Enzyme immunoassay is a relatively recent approach to quantitative analysis for drugs in biological fluids (1). One type of homogeneous enzyme immunoassay.
in a functional neural tumor. J. Clin. Invest. 39, 1729-1736 4. LaBrosse, E. H., Com-Nougue, C., Zucker, J. -M., et al., excretion of 3-methoxy-4-hydroxyphenylacetic acid by 288 with neuroblastoma and related neural crest tumors. Cancer 1995-2001(1980). 5. Muskiet, F. A. J., Fremouw-Ottevangers,
D. C., Wolthers,
(1960). Urinary patients Res. 40, B. G.,
and deVries, J. A., Gas-chromatographic profiling of urinary acidic and alcoholic catecholamine metabolites. Clin. Chem. 23, 863-867 (1977).
with cellulose acetate membrane. Am. J. Clin. Pathol. 41, 373-376 (1964). 11. Addanki, S., Hinnenkamp, E. R., and Sotos, J. F., Simultaneous quantitation of 4-hydroxy-3-methoxymandelic (vanilmandelic) and 4-hydroxy-3-methoxyphenylacetic (homovanillic) acids in human urine. Clin. Chem. 22, 310-314 (1976). 12. Van de Calseyde, J. F., Scholtis, R. J. H., Schmidt, N. A., and Leyten, C. J. J. A., Gas-chromatography in the estimation of urinary metanephrines and VMA. Clin. Chim. Acta 32, 361-366 (1971).
6. Knight, J. A., and Haymond, R. E., Improved colorimetry of urinary 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid). Clin. Chem. 23, 2007-2010 (1977). 7. Pisano, J. J., Crout, J. R., and Abraham, D., Determination of 3methozy-4-hydroxymandelic acid in urine. Clin. Chim. Acta 7, 285-291(1962). 8. Muskiet, F. A. J., Fremouw-Ottevangers, D. C., Nagel, G. T., and Wolthers, B. G., Determination of 4-O-methylated catecholamine metabolites in urine by mass fragmentography. Clin. Chem. 25,
13. Schwedt, G., Blodorn, A., and Bussemas, H. H., Gas-chromatographische Routineanalyse von Metanephrin/Normetanephrin, 3-
1708-1713
(1977).
(1979).
9. Williams, C. M., and Greer, M., Diagnosis of neuroblastoma by quantitative gas-chromatographic analysis of urinary homovanillic and vanilmandelic acid. Clin. Chim. Acta 7,880-883 (1962). 10. Hermann, G. A., The determination of urinary 3-methoxy-4hydroxymandelic (vanilmandelic) acid by means of electrophoresis
CLIN.
Cl-EM.
27/12.
2032-2034
Methoxy-4-hydroxyphenylglykol
als TMS-Vanillin
und Vanillinmandelsaure
bzw.-vanillylalkohol.
im Urin
Clin. Chim. Acta 65,309-318
(1975).
14. Melchert, Von H. -U., and Hoffmeister,
H., Determination
of the
urinary metabolites hydroxyindoleacetic acid, vanillylmandelic acid and homovanillic acid by means of lipophilic gel chromatography and gas chromatography.
J.
Clin. Chem.
Clin. Biochem.
15, 81-87
15. Brewster, M. A., Berry, D. H., and Moriarty, M., Urinary 3-methoxy-4-hydroxyphenylacetic (homovanillic) and 3-methoxy-4hydroxymandelic (vanilmandelic) acids: Gas-liquid chromatographic methods and experience with 13 cases of neuroblastoma. Clin. Chem. 23, 2247-2249 (1977).
(1981)
Antibody Selectivity of a Quantitative Immunochemical Lidocaine
Assay for Serum
Brian E. Pape I found the cross reactivity of an enzyme immunoassay for lidocaine towards a test drug, mepivicaine, to be inversely related to the concentation of mepivicaine. A simple quantitative method for the investigation of antibody se-
tivity
lectivity is presented, and a more rigorous approach towards “screening” for immunoassay cross reactivity is proposed.
co-administration or metabolic formation of the cross-reactive constituent, or the qualitative identification of “outlier” effects. The most potentially cross-reactive compounds are
AddItIonal Keyphrases: assessing antibody selectivity screening
#{149}
enzyme immunoassay
regarding
assay
(EMIT
EMIT
immunochemical)
selec-
Department of Pathology, Health Sciences Center, University Missouri, Columbia, MO 65212. Received March 24, 1981; accepted 2032
July
12, 1981.
CLINICAL CHEMISTRY, Vol. 27, No. 12, 1981
chemical cross reactivity
of
would require a significant
level of
usually structural analogs of the reference compound. The quantitation of EMIT immunochemical selectivity the drug being assayed
Enzyme immunoassay is a relatively recent approach to quantitative analysis for drugs in biological fluids (1). One type of homogeneous enzyme immunoassay (EMIT#{174}) is commercially available from Syva Co., Palo Alto, CA 94304. The EMIT methodology has currently been applied to drug assays, which include antiarrhythmic agents (2). Clinical studies have generally supported the manufacturer’s claims regarding the advantages of EMIT: technical simplicity, speed of analysis, and specimen requirements (3,4). Comparisons of EMIT with older methods have generally concluded that EMIT assays are sufficiently sensitive, selective for the drug-of-interest, and accurate (5). As a result of these comparisons, independent
conclusions
are based almost exclusively on regression analysis of vs the comparison procedure. While this is an accepted scientific approach, the identification of specific immunoEMIT
vs other
test drugs,
for
drug metabolites,
or endogenous biochemical factors has largely been a result of the manufacturer’s “in-house” screening as part of antibody production and quality control (2). One approach used by the manufacturer of EMIT is to determine the concentration of the test drug required to increase the apparent concentration of the assayed drug when added to a commercial “calibrator” material and analyzed by EMIT. As part of the EMIT lidocaine product disclosure, Syva reports the concentration of selected
drugs and metabolites necessary to produce a 30% quantitation error (increase) in a “calibrator” containing 3.0 mg of lidocaine per liter (2). A different approach to the quantitation of EMIT antibody selectivity is to determine the apparent molar selectivity ratio of antibody-enzyme reactivity towards the assayed drug as compared with the test drug (5). This approach can conveniently allow a more quantitative investigation of the chemistry as well as the stoichiometry of antibody selectivity in the presence or absence of the assayed drug. The EMIT assay for lidocaine has been reported to be most
sensitive (non-selective) towards mepivicaine (I) as compared with other structural analogs of lidocaine (II), with the exception of the N-dealkylated metabolite of lidocaine (2-5). Syva currently treats EMIT lidocaine antibody reagent with N-desalkyl-lidocaine to decrease cross reactivity to this metabolite (2). Therefore, EMIT lidocaine antibody cross reactivity to mepivicaine was investigated in order better to understand potential cross-reactivity problems that might be associated with this and other EMIT assays based on the same
mepivicaine, and [lidocainelnQulv is the concentration of lidocaine (in mg/L) equivalent to solutions of lidocaine equimolar with the mepivicaine standards. MSR’ is used to designate molar selectivity ratios calculated at different test drug concentration ratios [in the original report of mepivicaine cross reactivity the selectivity ratio MSR was used, calculated to quantitate cross reactivity at 1000 smolfL (5)1.Calculated MSR’s depend on the concentration of test drug (see Re-
antibody-enzyme
drug-antibody-enzyme-substrate process were recorded as .A .Ao by the CP-5000 timer-printer. All analyses of test and assay drug standards were done in triplicate and the mean was used when rate data were within a range of six LA LA0 units.
reagent
principle.
sults). Rate
-
or kinetic
data
for the
EMIT
-
CH3 0
-
II
Results
-
\/
c-,1
Standardization of the EMIT lidocaine assay was validated by quantitation of a 10 izmol/L plasma standard of lidocaine . HC1 (expected concentration = 2.34 mg/L lidocaine base, determined concentration = 2.21 mg/L).
H
CH3
Figure
1 summarizes
calculatedEMIT
\
I II N-C----C-N
I H
-
II
CH3
standards curves tmol/L system tmol/L
Materials and Methods Immunochemical (EMIT) Analysis Apparatus. The semi-automated equipment consisted of a Model 300-N microsample spectrophotometer equipped with a thermally regulated flowcell set at 30 #{176}C (Gilford Instrument Laboratories, Inc., Oberlin, OH 44074), a CP-5000 Clinical Processor modified to function as an EMIT printercalculator, and a pipettor-dilutor, the last two supplied by Syva. Reagents. The reagents, calibrators (1,2,3,5, and 12 mg/L), and controls were those commercially available from Syva. Procedure. The EMIT lidocaine procedure as established by Syva (2) was followed.
lmmunochemical
results,
and
5 mol/L, Figure 1). Additionally, the difference in mepivicaine rate data at the usual extremes of serum concentration encountered with therapeutic use of mepivicaine (0.5 to 5.0 mg/L or about 2-20 Mmol/L) (8) is approximately 90 A - A0 units; this range is compared with an equivalent reaction rate difference for usually-encountered lidocaine concentrations of approximately 175 LA A0 units (concentration range: 5-50 mol/L). Since mepivicaine is metabolized by aromatic hydroxylation, conjugation, and N-demethylation, assay of it with use of commercial EMIT lidocaine antibody-enzyme reagents would also require investigation of antibody cross reactivity towards relevant concentrations of mepivicaine metabolites (8). More important than the consideration of the development of assays for structural analogs of EMIT test analytes, these
2034 CLINICALCHEMISTRY, Vol. 27, No. 12, 1981
mepivicaine data clearly demonstrate that EMIT lidocaine antibody specificity is concentration-dependent. Any quantitative approach to determination of assay selectivity will depend on the nominal concentrations of assay and test drug chosen for comparison of EMIT assay response. Recently, analytical protocols have been developed for the quantitation of low concentrations of therapeutic agents in plasma ultrafiltrates by EMIT. “Free-drug” concentrations in plasma ultrafiltrates are typically much lower than the total drug concentration in plasma, and concentration ratios between common therapeutic agents and metabolites may be quite different in recovered ultrafiltrate. In cases where the concentration ratio between drug and cross-reactive substance decreases in plasma ultrafiltrate, concentration-dependent antibody selectivity could result in a clinically significant bias. In characterizing EMIT lidocaine assay selectivity as apparent cross reactivity at the 3.0 mgfL lidocaine concentration (EMIT number three calibrator), a “best-case” result is obtained. EMIT response to assay drug is typically most sensitive (slope of LoA - A0 rate data vs drug concentration) at drug concentrations between the number two and number four EMIT calibrators. At the same type, MSR’s for drug vs other compounds (mepivicaine) have been shown to decrease with decreasing concentration. As a general rule, I believe that EMIT assay response to assay drug vs “other” drug should be determined initially at an assay drug concentration equal to that of the number one EMIT calibrator and at “other” drug concentrations equal to two times the generally accepted mafimum therapeutic concentration of the “other” drug (or the concentration of the number one EMIT calibrator, whichever is greater). If the cross reactivity to “other” drug is below an arbitrarily set level, assay inhibition (decreased i.A - A0) due to high concentrations of “other” drug should be determined at toxic concentrations of the “other” drug. If cross reactivity towards “other” drug is substantial, EMIT antibody selectivity should be determined over the relevant concentration range of the “other” drug. If the quantitative EMIT assay is modified to measure effective in vitro concentrations of drug below the manufacturer’s specifications, analytical selectivity should be determined for the effective concentration ranges for assay drug and “other” drug.
References I. Bakerman,
S., Enzyme
immunoassays.
Lab. Man.
18(8), 21
(1980).
Lidocaine Assay, Syva, Inc., Palo Alto, CA, 1980. K. E., Schneider, R. S., and Ullman, E. F., “Homogeneous” enzyme immunoassay. A new immunochemical technique. Biochem. Biophys. Res. Cornmun. 47, 846 (1972). 4. Bastiani, R. J., Phillips, R. C., Schneider, R. S., and UIlman, E. F., Homogeneous immunochemical drug assays. Am. J. Med. Technol. 2. EMIT-CAD,
3. Rubenstein,
39, 211 (1973).
5. Pape, B. E., Whiting, R., Parker, K. M., and Mitra, R., Enzyme immunoassay and gas-liquid chromatography compared for determination of lidocaine in serum. Clin. Chem. 24, 2020 (1978). 6. EMIT-Benzodiazepine Assay, Syva, Inc., Palo Alto, CA, 1980. 7. EMIT-Barbiturate Assay, Syva, Inc., Palo Alto, CA, 1980. 8. Baselt, R. C., Disposition of Toxic Drugs and Chemicals in Man, 2, Biomed. Pub., Canton, OH, 1978, pp 84-86.
