a polarization signal that is the average of the individual signals of the derivatives. One adds urine to the pre-mixed reagent, incubates at room temperature for a few minutes, then measures fluorescence polarization. The presence of any of several abused drugs, at concentrations of 1 mg/L or more, noticeably decreases the average signal. Although other combinations are possible, the present assay detects the presence of a cocaine metabolite, amphetamine, and (or) several barbiturates.

Single-reagent polarization fluoroimmunoassay for theophylline in serum.

A polarization fluoroimmunoassay for theophylline was developed employing fluorescein-labeled drug and antiserum precombined in a single reagent. Assay was performed simply by addition of sample to an aliquot of the single reagent, incubation, and determination of fluorescence polarization. Because of the relatively rapid dissociation kinetics of the labeled drug from antibody binding, added unlabeled theophylline caused displacement within a practical time period. The precision, accuracy, and specificity of the simplified single-reagent assay were similar to those obtained by a conventional immunoassay procedure using the same reagents. Results for the assay of patients' serum specimens correlated well with those by an established enzymoimmunoassay.

Quality control of aminoglycoside assays using an enzyme immunoassay and a polarization fluoro-immunoassay

Quality control of aminoglycoside assays using an enzyme immunoassay and a polarization fluoro-immunoassay

Single-reagent polarization fluoroimmunoassay for amphetamine in urine.

Abstract This simple polarization fluoroimmunoassay for detection of amphetamine in urine involves the use of a single reagent: suitably diluted antiserum plus fluorescein-labeled amphetamine as tracer. To this, one adds 10 microL of urine, incubates for a few minutes at room temperature, and measures the fluorescence polarization. By using an antiserum obtained against an immunogen conjugated via the para position of the drug's phenyl ring, we have attained greater specificity than that of most existing immunoassays. As little as 1 mg of amphetamine per liter was easily detected. Results for 266 patients' urines agreed completely with those by gas-liquid chromatography. When a nonseparation enzymoimmunoassay (EMIT-d.a.u., Syva) was applied to 62 specimens chosen without conscious bias, three results were falsely negative, three falsely positive.

Dual-label fluoroimmunoassay for simultaneous determination of primidone and phenobarbital.

A dual-label magnetizable solid-phase fluoroimmunoassay for direct determination of serum levels of primidone and its main metabolite, phenobarbital, in a single tube was developed and optimized. Fluorescein isothiocyanate was used to label phenobarbital and a rhodamine 101 derivative (XRITC) to label primidone; these could be independently quantitated with no fluorimetric "cross-talk." The dual-label assay employs mixed immunochemical reagents but is otherwise similar in performance to conventional, single-drug, magnetizable solid-phase fluoroimmunoassays. The ability to measure simultaneously two related analytes, such as a drug and its metabolite, represents a useful extension of immunoassay. This was illustrated in the present work by assay of sera from patients on primidone therapy. Dual-label assay results correlated well with those by a conventional enzymoimmunoassay (for primidone) and by a polarization fluoroimmunoassay (for phenobarbital).

Single-reagent polarization fluoroimmunoassay for barbiturates in urine.

We describe a rapid polarization fluoroimmunoassay for screening for the presence of barbiturates in urine. The single reagent is prepared by pre-mixing antiserum with a fluorescein-labeled barbiturate derivative as tracer. Use of a mixture of two sheep antisera, raised against different barbiturate immunogens, results in an assay with a broad cross-reactivity spectrum for most common barbiturates. One adds urine to the pre-mixed reagent, incubates the solution for a few minutes at room temperature, and measures the fluorescence polarization. The tracer/antiserum reagent is stable for at least six months at 4 degrees C. Results compare favorably with two other commonly used screening techniques for barbiturates, thin-layer chromatography and the EMIT-stTM (Syva) system. Although designed as a qualitative screen, some drugs can be quantified by means of a standard curve of the relevant barbiturate.

Polarization fluoroimmunoassay of biopterin and neopterin in human urine

A polarization fluoroimmunoassay has been developed for the routine determination of biopterin and neopterin levels in human urine. The method employs fluorescein-labeled biopterin and neopterin. The assay is fast (incubation time: 2 min) and no separation step is required prior to measurement of fluorescence polarization. Linearity, recovery and precision were satisfactory. Estimations of biopterin and neopterin levels in human urine samples closely correlated with those obtained by radioimmunoassay.

Polarization fluoroimmunoassay of 11-deoxycortisol in serum and saliva.

We developed polarization fluoroimmunoassays for 11-deoxycortisol in serum and saliva. To avoid interfering factors, the steroid was initially extracted from the biological fluids with dichloromethane. Assays could then be completed without any further separation procedures or need to correct for blank signals. The serum assay was suitable for following the response to the metyrapone test and results correlated acceptably with those by an established, specific, direct 125I-radioimmunoassay. The method is not sufficiently sensitive to detect 11-deoxycortisol in normal saliva, but greatly increased concentrations were found in post-metyrapone saliva and results agreed well with those by the radioimmunoassay as modified for salivary assay. Salivary 11-deoxycortisol assay would provide a convenient means of monitoring results of the metyrapone test.

Direct determination of phenobarbital in serum or plasma by polarization fluoroimmunoassay.

A polarization fluoroimmunoassay for the direct determination of phenobarbital in serum or plasma was developed, optimized, and validated. The drug was derivatized by the introduction of a p-amino function in the phenyl group to enable coupling to carrier protein (used to raise antiserum in sheep) and to fluorescein label. The assay required simply the mixing of prediluted sample, labeled drug, and antiserum, followed by a short incubation and measurement of fluorescence polarization. Normal reliability criteria were satisfied and there was good correlation with established chromatographic and radioimmunoassay methods. Nonseparation, nonisotopic immunoassays are inherently susceptible to sample interferences, but it was shown that the polarization fluoroimmunoassay was subject to inaccuracy only in the presence of markedly elevated levels of bilirubin (above 500 mumol/L), or with gross hemolysis or lipemia.