Measurement of Gentamicin by Radioimmunoassay

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIEN CE, Vol. 11, No. 1 Copyright 1981, Institute for Clinical Science, Inc. Measurement of Gentamicin by Radioimmunoassay MING-CHUNG WANG, Ph.D., PH ILIP R. WALSH, Ph.D., and SURIGENIA D. ABARCA, M.S. Department o f Chemistry, Laboratory Procedures, Inc., Subsidiary o f The Upjohn Co., King o f Prussia, PA ABSTRACT A sensitive, specific and precise procedure for the measurement of serum gentamicin by radioimmunoassay is presented. The method is rapid, convenient, and highly reliable for this very important measurement. Studies designed to evaluate the validity and reproducibility of the assay are presented and discussed. Introduction Gentamicin is an aminoglycoside antibiotic which has been demonstrated to be effective in the treatment of infection by aerobic Gram-negative bacteria. However, one of the potential dangers associated with the use of gentamicin in clinical practice is the narrow range which exists between therapeutic and toxic concentrations of the antibiotic. Serum gentamicin concentrations between 4 and 12 mg per liter are generally considered to be optimum. Serum drug concentrations which exceed 12 mg per liter may lead to ototoxicity and nephrotoxicity,20,21 while concentrations below 4 mg per liter may be inadequate for treatment. As with many drugs, the concentration of serum gentamicin is very difficult to predict without a reliable quantitative method in patients with im paired renal function.6,8 As a result of the potential toxicity associated with the administration of gentamicin, the routine monitoring of serial serum gentamicin concentrations is of great clinical significance. The microbial assay1,23 was one of the earlier m ethods developed for the determination of serum gentamicin concentrations. This assay requires a long incubation time and is potentially less accurate owing to interferences by other antibiotics present in the sample. Over the past six years, other more accurate and precise methods have been published w hich include the enzym atic assay,9,12,15,25 radioimmunoassay 3>4>16>17 fluorescence polarization immunoassay,26 fluoroimmunoassay,24 high pressure liquid chromatography,2,18 and enzyme immunoassay.19 All of these newer methods have dem onstrated the acceptable accuracy and precision required for the serial monitoring of patients receiving gentamicin. Here are reported the data collected during the validation of a pre-precipitated double antibody radioimmunoassay (RIA) method for serum gentamicin, which uses /81/ $00.90 Institute for Clinical Science, Inc.

2 48 WANG, W ALSH, AND ABARCA 125I-gentamicin as the tracer. The assay is a modification of the pre-precipitated double antibody radioim m unoassay originally described by Hales and Randle.10 The method is simple, fast, sensitive and precise. Materials and Methods G entam icin Radioimmunoassay Kit RIANEN which was purchased contains all of the reagents necessary for performing the assay. Principle The RIANEN * gentamicin radioimmunoassay is a pre-precipitated, double antibody radioimmunoassay procedure. The primary antiserum (sheep), generated against a gentamicin-bovine serum albumin conjugate, is pre-reacted with anti-sheep (goat) gamma globulin to form a pre-precipitated primary and secondary antibody complex. A sample of patient serum or gentam icin standard and a known amount of 125I-labelled gentamicin derivative are placed in a test tube to which a known quantity of the preprecipitated antibody complex is added to initiate the reaction. During the specified incubation period, the 125I-labelled gentamicin derivative and the unlabelled gentamicin present in the patient serum or standards compete for the available antibody b inding sites on the preprecipitated antibody complex. After incubation, the 125I-labelled gentamicin derivative and the unlabelled gentamicin not reacting with the anti-gentamicin serum are separated from the antibodybound gentamicin by centrifugation. As the concentration of the unlabelled gentamicin in standard or patient specimens increases, the am ount of labelled gentamicin bound to the pre-precipitated complex decreases, and vice versa. If the concentrations of the labelled gentamicin and antibody complex are constant, by using several known concentrations of unlabelled gentamicin, a standard curve can be generated which can be used for quantifying unknown specimens. * Purchased from New England Nuclear, North Billerica, MA G e n t a m i c i n A n t i s e r u m C o m p l e x Gentamicin antiserum, produced in sheep against gentamicin-bovine serum albumin conjugate, is pre-precipitated with goat anti-sheep gamma globulin in 10 mm phosphate buffer, ph 7.4. R a d i o i o d i n a t e d G e n t a m i c i n The iodinated derivative of gentamicin has a structure similar to that described previously.4 The tracer is prepared in 10 mm phosphate buffer, ph 7.4. The specific activity is approximately 100 c per g. B l a n k A n t i s e r u m C o m p l e x Normal sheep serum pre-reacted with an antiserum to sheep gamma globulin. This blank antiserum complex is used to determ ine the non-specific binding. G e n t a m i c i n S t a n d a r d s The standards are prepared in gentamicin-free serum at concentrations of 1.0, 2.0, 4.0, 8.0 and 16.0 mg per liter. G e n t a m i c i n, T o b r a m y c i n, a n d A m ik a c in Gifts were received of gentamicin sulfate (Garamycin ),* Tobramycin sulfate (Nebcin ) standard solution (1000 mg per liter),f and Amikacin sulfate (Amikin ).$ * Gift from Schering Corp., Kenilworth, NJ f Gift from Lilly Research Laboratories, Division Eli Lilly and Co., Indianapolis, IN t G ift from Bristol L aboratories, D ivision of Bristol-Myers Co., Syracuse, NY

3 Radioimmunoassay Procedure MEASUREM ENT O F GENTAM ICIN BY RADIOIMMUNOASSAY 49 Aliquots (50 /u,l) of the individual gentamicin standards (0, 1.0, 2.0, 4.0, 8.0, and 16.0 mg per liter), controls, and unknowns were diluted with 5.0 ml of deionized water. Duplicate disposable polypropylene percent B = ( were averaged. The standards, patient specimens, and controls were calculated as a percentage of the zero standard in order to determine the percent binding of the antiserum for each of the other standards, patient specimens, and controls. The calculations were performed as follows: Average counts of standard or specimen-blank Average counts of zero standard-blank ) x 100 tubes (12 x 75 mm) were marked for each standard, control and unknown. Fifty /xl of each 1:101 dilution were pipetted into appropriately marked tubes followed by the sequential additions of 500 /xl of 125Igentam icin and 500 /a1 of the p reprecipitated anti-gentamicin serum. The tubes were vortex-mixed for three to five seconds between additions. Duplicate non-specific binding tubes were prepared by adding 50 (A of deionized water, 500 /xl of 125I-gentamicin, and 500 A of the blank antiserum complex to duplicate tubes. The total counts were determ ined by counting duplicate 500 d aliquots of the 1Z5I-gentamicin. All of the tubes were vortex-mixed gently for three to five seconds and incubated for 10 minutes at room temperature. Following this incubation period, all of the tubes (with the exception of the total count tubes) were centrifuged at 1600 x g for 10 minutes at 4, and the supernates were discarded. The tubes were blotted on absorbent paper to remove residual radioactivity from the lips of the tubes, and the radioactive counts in the individual precipitates were determined by counting in an automatic gamma scintillation spectrom eter 1 for two minutes. Calculations The duplicate counts for the total counts, standards, and patient specimens 1 Searle Model 1285, Searle Analytic, Inc., Des Plaines, IL The standards were subjected to linear regression analysis of the logit transformation of the percent bound versus the log10 of the gentamicin concentration.22 From this analysis, the slope and the y-intercept of the standard line were obtained. Using the formula for a straight line (where y = logit of the percent bound and x = log10 of the gentamicin concentration), the concentration of gentamicin in each patient specimen and control was computed. Results S e n s i t i v i t y a n d S p e c i f i c i t y o f t h e A n t i - G e n t a m i c i n S e r u m In figure 1 is illustrated a typical semilogarithmic standard curve for the gentamicin radioimmunoassay. The assay demonstrated a detection limit, defined as the smallest quantity of gentamicin which could routinely be distinguished from the zero standard, of approximately 900 pg. This detection lim it corresponds to a serum concentration of approximately 1.0 mg per liter. The binding of the 125Igentam icin derivative to the antigentam icin serum in the absence of unlabelled gentamicin averaged 55 ± 5 percent of the total radioactivity. The non-specific binding (125I-gentamicin, zero standard, and blank antibody complex) averaged 5.0 ± 0.5 percent of the total radioactivity. Although the standard curve shown in figure 1 indicates a high standard of 16.0 mg per liter, all patient and control specimens 3= 14.0 mg per liter were routinely diluted with an equal vol-

4 50 W ANG, W ALSH, AND ABARCA FIGURE 1. Sem i-logarithm ic standard curve for the gentam icin assay. The percent antibodybound 125I-gentam icin is plotted as a function of the gentamicin concentration (mg per liter). ume of gentamicin-free serum and reassayed. The final value is corrected for dilution. Evaluations of the cross-reactivities of tobramycin and amikacin in the gentamicin assay are depicted in figure 2. Tobramycin at a concentration as high as 1000 mg per liter did not result in a demonstrable displacement of the 125I-labeled gentam icin derivative from the antibody complex. Similarly, amikacin at a concentration of approximately 2000 mg per liter did not affect the accuracy of the gentamicin assay. Furthermore, the following antibiotics were assayed individually with the radioimmunoassay procedure for gentamicin at a concentration of 1280 mg per liter and were found not to cross-react with the anti-gentamicin serum: streptomycin, clindamycin, penicillin, erythrom ycin, lincom ycin, tetracycline, kanam ycin, natidixic acid, chloram phenicol, vancomycin, polymixin B, dicloxacillin, methicillin, and cephalothin. The specificity of the anti-gentamicin serum was further evaluated by determ ining the parallelism of the radioimmunoassay. In figure 3 are illustrated the data obtained following the analysis of serial dilutions of three specimens collected from patients known to have received gentamicin as part of their therapy. The excellent proportionality observed (figure 3) betw een the amount of gentamicin quantified and the quantity of specimen analyzed for all three specimens documents the immunochemical similarity between the gentamicin measured in serum and the gentamicin used as the reference standard. AMINOGLYCOSIDE CONCENTRATION, mg/taf F ig u r e 2. C ross reactivity of anti-gentam icin serum w ith g entam icin (o), tobram y cin (A), and a m ik a c in ( ). P e r c e n t a n tib o d y -b o u n d 125Igentam icin is plo tted as a function of th e am inoglycoside concentration (mg p e r liter). F ig u re 3. T he m easured concentration of gentam icin (mg p e r liter) p lo tted as a function o f the volum e (/u.1) of serum added p er tube.

5 M EASUREM ENT O F GENTAM ICIN BY RADIOIMMUNOASSAY 51 A n a l y t i c a l R e c o v e r y The accuracy of the gentamicin assay was determ ined by adding known quantities of crystalline gentamicin (5.0, 10.0, and 20.0 mg per liter) to sera collected from patients receiving gentamicin as part of their therapy. The data presented in table I indicate that the analytical recovery of gentamicin from the sera of these patients averaged 102 percent. I n t r a -a s s a y a n d I n t e r -a s s a y V a r i a t i o n The intra-assay variation of the method was determ ined by assaying three different quality control pools on the same day (n = 20). The inter-assay variation was determined by assaying the same control pools over a period of 50 consecutive assays. The data accumulated from both studies are summarized in table II. The intra-assay variation was generally < 10 percent at a gentamicin concentration of approximately 3.2 mg per liter and < 5 percent for values between 10 and 16 mg per liter. Similar precision data were observed for the inter-assay variations. Discussion The radioimmunoassay technique described is an excellent method for the determination of gentamicin by the clinical chemistry laboratory. The rapidity, sensitivity and specificity characteristics of the method render it useful both as an aid to the clinician in arriving at accurate management decisions and as a tool for investigators studying the pharmacodynamics of gentamicin. Sources of Error Serum or EDTA plasma collected by standard procedures may be used in this assay. If plasma samples are used, they should be fresh, not previously frozen. Serum samples may be stored between 2 and 8 C for assay within 48 hours or TABLE I Analytical Recovery of Gentamicin Added to Human Serum Gentamiein (mg/iiter) A B C Recovery Specimen Present Added Assayed (Percent)* C-A Recovery (percent) = B x 100. frozen for longer periods of time. Specimens must be free of particulate matter, such as red cells, fibrin strands, or insoluble proteinates, because they may interfere with the method. The gentamicin standards, control sera and all patient specimens must be diluted in polystyrene or polypropylene tubes to minimize adsorption to container walls. Dilution of sera containing gentamicin in glass containers results in substantial adsorption of the antibiotic to the surface of the container.14 In order to obtain an assay value which represents the steady-state physiological level, peak gentamicin levels should be obtained within one-half hour at the end of the infusion for intravenously administered gentamicin or one hour after intram u scu lar injection. T rough levels TA BLE I I Intra- and Inter-Assay Variations of the Gentamicin Measurement Control Number 1 Number 2 Number 3 Intra-Assay Variation Mean (mg/liter) S.D C.V. (percent) Inter-Assay Variation Mean (mg/liter) S.D C.V. (percent)

6 52 W ANG, W A LSH, AND ABARCA should be obtained just prior to the subsequent dose. Resumé of Clinical Investigations Peak gentamicin levels of 4 to 8 xg per ml are generally considered acceptable for treating serious Gram-negative bacillary infections.13 Trough values of 2 /xg per ml or greater7 and peak values of 12 /xg per ml or greater11,20,21 have been associated with toxicity. Peak and trough gentamicin levels should be collected during the first day of treatment and every three to four days thereafter in patients with stable renal function. Elderly patients and those w ith pre-existing renal im pairm ent should be monitored prior to and after every dose to avoid toxicity. Acknowledgm ents Thanks are extended to Dr. Charles D. Hawker for his review of the m anuscript and his many helpful suggestions. A special thanks is extended to Ms. Susan C. Schoenberger for the preparation of the manuscript. References 1. ALCID, D. V. and SELIGMAN, S. J.: Simplified assay for gentam icin in the presence of other antibiotics. A ntim icrob. A gents C hem other. 3: , A n h a l t, J. P.: Assay of gentam icin in serum by high-pressure liquid chromatography. Antimicrob. Agents Chemother. 11: , B e rk, L. L ew is, J. L., and N e ls o n, J. C.: Onehour radio-immunoassay of serum drug concentrations, as exem plified by digoxin and gentamicin. Clin. Chem. 20: , B r o u g h t o n, A. and S t r o n g, J. E.: Radioimm unoassay o f io d in ated gentam icin. C lin. Chim. Acta 66: , B u rd, J. F., W o n g, R. C., F e e n e y, J. E., C a r r ic o, R. J., and BOGUSLASKI, R. C.: H om ogeneous reactant-labeled fluorescent im m unoassay for therapeutic drugs exem plified by gentam i c in d e te rm in a tio n in h u m a n se ru m. C lin. C hem. 23: , C h a n, R. A., B e n n e r, E. J., and H o e p r ic h, P. D.: G entam icin therapy in renal failure: A nonogram for dosage. A nn. In te rn. M ed. 76: , D a h l g r e n, J. G., A n d e r s o n, E. T., a n d H e w it t, W. L.: G entam icin blo o d levels: A guid e to nephrotoxicity. A ntim icrob. A gents C hem other. 8 :58-6 2, G i n g e l l, J. C. and W e l t e r w o r t h, P. M.: Dose of gentam icin in patients with normal renal function and renal impairment. Brit. Med. J. 12:19-22, H a a s, M. J. and D a v ie s, J.: Enzymatic acetylation as a means of determ ining serum aminoglycoside concentrations. Antimicrob. Agents Chemother. 4: , H a le s, C. N. and R a n d le, P. J.: Immunoassay of insulin w ith insulin-antibody precipitate. Biochem. J. 88: , H ew IT T, W. L.: G entam icin: T oxicity in perspective. Postgrad. Med. J. 50:55-58, H o lm e s, R. K. and S a n f o r d, J. P.: Enzymatic assay for g entam icin and re la te d am inoglucoside antibiotics. J. Infect. Dis. 129: , J a c k s o n, G. G. and R if f, L. ].: Pseudomonas bacteremia: Pharmacologic and other bases for failure of treatm ent with gentamicin. J. Infect. Dis.. 124: , Jo s e p h s o n, L., H o u le, P., and H a g g e r t y, M.: Stability of dilute solutions of gentamicin and tobramycin. Clin. Chem. 25: , K r o o d e n, E. and DARRELL, J. H.: Rapid gentam icin assay by enzymatic adenylation. J. Clin. Pathol. 27: , L ew is, J. E., N e ls o n, J. C., and E l d e r, H. A.: Radioimmunoassay of an antibiotic: Gentamicin. Nature New Biol. 239: , M a h o n, W. A., E z e r, J., and W ils o n, T. W.: Radioimmunoassay for m easurem ent of gentam icin in blood. A ntim icrob. A gents Chemother. 3: , M a i tr a, S. K., Y o sh ia w a, T. T., H a n s e n, J. L., ET AL: Serum gentam icin assay by high performance liq u id chrom atography. C lin. Chem. 23: , M a t ti a s s o n, B., S v e n ss o n, K., B o r r e b a e c k, C., J o n s s o n, S., and K r o n v a l l, G.: N onequilibrium enzyme immunoassay of gentamicin. Clin. Chem. 24: , R e e v e s, D. S.: G entamicin therapy. Brit. J. Hosp. Med. 12: , R if f, L. J. and J a c k s o n, G. G.: Pharmacology of g en tam icin in m an. J. In fect. D is. 124 (suppl): , Ro d b a r d, D., B r id s o n, W., and Ra y f o r d, P. L.: R apid calculation o f radioim m unoassay results. J. C lin. L ab M ed. 74: , S a b a th, L. D., C a se y, J. I., R u c h, P. A., e t a l : Rapid micro-assay of gentamicin, kanamycin, neom ycin, streptom ycin and vancomycin in serum or plasma. J. Lab. Clin. Med. 78: , S h a w, E. J., W a ts o n, R. A., L a n d o n, J., and S m ith, D. S.: Estimation of serum gentamicin by quenching fluoroim m unoassay. J. Clin. Path. 30: , S m ith, D. H., V an O t t o, B., and S m ith, A. L.: A rapid chemical assay for gentamicin. New Eng. J. Med. 286: , W a ts o n, R. A. A., L a n d o n, J., S h a w, E. J., and S m ith, D. S.: Polarization fluoroimmunoassay of gentamicin. Clin. Chim. Acta 73: ,1976.