Dry-ReagentStripsUsed for Determinationof ... - Clinical Chemistry

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We appreciatehelpfuldiscussionswith Drs.JohnBurd and David. Morris and the excellent technical assistanceof Denise Clay,. Christine Nelson,Paula Smith, and ...
CLIN. CHEM.

31/5, 737-740

(1985)

Dry-ReagentStripsUsed for Determinationof Theophyllinein Serum Patricia Rupchock, Ronald Sommer, Alfred Greenqul$, Richard Tyhach, Bert Walter, and Adam Zlpp A reagent strip for quantifying theophylline in serum or plasma has been developed for use with the Apoenzyme activation knmunoass’ System (ARIs) with the Ames Seralyzer reflectance photometer. The test takes 80 s and involves comparison with a two-point calibration line, which can validlybe storedinthe instrumentfor two weeks. Results for theophylline in clinical serum samples correlate well (r >0.98) with results by liquid chromatography, fluoroimmunoassay, and enzyme immunoassay procedures. The withinrun 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.

Materials and Methods

Apparatus. Routine reflectance measurements were made in a Seralyzer reflectance photometer, with use of a 740-nm, three-cavity interference ifiter (Ditric Optics, Hudson, MA 0174w. Measurements of reflectance spectra were obtained with a rapid scanning reflectometer, as previously described (6). Reagents. Theophylline monoclonal antibody was obtained from murine ascites fluid. Antiserum to glucose oxidase was prepared by immunization of goats with glucose oxidase. Glucose oxidase was obtained from Miles Scientific, Naperville, IL 60566. Apoglucose oxidase and theophyllineFAD were prepared as previously described (5). Peroxidase (EC 1.11.1.7, Grade VI) and theophylline were obtained from Sigma Chemical Co., St. Louis, MO 63178. Glucose was obtained from Mallinckrodt, Inc., St. Louis, MO 63147. AddItIonal Keyphrases: reflectance photometry immunoasThe Ames TDAtm theophylline kit was from Ames Divisay dry-reagentchemistry methodsforthesmalllaboratory sion. The EMIT-sad” theophylline kit was from Syva Co., Palo Alto, CA 94304. The PipDiltm pipettes were from Gilson The effective therapeutic range for theophylline (1,3Medical Electronics, Middleton, WI 53562; the MLA”' pidimethyixanthine), a potent bronchodilator used to treat pettes were from Medical Laboratory Automation, Inc., Mt. acute and chronic asthmatic symptoms, is 10 to 20 mg/L (1). Vernon, NY 10550; the Dip-Stattm pipettes were from Godax Greater concentrations in serum are often associated with Laboratories, Inc., New York, NY 10013. toxic symptoms such as nausea, vomiting, headaches, or, in Procedures. The reagent papers were prepared in two extreme cases, convulsions and death (2, 3). impregnations. First, the filter paper was passed through an Current methods to detect theophylline in serum include ultraviolet spectrophotometry, gas-liquid and “high-peraqueous solution containing antitheophylline ascites fluid, apoglucose oxidase complexed with goat antiserum to gluformance” liquid chromatography (HPLC), radioimcose oxidase, peroxidase, glucose, and buffer. The antimunoassay, enzyme immunoassay, fluorescence polarizaglucose oxidase enhances the activity of apoglucose oxidase tion immunoassay, and substrate-labeled fluorescent immuduring reaction at 37#{176}C (7). After drying, the paper was noassay. Unlike many of these methods, the theophylline passed through a second solution containing theophyllineimmunostrip procedure we describe here can be performed FAD and 3,3’,5,5’-tetramethylbenzidine in organic solvent. quickly, requires relatively inexpensive instrumentation, Sections of reagent paper (0.5 x 1 cm) were mounted on 0.5 and needs no reagent manipulation. x 8.3 cm polystyrene supports by using double-face adheOur procedure involves the apoenzyme reactivation imsive. munoassay system (Allis) for measuring drug concentrations Serum and control standards were dispensed with a 30-tL (4, 5); in a competitive protein binding reaction, theophylMLA pipette into 800 pL of distilled water dispensed from a line competes with a theophylline conjugate labeled with flavine adenine dinucleotide (FAD) for a limited number of Dip-stat or PipDil. A 30-pL MLA pipette was again used to apply the diluted sample to the reagent pad to start the antibody binding sites. A monoclonal antibody to theophylreaction. Reflectance was measured every 5s in a Seralyzer line is incorporated into the reagent strip to control reproreflectance photometer and the results were converted into ducibility of the immunochemical response. In the absence of theophylline, the conjugate binds to antibody and is the corresponding Kubelka-Munk ratio (8), K/S [where K/S unavailable for fUrther reaction with apoglucose oxidase. In = (1 R)2/2R; K is the absorption coefficient, S is the the presence of theophylline, conjugate that is not bound by scattering coefficient, and R is reflectance], and used to antibody is available for reactivation of apoglucose oxidase, determine the rate of color development from 60 to 80 a. For comparison, we measured theophylline in the sample the protein component of glucose oxidase (BC 1.1.3.4) remnining after FAD is removed. Active enzyme is detected by by enzyme immunoassay (EMrr-aad) and the substratelabeled fluorescent immunoassay (Ames TDA) as described a colored product produced through a coupled reaction with in the respective product inserts. Liquid-chromatographic peroxidase. We describe here the preparation and performance of an measurements of theophylline were done as previously ais theophylline reagent strip for use with theiSeralyzer#{174} reported (9). reflectance photometer (both from Ames Division, Miles Laboratories, Elkhart, IN 46515). ‘

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Results Ames Division, Miles Laboratories, Inc., Elkhart, IN 46515. Received October 15, 1984; acceptedFebruary 12, 1985.

Reflectance measurements on paper. Figure 1 shows the reflectance spectrum of a theophylline immunostrip, meaCLINICALCHEMISTRY, Vol. 31, No. 5, 1985 737

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Fig. 1. Reflectancespectrum of a theoph1line dry reagent stiip, 90s after a theophyllinecontrol sera (15 mg/I.)was diluted27.7-foldand 30 L applied to the reagent pad at 37#{176}C sured after reaction for 90 s. To keep the intensity of color development of the oxidized tetramethylbenzidine product within the usable range of the Seralyzer reflectance photometer, we routinely measure reflectance at 740 nm. Strip response. The kinetic responses of theophylline immunostrips measured at several concentrations of theephylline are shown in Figure 2. An initial lag period occurs at all theophylline concentrations while the apoglucose oxidase is reactivated by unbound theophylline-FAD; at higher theophylline concentrations, more conjugate is available for reactivation and consequently the rate is increased. The amount of apoglucose oxidase in the strips is designed to yield a optimal reactivationrate in the 80-s test period. Figure 3 shows inhibition of enzyme reactivation by increasing amounts of theophylline monoclonal antibody impregnated in the strip. The antibody inhibits reactivation by binding to the theophylline moiety of the conjugate. Inhibition exceeds 90% at higher antibody concentrations. A standard curve for the theophylline strip is shown in Figure 4. Strip response was measured as the rate of change in K/S from 60 to 80 s, for theophylline samples in the concentration range from 0 to 40 mg/L. The strip response is linear up to 40 mg of theophylline per liter, well beyond the therapeutic range. ThEOPHYLUNE mg/I

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Fig.3. Titeringof theophylline monoclonal antibody on reagentstrips Thereagentpads (0.5 x 1 cm) ccntainedapoglucoseoxidase,theophyllineFAD, and the volumeof ascitesfluid shown. Adding water to the pad initiatedthe reaction,wntcl,was monitoredby reflectancechangesat 740 nm aftera 90-s

ThEOPHYLUNE

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FIg.4. Dose-response curve formis theophyllinereagentstrips Precision. Within-run precision was determined from 45 replicate analyses of theophylline controls in the range of 10 to 25 mg/L. Between-nm precision was determined by running a calibration line and three replicates of each control as unknowns for a total of 15 runs. Table 1 sumrnarizes the precision and accuracy of the strip at four different concentrations of theophylline. In a separate study the within-run precision at 5 mg/L theophylline was ±0.43 (SD, n = 20). The bias at all concentrations was 6700

Hypoxanthine

Concentrationof compoundrequired to increase the apparent valueof a 15 mg/L theophylline control sample by 20%. bMalor theophylline metabolite. C

Major caffeinemetabolftesalongwith theophylline.

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FAD conjugate from binding prematurely either to antitheophylline or to apoglucose oxidase. Accordingly, the theophylline-FAD conjugate is introduced into the paper in a solvent that prevents these interactions. Because coffeeand many soft drinks contain caffeine, they represent a potential source of interference in the theophylline assay. Low cross reactivity with caffeine is maintained by use of a monoclonal antibody. At the concentrations of caffeine anticipated in serum, the low caffeine cross reactivity is not significant (17-23). Even though the expected concentrations of 1,3-dimethyluric acid are low, they may be high in the serum of uremic patients who are being treated with theophylline (24). No interference is observed with either hemolyzed samples or with samples containing abnormally high concentrations of uric acid, bilirubin, or lipids. The anticoagulants tested-EDTA, heparin, and oxalate-did not affect strip response. This demonstrates that plasma specimens can also be tested with the immunostrip. The formulation has been optimized to achieve a linear response over the entire test range. The system requires only a two-point calibration, which can be stored up to two weeks, thus making the assay both convenient and cost effective. Incorporation into a dry reagent format eliminates all reagent manipulations, and no separation steps are required. A single dilution of a 30-ML sample is all that ordinarily is required to perform the assay. The assay is rapid, accurate, precise, and exceptionally easy to perform. The format for the theophylline immunostrip is particularly amenable to those conditions where rapid testing is desirable, such as the emergency room or in the physician’s office (25). We appreciatehelpfuldiscussionswith Drs.JohnBurd and David Morris and the excellent technical assistanceof Denise Clay, Christine Nelson,Paula Smith, and OsceolaSkinner.

References 1. HendelesL, Weinberger M. Theophylline: A “stateof the art” review. Phannacotherapy 3,2-44(1983). 2. Bresnich E, Woodard WK, Sageman EB. Fatal reactions to intravenous administration ofaminophylline. JAm MedAssoc 136, 397-398 (1948). 3. Zwillich CW, Sutton FD, Neff TA, et al. Theophylline-induced seizures in adults: Correlation with serum concentrations. Ann Intern Med 82, 784-787 (1975). 4. Morris DL, Ellis PB, Hornby WE, et al. Flavin adeninedinucleotide as a label in homogeneouscoloriznetricimmunoaasays.Anal Chem 53, 658-665 (1981). 5. Tyhach RJ, Rupchock PA, PendergrassJH, et al. Adaptation of prostheticgroup-label homogeneousimmunoassay to reagent strip format. Clin Chem 27, 1449-1504 (1981).

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6. Genshaw MA, Rogers RW. Rapid scanningreflectance spectrophotometer.Anal Chem 53, 1949-1952 (1981). 7. Morris D. Submittedfor publication. 8. Kortum G. Reflectance Spectroscopy: Principles, Methods and Applications, Springer-Verlag, Inc., New York, NY, 1969, p 178 if. 9. Patel C, Benovic J, Chong R. Comparisonof EMiT, TDA and HPLC methodsin the determination of serum theophylline.Clin Chem 26, 1002 (1980). Abstract. 10. Midha KK, Sved 5, HossieRD, McGilveray LI. High performanceliquid chromatographic and mass spectrometric identification of dimethylxanthine metabolites of caffeine in human plasma. Biorned Mass Spectrum

4, 172-177 (1977).

11. Zipp A. Developmentof dry reagent chemistry for the clinical laboratory. J Autom Chem 3, 71-74 (1981). 12. Walter B. Dry reagent chemistries in clinical analysis. Anal Chem 55, 498A-514A (1983). 13. Curme HG, ColumbusRL, Dappen GM, et al. Multilayer film elements for clinical analysis: General concepts.Clin Chem 24, 1335-1342 (1978). 14. Spayd RW, Bruachi B, Burdich B, et a!. Multilayer film elementsfor clinical analysis: Applications to representative chemical determinations.Clin Chem 24, 1343-1350 (1978). 15. Greenquist AC, Walter B, Li TM. Homogeneousfluorescent immunoassay with dry reagents.Clin Chem 27, 1614-1617(1981). 16. Walter B, GreenquistAC, Howard WE III. Solid-phasereagent strips for detectionof therapeutic drugs in serum by substratelabeled fluorescent immunoassay. Anal Chem 55, 873-878 (1983). 17. Axelrod J, Reichenthal J. The fate of caffeinein man and a method for its estimation in biological material. J Pharmacol Exp Thor 107, 519-523 (1953). 18. Grab FL, Reinstein JA. Determination of caffeine in plasma by gaschromatography. J Pharm Sci 57, 1703-1706 (1968). 19. Sved S, HoesieRD, McGilveray LI. The human metabolism of caffeine to theophylline.Res Commun Chem Pathol Pharmacol 13, 185-192 (1976). 20. Merriman RL, Swanson A, Anders MW, Sladek NE. Microdetermination of caffeine in blood by gas chromatography mass spectrometry.J Chromatogr 146, 85-90 (1978). 21. CookE, Tallent CR, Amerson EW, et al. Caffeinein plasma and saliva by a radioimmunoassay procedure. J Pharmacol Exp Thor 199, 679-686 (1976). 22. RouthJI, Shane NA, ArredondoEG, Paul WD. Determination of caffeine in serum and urine. Clin Chem 15, 661-668 (1969). 23. ThompsonRD, Nagasawa HT, Jenne JW. Determination of theophylline and its metabolites in human urine and serum by high-pressure liquid chromatography. J Lab Clin Med 84, 584-593 (1974). 24. Opheim K, Ainardi V. Increase in apparenttheophyffineconcentrationin the serumoftwo uremic patients as measuredby some immunoassay methods (caused by 1,3-dimethyl uric acid). Clin C/tern 29, 1698-1699 (1983). Letter. 25. Pippenger CE. The future role of the clinical laboratory in therapeuticdrugmonitoring. Am J Med Technol 49,59-565(1983).