Apoenzyme Reactivation Immunoassay System Research Articles

Dip-and-read test strips for the determination of trinitrotoluene (TNT) in drinking water

A test strip to quantify trinitrotoluene (TNT) in water has been developed using a homogenous apoenzyme reactivation immunoassay system (ARIS). In comparison with other test kits for the detection of TNT, the novel test strip is much simpler in application. The test strip has only to be dipped into the aqueous sample. A blue color develops on the reagent strips proportional to the TNT concentration. The concentration of TNT is determined either by visual comparison with a color card, or more precisely using a reflectometer. A measuring range of about 1–10,000 μg l −1 TNT in water was demonstrated. The reagent strips were prepared by sequential impregnation of a cellulose pad with two solutions, one based on acetone and the other based on aqueous buffer. All necessary components are included in the reagent strip in dry form.

Homogeneous immunoassay for the detection of trinitrotoluene (TNT) based on the reactivation of apoglucose oxidase using a novel FAD-trinitrotoluene conjugate

A flavin adenine dinucleotide-trinitrotoluene derivative (FAD-TNT) was synthesized by coupling N6-(2-aminoethyl)-FAD covalently to the N-hydroxysuccinimidyl ester of trinitrophenyl-γ-aminobutyric acid and characterized by negative-ion electrospray-ionization mass spectrometry (ESI-MS) after purification by reversed-phase HPLC. Free FAD-TNT can be detected at very low levels by recombination with apoglucose oxidase, since the FAD-TNT-glucose oxidase complex is enzymatically active. On the contrary, if FAD-TNT has been bound by an anti-TNT antibody, the conjugate cannot recombine with apoglucose oxidase any more. Based on these two phenomena, a homogeneous apoenzyme reactivation immunoassay system (ARIS) was developed for the detection of TNT. No separation step is needed in this assay. Proportionality between the TNT concentration and enzyme activity was demonstrated with a detection limit of 5 μg/L TNT.

Evaluation of the Ames Seralyzer for the Determination of Carbamazepine, Phenobarbital, and Phenytoin Concentrations in Saliva

The performance of the dry-phase apoenzyme reactivation immunoassay system (ARIS) for the measurement of carbamazepine (CBZ), phenobarbital (PB), and phenytoin (PHT) concentrations in saliva was compared with fluorescence polarization immunoassay (FPIA). Blood and saliva samples were collected from 163 adult and pediatric epilepsy patients, then analyzed using both methods. Regressions between ARIS saliva CBZ, PB, and PHT concentrations, and FPIA unbound and total serum concentrations were highly correlated, but the ARIS technique was somewhat less precise than the FPIA. Valproic acid co-medication did not affect the relationships between ARIS and FPIA saliva concentrations and unbound serum concentrations of PHT, but did disrupt the relationship between ARIS and FPIA saliva PHT and total serum PHT. The sensitivity, specificity, predicted value positive (PV+) of a therapeutic concentration, and predicted value negative (PV-) of a concentration outside the therapeutic range for the ARIS saliva technique compared very well with FPIA for CBZ, PB, and PHT. The ARIS technique for CBZ, PB, and PHT saliva determination provides acceptable accuracy, precision, and sensitivity for therapeutic monitoring. In practice, the benefits of the ARIS saliva technique, including ease of collection, safety, patient/parent acceptance, and short analysis time, are striking.

An improved chemiluminescence-based liposome immunoassay involving apoenzyme.

An improved liposome immunoassay system (LIS) combining the chemiluminescence-based LIS with an apoenzyme reactivation immunoassay system (ARIS) was developed. A low-molecular-weight co-factor, FAD (flavin adenine dinucleotide), was incorporated into liposomes instead of the high-molecular-weight enzyme GOD (glucose oxidase). FAD released from liposomes by cytolysin-hapten conjugates bound to Apo-GOD and regenerated GOD. The system allowed detection of 10 pM digoxin, the model analyte and was linear over 10 pM to 13 nM digoxin. This sensitivity was about 300 times higher than that of the homogeneous system using GOD-containing liposomes and 30 times higher than that of the heterogeneous system which we reported previously. The time required for incubation during the detection of digoxin was reduced from 20 to 3 h.

The Effect of Vitamin B2 and Its Derivatives on a Dry-Reagent Strip Theophylline Assay

This report describes the effect of flavin adenine dinucleotide (FAD) and its precursor vitamin B2 on an apoenzyme reactivation immunoassay system (ARIS) both in vitro and in vivo. It was observed that 10 nM/L FAD in the buffer causes a false-positive result in ARIS for theophylline by the value of 94 microM/L, whereas vitamin B2, flavin mononucleotide, adenosine, adenosine monophosphate, and adenosine diphosphate (ADP) at the same concentration caused no such effect. Nevertheless, ADP at 2 microM/L can cause a drop in theophylline reading of 55 microM/L. However, the sera from four volunteers with normal liver functions who had taken 30 mg of vitamin B2 daily for a week showed no effect on theophylline reading.

Dry reagent immunoassay for measuring phenobarbital in serum and plasma.

A reagent strip has been developed for measuring phenobarbital concentrations in serum or plasma. This strip utilizes the apoenzyme reactivation immunoassay system (ARIS) and is designed for use with the Ames Seralyzer reflectance photometer. The test takes 85 s and compares strip reactivities with a two-point calibration line that is stored in the instrument. Within-run precision coefficient of variation ranges from 2.4 to 4.1% over five concentrations while the between-run precision ranges from 2.3 to 4.9%. Results obtained with clinical serum samples correlated well (r greater than 0.99) with those obtained by a fluorescence polarization immunoassay (TDx). The strips crossreact with other barbituric acid derivatives that are not coadministered with phenobarbital. Crossreactivity to the metabolite p-hydroxyphenobarbital is sufficiently low as to not interfere in the usual specimens. Samples that are hemolyzed, highly icteric, or from uremic patients should be avoided. This assay is a rapid and convenient method for monitoring serum or plasma phenobarbital concentrations. It is particularly well suited for decentralized test sites, such as emergency rooms, urgent care centers, and physician's offices.

A dry-reagent strip for quantifying carbamazepine evaluated.

We examined a new colorimetric homogeneous immunoassay for carbamazepine based on the apoenzyme reactivation immunoassay system (ARIS) principle. The test, in dry-reagent strip format, is to be used with the Ames Seralyzer reflectance photometer. Within-run CVs (n = 20) were 3.0%, 2.7%, and 2.8% at 3.0, 6.1, and 12.1 mg/L; between-run CVs (n = 15, in 15 days) were 4.1%, 2.7%, and 1.9% at 6.0, 9.1, and 12.1 mg/L. Mean analytical recovery was 99.9 (SD 2.3)%. Results by this test (y) for clinical plasma specimens (n = 96) compared very well with those obtained by fluorescence polarization immunoassay (y = 1.01 x - 0.02, r = 0.995) and by liquid chromatography (y = 0.99 x + 0.14, r = 0.990). Bilirubin (45 mg/L), uric acid (145 mg/L), and various anticoagulants at about fourfold the usual concentrations did not interfere. High concentrations of cholesterol (4.9 g/L), triglycerides (3.8 g/L), and hemoglobin (4 g/L) caused slight positive interference. Carbamazepine-10,11-epoxide cross reacted only at greater than or equal to 5 mg/L. The two-point calibration line was validly stored for at least three weeks. Free carbamazepine also can be measured. The test is convenient and rapid (test time 80 s), and thus is particularly useful in all clinical settings where prompt testing is needed.

Quantitative determination of phenobarbital and phenytoin by dry-phase apoenzyme reactivation immunoassay system (ARIS).

We assessed the performance of the apoenzyme reactivation immunoassay system (ARIS) reagent strip tests for determination of phenobarbital (PB) and phenytoin (PHT) with the Seralyzer reflectance photometer. In the assay, the drug of the sample competes with a flavine adenine dinucleotide (FAD)-drug conjugate for binding to a specific antibody; the unbound conjugate then activates apoglucose oxidase to reconstitute glucose oxidase, whose activity is kinetically monitored by a coupled chromogenic reaction. Within-run coefficients of variation (CVs) were less than or equal to 5.0% for PB and less than or equal to 5.6% for PHT; between-run CVs were less than or equal to 6.1% for PB and less than or equal to 6.5% for PHT. Mean analytical recoveries were 100.3% for PB and 100.2% for PHT. Test results were not significantly affected by bilirubin (5 mg/dL), hemoglobin (25 mg/dL), triglycerides (500 mg/dL), uric acid (15 mg/dL), or elevated levels of other antiepileptic drugs. Reagent strip tests correlated very well with substrate-labeled fluorescent immunoassay (r = 0.9923 and 0.9944 for PB and PHT, respectively), enzyme multiplied immunoassay technique (r = 0.9941 and 0.9919), and gas-liquid chromatography (r = 0.9980 and 0.9960). These homogeneous competitive colorimetric immunoassays are particularly suitable for emergency use, for testing small batches of samples, wherever prompt results are needed.

抗てんかん薬の血中濃度測定におけるＡｐｏｅｎｚｙｍｅ Ｒｅａｃｔｉｖａｔｉｏｎ Ｉｍｍｕｎｏａｓｓａｙ Ｓｙｓｔｅｍ（ＡＲＩＳ）法の信頼性の検討

In an attempt to evaluate the reliability of apoenzyme reactivation immunoassay system (ARIS) method, phenobarbital (PB) and phenytoin (PHT) concentrations in patient's serum were measured by ARIS method, and were compared with those obtained by fluorescence polarization immunoassay (FPIA) method and enzyme multiplied immunoassay (EMIT) method.The within-run precision of ARIS method was 2.56-5.26% as the coefficient of variation (C. V.). The C. V. value of between-run precision was less than 6%. There were significant correlations between the serum concentrations determined by ARIS method and each method (FPIA; EMIT). The interference from hemoglobin, bilirubin, ascorbic acid, flavin adenine dinucleotide (FAD) and cross-reacting substances was also investigated and discussed.ARIS method was found to be reliable, simple and rapid one. Therefore, ARIS method seemed to be a useful technique for the rapid determination of drug concentration.

血清中抗てんかん薬濃度測定へのセラライザーシステムの適用 フェニトイン，フェノバルビタール

Apoenzyme reactivation immunoassay system with Seralyzer reflectance photometer for the determination of serum phenytoin (PHT) and phenobarbital (PB) concentration (Seralyzer method) was evaluated. Each coefficient of variation of within-run and between-run precision for 3 concentrations of PHT or PB in serum was less than 4.0%. The serum PHT (33 samples) or PB (31 samples) concentrations were determined by Seralyzer method and compared with those determined by high-performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA method). There were good agreements between HPLC and Seralyzer method results and FPIA method and Seralyzer method results, where all correlation coefficients were more than 0.992. The cross-reactivities of p-hydroxyphenylphenylhydantoin in PHT reagent strips, and p-hydroxyphenobarbital and several barbiturates in PB reagent strips were found. However, these cross-reactivities are not serious problems in clinical condition. Therefore, the Seralyzer method may be useful for serum drug-level monitoring of patients under PHT or PB therapy.

Homogeneous dry reagent immunoassay strips for the determination of therapeutic drugs in human serum or plasma

AbstractSpecific dry reagent immunoassay strip tests for therapeutic drugs have been developed with the apoenzyme reactivation immunoassay system (ARIS). This simple and convenient method for measuring such analytes in human serum or plasma is described with assays for theophylline, phenytoin, and phenobarbital.

抗てんかん薬の血中濃度測定におけるＡｐｏｅｎｚｙｍｅ Ｒｅａｃｔｉｖａｔｉｏｎ Ｉｍｍｕｎｏａｓｓａｙ Ｓｙｓｔｅｍ法の信頼性 ＥＭＩＴ法およびＦＰＩＡ法との比較

抗てんかん薬の血中濃度測定におけるＡｐｏｅｎｚｙｍｅ Ｒｅａｃｔｉｖａｔｉｏｎ Ｉｍｍｕｎｏａｓｓａｙ Ｓｙｓｔｅｍ法の信頼性 ＥＭＩＴ法およびＦＰＩＡ法との比較

Dry-reagent strips for measuring phenytoin in serum.

A new reagent strip has been developed for measuring phenytoin in serum or plasma. It utilizes the chemistry of the Apoenzyme Reactivation Immunoassay System (ARIS) and is designed for use with the Seralyzer reflectance photometer. The 90-s test relies on comparison of strip reactivities with a two-point calibration line, which can be validly stored in the instrument for two weeks. Within-run CVs for controls at five concentrations ranged from 2.7 to 5.6%, between-run CVs from 2.3 to 6.3%. Phenytoin values obtained for clinical serum samples correlated well (r greater than 0.99) with those obtained by liquid chromatography or substrate-labeled fluorescent immunoassay. The speed and convenience of this assay for monitoring phenytoin in serum make it well suited for decentralized test sites such as emergency rooms, urgent-care centers, and physician's offices.

Homogeneous apoenzyme reactivation immunoassay for thyroxin-binding globulin in serum.

In this automated apoenzyme reactivation immunoassay system (Ames Optimate) for thyroxin-binding globulin (TBG), the sample and N6-aminohexylflavin adenine dinucleotide-labeled TBG react sequentially with antiserum. Then apoglucose oxidase is added to combine with the free fraction and generate glucose oxidase activity, which is measured colorimetrically. The assay requires 100 microL of sample and covers the clinically significant range for TBG (less than 2.5 to 55 mg/L). The first result is obtained in 16 min; assay of 29 samples and their blanks is completed in less than 1 h. The lower limit of detection is about 2.5 mg/L. Between-assay CVs (n = 9) were less than 9%, within-assay CVs (n = 5) were less than 6%, and analytical recovery of TBG was 103-112%. Reagents are stable at 4 degrees C for at least five months. Results by this method for serum TBG (y) compared well with those determined by radioimmunoassay (x): y = 1.029x--0.352 (r = 0.990, n = 49, Syx = 1.165 mg/L). In addition, with 39 other sera the ratio of total thyroxin (by RIA) to TBG compared well with free thyroxin measured by equilibrium dialysis (r = 0.930) and the free thyroxin index (r = 0.970).

血清中テオフィリン濃度測定のためのセラライザー法の評価

Seralyzer system applying apoenzyme reactivation immunoassay system for determination of serum theophylline concentration (SERALYZER method) was evaluated. The coefficients of variation of within-run and between-run precisions for 3 concentrations of theophylline in serum were less than 4%. The serum theophylline concentrations determined by SERALYZER method for 40 samples were compared with those determined by high-performance liquid chromatography (HPLC), substrate-labeled fluorescent immunoassay method-fluorostat (SLFIA method-A), SLFIA method-OPTIMATE (SLFIA method-B) and fluorescence polarization immunoassay method (FPIA method). There were good agreements between HPLC and SERALYZER method results, SLFIA method-A and SERALYZER method results, SLFIA method-B and SERALYZER method results, and FPIA method and SERALYZER method results, where all correlation coefficients were 0.996. The cross-reactivities of caffeine, 8-chlorotheophylline, 1, 3-dimethyluric acid, 1, 7-dimethylxanthine, 3-methylxanthine, theobromine, and 1, 3, 7-trimethyluric acid in SERALYZER method were found. However, these cross-reactivities are not serious problems in clinical condition. Therefore, the SERALYZER method may be useful for the drug-level monitoring of patients under theophylline therapy.

Effect of antibodies to glucose oxidase in the apoenzyme reactivation immunoassay system

The use of antiserum to glucose oxidase in the apoenzyme reactivation immunoassay system (ARIS) is described. Formation of an immune complex between apoglucose oxidase and antibodies to native glucose oxidase enables FAD conjugates to be detected with greater sensitivity and at higher temperatures. In the absence of antiserum to glucose oxidase, optimum activity with theophylline-FAD was at 23°C whereas in the presence of the antiserum the optimum shifted to 32°C and was accompanied by a sixfold increase in activity. At 37°C in the absence of antiserum very little activity is found. The use of the antiserum makes it possible to perform ARIS assays at 37°C and this is illustrated by description of an assay for theophylline.

Coupling aminohexyl-FAD to proteins with dimethyladipimidate.

We used an efficient method having general applicability to couple N6-aminohexyl-flavin adenine dinucleotide (AHFAD) to several proteins for use in an apoenzyme reactivation immunoassay system (ARIS). AHFAD is first activated with 40-fold molar excess of dimethyladipimidate, excess imidate is removed rapidly by gel filtration, the activated product is incubated with the protein, and the conjugate formed is purified. This labeling technique permits incorporation of a controlled amount of amino-label into a protein, and eliminates the possibility of self-crosslinking, which would reduce the immunoreactivity of the conjugate. Here we demonstrate the utility of such a conjugate in a totally automated ARIS assay for thyroxin-binding globulin (TBG). After a competitive protein-binding reaction, apoglucose oxidase is added to combine with free TBG-AHFAD conjugate and produce active glucose oxidase, which is measured colorimetrically in a peroxidase-linked reaction. The assay covers the clinically significant range for TBG from 0 to 60 mg/L and has a throughput of 60 reactions in 75 min. Comparison with an RIA method (x) by regression analysis yielded the equation y = 0.890x + 1.217 (r = 0.975, n = 47, Syx = 1.906 mg/L).

Dry-reagent strips used for determination of theophylline in serum.

A reagent strip for quantifying theophylline in serum or plasma has been developed for use with the Apoenzyme Reactivation Immunoassay System (ARIS) with the Ames Seralyzer reflectance photometer. The test takes 80 s and involves comparison with a two-point calibration line, which can validly be stored in the instrument for two weeks. Results for theophylline in clinical serum samples correlate well (r greater than 0.98) with results by liquid chromatography, fluoroimmunoassay, and enzyme immunoassay procedures. The within-run CV for four concentrations of controls ranged from 3.5 to 6%; the between-run CVs ranged from 3 to 5%. This assay for use in therapeutic drug monitoring is convenient, rapid, and simple, and thus is appropriate for use in emergency rooms, physician's offices, and small laboratories.