Rapid, Simple Enzyme Immunoassay for Gentamicin

Radioimmunoassay and enzyme immunoassay methods for analysis of serum gentamicin levels have been shown to be comparable. The purpose of this study was to determine if serum concentration-time data from the same patient assayed by radioimmunoassay and enzyme immunoassay would provide the same estimates for half-life, elimination rate constant, distribution volume, drug clearance, and gentamicin dose. A total of 103 pre-and postinfusion serum samples were obtained from 32 patients. The samples were divided and assayed by radioimmunoassay and enzyme immunoassay. Serum concentration-time data were fitted to a one-compartment model, and kinetic calculations were performed using the method of Sawchuk et al. (Clin. Pharmacol. Ther. 21:362-369, 1977). While good correlation was established between the two assay methods, significant (P < 0.05) mean differences were seen in distribution volume (25%), gentamicin clearance (15%), and half-life (11%), using the quantitative data from both methods. Because of differences noted in these pharmacokinetic parameters, significant differences were also noted in dosage calculations. We conclude that there are differences in the pharmacokinetic parameters obtained using results from the radioimmunoassay and enzyme immunoassay. These differences also translate into significant differences between dosage recommendations when individualization of the gentamicin regimen is attempted.

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Comparison of radioimmunoassay and enzyme immunoassay methods in determining gentamicin pharmacokinetic parameters and dosages

Rotschafer¹,

Morlock²,

Strand³

et al. 1982

mentioning

confidence: 99%

“…However, since there is only a narrow margin between effective and toxic blood concentrations (8), it is necessary to monitor serum levels. Assay methods include microbiological assays (2), enzymatic assays (3, 11), radioimmunoassay (RIA) (4), fluoroimmunoassay (5, 13), enzyme-mediated immunoassay (6,12,14), hemagglutination inhibition (7), and chromatographic (1, 9) methods.Homogeneous EMIT tests (Syva Corp., Palo Alto, Calif.) are used extensively for drug monitoring, having the advantages of extreme speed, technical simplicity, and high specificity, and EMIT gentamicin and tobramycin assays are now available. In EMIT, enzyme-labeled drug competes with free drug in the test sample for antidrug antibodies.…”

mentioning

confidence: 99%

Serum Aminoglycoside Assay by Enzyme-Mediated Immunoassay (EMIT): Correlation with Radioimmunoassay, Fluoroimmunoassay, and Acetyltransferase and Microbiological Assays

White

Scammell

Reeves

1981

Enzyme-mediated immunoassay (EMIT) serum aminoglycoside assay results were accurate and precise and correlated well with radioimmunoassay, fluoroimmunoassay, and acetyltransferase and microbiological assay determinations.The aminoglycosides gentamicin and tobramycin are commonly used for therapy of serious bacterial infections. However, since there is only a narrow margin between effective and toxic blood concentrations (8), it is necessary to monitor serum levels. Assay methods include microbiological assays (2), enzymatic assays (3, 11), radioimmunoassay (RIA) (4), fluoroimmunoassay (5, 13), enzyme-mediated immunoassay (6,12,14), hemagglutination inhibition (7), and chromatographic (1, 9) methods.Homogeneous EMIT tests (Syva Corp., Palo Alto, Calif.) are used extensively for drug monitoring, having the advantages of extreme speed, technical simplicity, and high specificity, and EMIT gentamicin and tobramycin assays are now available. In EMIT, enzyme-labeled drug competes with free drug in the test sample for antidrug antibodies. Typically, when the enzyme-labeled drug binds to antibody, the enzyme's activity is reduced; thus, enzyme activity correlates with the concentration of drug in the test sample. The enzyme label is usually glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides since its activity may be simply determined spectrophotometrically. We have compared EMIT with the microbiological assay, acetyltransferase assay, RIA, and fluoroimmunoassay.All assays were done with five or six calibrators (0, 1, 2, 4, 8, and 16 ,ug/ml) prepared in pooled human serum, except for the quenching fluoroimmunoassay, for which the top calibrator was 12 ,ug/ml, and most EMIT assays, for which the 16-,ug/ml calibrator was excluded (see below). The microbiological assay was a large-plate method, with Klebsiella edwardsii subsp. atlantae (NCTC 10896) as the indicator organism (13). The acetyltransferase assay was performed as described by Broughall and Reeves (3).[125I]gentamicin RIA kits (Diagnostic Products Corp., Los Angeles, Calif.), quenching fluoroimmunoassay kits (Technia Diagnostics Limited, London, England), and substrate-labeled fluoroimmunoassay kits (Ames Laboratories, Elkhart, Ind.) were used according to the manufacturers' instructions. EMIT-AMD kits (lot J03; Syva) were used. Manual assays were performed according to the manufacturers' instructions in a Gilford Stasar III linked to a CP5000 clinical processor (Syva). Samples and reagents were simultaneously diluted and dispensed into autoanalyzer cups (Syva) with a pipettor diluter (Syva model 1500). Standard curves were plotted manually on the graph paper supplied with the kits.Preliminary studies with EMIT indicated that some autoanalyzer cups (from suppliers other than Syva) gave poor precision, as did any protocol which allowed insufficient time for the first dilutions of sample to equilibrate. All EMIT results reported here were obtained by using Syva cups and a protocol allowing first dilutions of at least 1-min equilibration. The 16-,ug/ml c...

“…Analytical performance of the homogeneous enzyme immunoassay (EIA) for the determination of serum gentamicin has been reported recently by several investigators (2,3,5). During our evaluation of EIA and correlation with a standard microbiological assay, we noted two facets which can significantly affect precision and accuracy of the assay.…”

mentioning

confidence: 99%

Potential Liabilities of Gentamicin Homogeneous Enzyme Immunoassay

Carlson

Delaney

Plorde

1982

We report two potential liabilities of the homogeneous enzyme immunoassay for the determination of serum gentamicin. One is a considerable loss of precision and accuracy at the ends of the calibration curve, and the other is an apparent loss of gentamicin with storage at -60°C.Analytical performance of the homogeneous enzyme immunoassay (EIA) for the determination of serum gentamicin has been reported recently by several investigators (2, 3, 5). During our evaluation of EIA and correlation with a standard microbiological assay, we noted two facets which can significantly affect precision and accuracy of the assay. We report here the apparent lack of specimen stability upon freezing and the nonuniformity of variance observed for the EIA. Serum samples were collected from patients hospitalized at the Veterans Administration Medical Center, Seattle, Wash., who were receiving gentamicin. For patients known to be concomitantly receiving penicillins or cephalosporins, sera were treated by the addition of 2 IU of P-lactamase II (EC 3.5.2.6) (International Enzymes, Fallbrook, Calif.) within 30 min of serum separation by centrifugation and were stored in glass tubes at 4°C until assayed. Twenty specimens were assayed immediately by the microbiological method, transferred to plastic tubes (Falcon no. 2027, Falcon Plastics, Oxnard, Calif.), and stored at -60°C until assayed by the EIA technique. Time of storage ranged from 1 to 63 days, with an average storage interval of 28 days. Seventy specimens were assayed on the day of collection by both methods.The EIA was performed with reagents obtained from Syva Co., Palo Alto, Calif. Sample and reagents were diluted and mixed with a pipetter dilutor (model 1500, Cavro Scientific Instruments, Sunnyvale, Calif.) in accordance with Syva's instructions (Emit-amd Gentamicin Assay; Syva Co.). The mixture was aspirated into a spectrophotometer (Gilford Stasar III, Gilford Instrument Laboratories, Inc., Oberlin, Ohio) equipped with a flow cell thermostatically maintained at 30°C (model 3017T thermal control unit; Gilford Instrument Laboratories). A two-point fixed-time kinetic measurement was made at 340 nm. Absorbance readings were taken at 15 and 45 s after aspiration of the sample, and the difference (A A) was printed out by a timer-printer (CP-5000 EMIT Clinical Processor; Syva Co.). The timer-printer also corrected A A values for the zero calibrator absorbance difference (A AO), performed a fiveparameter logit transformation on the stored calibrator data, and printed out computed gentamicin concentrations for specimens. Acceptance criteria for standard curve, control sample, and results of duplicate patient assays were those recommended by Syva. The lyophilized serumbased calibrators supplied by Syva were reconstituted in accordance with the manufacturer's instructions and used to standardize the EIA method at gentamicin concentrations of 0.0, 1.0, 2.0, 4.0, 8.0, and 16.0 ,ug/ml.A standard large-plate disk-diffusion technique described by Lund et al. (1) was used for the microbiolo...