Enzyme Multiplied Immunoassay Technique: A Review

December 1978 303

ENZYME MULTIPLIED IMMUNOASSAY TECHNIQUE: A REVIEW

Authors: Elvin G. Curtis Pharmacy Laboratories and College of Pharmacy University of Michigan Ann Arbor, Michigan

Jayant A. Patel Drug Analysis Laboratory University Hospital Ann Arbor, Michigan

Referee: John 0. Batsakir Department of Pathology and Clinical

University of Michigan Medical School Ann Arbor, Michigan

Laboratories

INTRODUCTION

The enzyme multiplied immunoassay technique (EMIT@)* is one of several immunoassays that have come into general use in many of today's clinical laboratories, primarily because of their relative simplicity, specificity, and accuracy. Similar enzyme immunoassays are the enzyme immunoassay (EIA) and the enzyme linked immunoassay (ELISA). Aside from the improved qualitative aspect which allows information on overdose cases to be provided on a stat basis, the quantitative or monitoring aspect of the EMIT system benefits those patients on maintenance therapy, particularly outpatients for whom a rapid monitoring system is now available to aid in the adjustment of dosage. A number of different EMIT assays are now marketed or under development for a rather wide range of clinical applications. A singular advantage, particularly for such drugs as anticonvulsants,

is that large numbers of samples from different patients can be run quickly and consecutively.

IMMUNOASSAY LABELING

Scharpe et al. * and Wisdom2 have reviewed the different enzyme immunoassay techniques. Therefore, it seems appropriate to review briefly the other immunoassay methods before examining the merits and limitations of the EMIT system. The basic principle of reactivity for all the different immunoassay techniques remains a com- petitive binding mechanism, but the differences between them lies in the ways in which they have been labeled, the molecular recognition properties of antibodies, and the methods used to detect the label (i.e., a radioisotope in the RIA or an enzyme in the EMIT). All use either labeled haptens, antigens, or antibodies, depending on the sensitivity and specificity re-

* EMIT is a trademark of the Syva C o . . Palo Alto. California.

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304 CRC Critical Reviews in Clinical Laboratory Sciences

quired. The most commonly used labels in immunoassay methodologies are

Label Name

Radioisotope Radioirnmunoassay or RIA Erythrocyte Hemagglutination inhibition or HI Bacter iophagc Viroimmunoassay or VIA Fluorescent group Fluoroimmunoassay Stable free radical.

Enzyme Enzyme immunoassay or EIA,

Spinimmunoassay or FRAT@ Free radical

EMIT, ELISA

Radioisotope labeled (radioimmunoassay or RIA)'-'" - Since Yalow and Berson' first published a method for measurement of insulin in a biological specimen, growth of radioimmunoassay and other immunoassay techniques resulted in the availability of drug assays for morphine, LSD, and such antibiotics as gentamicin and tobramycin; all had been difficult to ana- lyze in biological specimens due to nanogram or picogram concentrations. Radioirnrnunoas- say utilizes a radioactive isotope, e.g.,'H,''C, or I Z s I , as a label for the compound to be analyzed. The displacement of the quantity of labeled drug from &he antibody complex by the free drug in the sample is detected by measuring the radioactivity of the liberated labeled drug- using scintillation spectrometry or gamma counting. In order to detect the radioactive drug fraction that becomes detached from the antibody complex, it is necessary to separate the displaced labeled drug from the mixture of antibody complex, antibody, and labeled drug antibody complexes. This is achieved by precip- itation with ammonium sulfate. Radioactivity is then measured in a sample of the supernatant solution that contains the displaced radioactive drug. The quantity of displaced radioactive drug from the antibody compIex is proportional to the concentration of free drug present in the sample solution. This method for the detection and quantitative analysis of drugs has distinct advantages for most assays in that it is sensitive to very low concentrations in the range of 10-' to lo-'" Equipment and reagents are rather expensive and special technical skills are required. Digitoxin, dexamethasone, insulin, gentamicin, and tobromycin are radioim- munoassays of clinical importance. Assays for

drugs of abuse such as morphine, barbiturates, cocaine, amphetamines, and methaqualone, are also available.

Ertbrocyte labeled (hemagglutination inhibition or HI'2-'5 - This method utilizes red blood cells with drug molecules attached. Adler et al. have developed methods for detection of mor- phineaz~23 and methadone". When morphine is present in a urine or serum specimen, i t binds the antibodies of morphine and prevents agglutination of the red blood cells. The red blood cells which have been coupled with the morphine antigens of the red blood cells then settle down as a pellet in a disposable micro-titer well. In the absence of morphine, the antibodies to morphine will agglutinate the red blood cell- morphine antigen complex, and the cross- linked substance then remains in a suspended state. The reaction is observed and interpreted after 2 to 3 hr. This is a semiquantitative test, and sensitivity is adjusted over a wide range, since inhibition of agglutination is proportional to the amount of antibody used. The sensitivity of this system is 20 to 30 ng/ml. However, at very low concentrations, there is a danger of false positive readings due to loss of antibody binding capacity. The most advantageous fea- ture of this system is the relative low cost, as detection is performed by utilizing disposable titer trays and the results are manually read. The disadvantages of this system are that it takes more time than any other immuno system and interpretation of the results is subjective. Presently, assays for morphine, barbiturates, and methadone are commercially available. *

Bacteriophage labeled (viroimmunoassay or VIAY6-''' - This technique involves chemical bonding between antigens or haptens and a bacteriophage. The modified bacteriophage is via- ble but can be inactivated by specific antibodies for hapten or antigens attached to bacteriophage. Free hapten or antigen present in the specimen inhibits the inactivation.

Fluorescent labeled (fluoroimmunoassay or FIAY9-'" - The fluoroirnmunoassay method is similar to the radioimmunoassay technique except that there is replacement of the radioactive label by a fluorescent label which is then quan- titated in a fhorometer.

Stable free radical labeled (spinimmunoas-

Marketed by R.D. Products, Industrial Diagnostic Division. Victor, New York.

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December 1978 305

Antibodv

0 Ba~l.rml substrat. 1cI.Wl IC'I

FIGURE 1. Semiquantitative: EMIT@ assays for drugs of abuse.

say) or free radical assay technique (FRAT)3*- l3 - A stable free radical, such as a nitroxide, is attached to the drug, which gives off a very sharp electron spin resonance (ESR) spectrum, measured on a spin resonance spectrometer. The spectrometer measures the energy absorbed by the unpaired electrons when they tumble from a stable to a metastable state. However, when the free radical is complexed to an antibody, the tumbling is slowed, thus producing a broad electron resonance spectrum. During analysis, if a free drug is in the specimen, the complexed spin label is displaced and this gives off a sharp ESR spectrum. The displacement and the magnitude of the ESR peak intensity is proportional to the amount of drug present in the specimen. Thus, the concentration of the drug present in the specimen can be calculated by comparison with the peak height of the standards. Some advantages of the free radical assay technique (FRAT) are

1. ' After calibration of the instrument and sta- bilization of the reaction mixture, results are available in less than 1 min.

2. Results are interpreted objectively. 3. Phase separation of bound and free forms

of the spin label drug is not required as in other immunoassay systems except EMIT.

4. Sensitivity at the lower detection limit is 250 ng/mP (0.1 x IO-'M), and conjugates of the drug are detectable. Very little sample preparation is required, making the system suitable for mass screening.

Assays are available for opiates, barbiturates, cocaine, and methadone.

5 .

6. The system is nonradioactive. 7.

Prior to the advent of the FRAT assay, there was no other analytical system for quick mass screening for drugs of abuse. Enzyme labeled immunoassay technique'

- In this procedure, the active enzyme label is attached to the hapten or drug to be analyzed (Figure 1). This enzyme (lysozyme) serves as a catalyst in the lysis of the mucopolysaccharide component of the cell wall of the bacterial substrate solution. When the enzyme labeled drug (A) is complexed (as shown in Figure I ) with the drug specific antibody (B), the enzymatic activity is blocked and lysis of the bacterial cell wall (C) does not occur. However, i f the drug to be detected is present in the sample undergoing analysis, the unknown drug (D) competes with the enzyme drug for antibody binding site and displaces the enzyme from the antibody. The enzyme labeled drug is displaced (A') and

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TABLE 1

C r o s k r d v i t y of EMIT@ Assay For Amphetamines With Other Medi- cationr

Urine Relative concentration reactivity

Drug bcg/m!) ( R R 3 Ref.

&Amphetamine Methamphetamine Ephedrine Pseudoephedrine Phenmetrazine Methylphenidate Mephentermine Phcnylpropanolamine Benrphetamine

Tranylcypromine Triarninoheptane Phenylethylamine Tyramine HCL

I lndomethacin

1 .o 0.9 4.5 4.5 0.95

50.0 1.6 5.0

24.0 1.2

14.0 20.0 3.1

I .o I .08 0.22 0.22 I .05 0.02 0.62 0.2 0.04 0.83 0.07 0.05 0.32

34 34 34 34 34 34 34 34 36 36 36 ’

36 31

Relative reactivity (RR) is the ratio obtained by dividing the & amphetamines equivalent value of I p g h ! by the concentration in pg/mf of the cross-reacting drug in the urine.

rendered active, and lysis of the bacterial cell wall occurs (C’). The amount of a free or un- bound enzyme is proportional t o the amount of free drug present in the specimen. The reaction is then measured by a spectrophotometer over a fixed length of time (40 sec) and at constant temperature (37°C). The difference in optical density is a measurement of the amount of bacterial lysis that occurs.

The following enzyme multiplied immunoassays are commercially available:

semiquantitative Quantitativc

Amphetamines Phenytoin Barbiturates P henobarbi-

Benzodiazepines Primidone Cocaine Metabolites Ethosuxim-

Methadone Carbamaze-

Opiates (morphine, codeine) Digoxin Propoxyphene

tal

ide

pine

A critical narrative on each assay follows.

SEMIQUANTITATIVE EMIT ASSAYS

Amphetamines The EMIT amphetamine assay is designed to

detect d-amphetamine and methamphetamines with a detection sensitivity of 1 .O pg/mf of urine specimen. EMIT is less sensitive for detection of &amphetamine than is radioimmunoassay (RIA), which detects levels of 0.25 to 0.5 pg /mf of this drug.35 A limiting factor to RIA is that it fails to detect levels of methamphetamine less than 22 pg/mf of urine and therefore, most samples would depict a false negat i~e.’~ EMIT detects amphetamine in the urine only, and the results are semiquantitative. To obtain accurate serum levels, RIA or gas chromatography (GC) should be

Another shortcoming of the EMIT assay for amphetamines is that false positives are obtained due to cross-reactivity with drugs commonly present in cold remedies, diet medica- tions, or other prescribed drugs (Table l). This probably accounts for the relatively high percentage of false positives reported in the Toxi- cology Proficiency Testing Program conducted by the Center for Disease Control (CDC), At- lanta, G e ~ r g i a . ‘ ~ . ~ ~ The presence of lysozymes

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December 1978 307

in the urine specimen may also produce a false positive. However, this can be easily ruled out by performing a determination without using antibody and enzyme-labeled drug reagents. The presence of lysozymes is indicated by lysis of the bacterial wall in the substrate solution which results in a decrease in optical density.

The pH of the specimen is important because EMIT assays perform best in the pH range of 5.5 to 8.0. Older specimens may become more alkaline due to storage in soft glass containers or the presence of preservatives or alkalizing drugs, which results in an inactivation of the enzyme displaced from the antibody complex by the free drug. A false negative then results. In such cases, the pH of the urine specimen is adjusted by using 1 .O N hydrochloric acid. Ex- tremely turbid specimens must be centrifuged prior to analysis to avoid false negatives. This extraneous turbidity may be due to other pre- cipitated urine wastes.

In the 1975 CDC Drug Abuse Proficiency Testing Programs Numbers I through IV, where spiked unknowns containing 1.5 pg/ml of d-amphetamines were used, 8 to 10 (70 of the participating laboratories reported false negatives by immunoas~ay."-'~At higher concentrations of the unknowns, less than 5% of the par- ticipants reported false negatives. Because this survey included laboratories using radioimmunoassay techniques as well as EMIT, the ob- vious inability of detecting methamphetamines at low concentrations by RIA may have con- tributed to the higher percentage of false negatives. Owing to the inherent problem of obtain- ing false positives due to cross-reactivity with other drugs, it is imperative to confirm positive results by alternative nonimmunoassay te,ch- niques, such as GC or thin layer chromatography (TLC). Primary detection of amphetamines by EMIT and confirmation by gas liquid chromatography in the authors' laboratory have resulted in neither false negatives nor false positives in the CDC Proficiency Testing Pro- grams.

Barbiturates The EMIT barbiturate assay is capable of

detecting free, commonly abused barbiturates from three catagories: short-acting (pentobarbital and secobarbital); intermediate-acting ( a m o b a r b i t a l ) ; 1 o n g - a c t i n g a n d

TABLE 2

Relative Reactivity of Barbiturates and Other Drugs

Drug

Secobarbital Phenobarbital Pentobarbital Amobarbital Thiopental Butabarbital Glutethimide Mephobarbital Tal bu tal Aprobarbital Thiamylal Metharbital Barbital Probarbital Dilantin 6

vcg/ml

1 .O 2.0 1.1 1.8 0.7 I .5

0.65 4.2

> 50

15 > 50 > 50 > 50 > 50 > 50

RR

1 .O 0.5 0.9 0.55 1.42 0.66 0.02 1.53 0.24 0.02 0.02 0.02 0.02 0.02 0.02

Ref.

34 34 34 34 34 34 34 52 52 52 52 52 52 52 52

(phenobarbital). Other barbiturates may be detected at higher concentrations (Table 2). The assay is not specific for individual barbiturates; however, if any of the above drugs are present, a positive reaction will be obtained.

The CDC barbiturate surveys of 1974 and 197540-4' (Table 3) reported a higher percentage (35%) of false negatives at a concentration of 1 pg/mP by immunoassays. However, TLC, gas liquid chromatography (GLC), and ultraviolet (UV) spectrophotometry reported 19%, 20%. and 47% false negatives, respectively. Immu- noassays have a much better overall record for false positives (2.0%) in comparison to other methods (3 to 4%). Because of a sensitivity at the 2 pg/mP level, immunoassays also have greater detectability than TLC, GLC, and UV. The EMIT assay for barbiturates has less problem with cross-reactivity than does the EMIT amphetamine assay.

Walbergl' reported a correlation between blood and urine barbiturate levels using enzyme immunoassay and spectrophotometry. In his study, a 1 to 5 pg/ml urine level correlated to 3 pg/ml of blood and, thus, eliminated the need for further analysis of blood. if the urine specimen was found negative by immunoassay. Nonagreement between the two methods was due to an inappropriate sample or to the presence of short-acting barbiturates. The sensitivity of the barbiturate enzyme imrnunoassay correlated well with the radioimniunoassay for this same category of drugs.'" However, both im-

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\

TABLE 3

Barbiturates - Center for DisuuK Control Survey (1974-1975)

False negatives (average To) False Concentration positives

(pglm!) 1.0 1.5 1.6 2.0 2.5 3.0 3.5 5 (average%)

TLC 1 9 9 2 4 6 6 3 2 1 4 GLC 2 0 1 1 2 3 5 8 6 2 4 3 uv 47 4 37 11 11 11 0 7 3 Immunoassay 35 10 29 5 2 4 0 0 2 (RIA, EMIT, HI)

TABLE 4

Concentration Relative reactivity Drug Ocg/ml)

0 x a z e p a m Diazepam Chlordiazepoxide Flurazepam Morphine

munoassays lack absolute specificity. Due to the cross-reactivity of immunoassays, it is desir- able to confirm any positive resutts by an alternative nonimmunoassay method.

Benzodiazepines The benzodiazepine enzyme immunoassay

utilizes enzyme-labeled oxazepam, a metabolite common to all of the benzodiazepines. Oxaze- pam is excreted in the urine of patients undergoing benzodiazepine therapy. The assay is highly specific for benzodiazepine metabolites and cross-reactivity with other drugs has not been reported (Table 4). Lately, diazepam has come to the forefront as one of the most abused drugs, not only because of indiscrimi- nate prescribing by health practitioners, but also due to its ready availability through illicit channels. Regent and WahlS4 examined 1076 patient samples, and 36.9% of these samples showed the presence of diazepam and chlordiazepoxide, along wih alcohol and other drugs.

Introduction of the EMIT benzodiazepine urine assay has thus made a significant contri- bution to drug abuse screening programs. This assay is sensitive (0.5 pg/mt) and specific for benzodiazepines. Because of the long halflife of these drugs and their metabolites and because of the sensitivity of the test, a single dose of 10

0.7 I .o I .5 0.46 1.5 0.46

100 0.007 >m 0.0035

mg diazepam can be detected 48 hr after inges- tion. Gas chromatography is equally sensitive for benzodiazepines and has the advantage of applicability to other biological fluids. How- ever, the procedure is more complex and time- consuming.

Cocaine Metabolites Cocaine is metabolized primarily to benzoy-

lecgonine and ecgonine, both of which appear in the urine. Due to the insolubility of these metabolites in organic extraction solvents, many urine screens do not detect the presence of cocaine and its metabolites, even though cocaine is widely used. Koontz et al.55 reported a gas chroniatographic method for detecting cocaine metabolites which involved purification by TLC, preparation of methyl derivatives, and GC. For routine identification purposes, this method seems lengthy and somewhat impracti- cal for a clinical laboratory.

Bastos, Jukofsky, and Mule56 studied cocaine metabolites by EMIT and TLC. Three thousand urine samples were analyzed by the EMIT system for the cocaine metabolites (ecgonine and benzoylecgonine). Of these, 259 samples were found to be positive. However, upon further analysis of these EMIT positives through use of TLC, 10% of the EMIT positive

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December 1978 309

TABLE 5

Cross-reactivity of Drugs with EMIT Cocaine Assay

Concentration Drug ( d m l ) RR

Benzoylecgonine Ecgonine Methylester of Ecgonine Benzoylnorecgonine Cocaine Atropine Nicotine Homatropine Scopolomine &amphetamine Morphine Methadone Secobarbital

I .o I .Ooo 9.5 0.105

380 0.003 335 0.003 460 0.002

NR 500 - NR 500 - NR 500 - NR 500 - NR 500 - NR 5 0 0 - NR 500 - NR 500 -

Ref.

56 56 56 56 56 56 56 56 56 56 56 56 56

Note: NR, no cross-reactivity detected at concentration indicated; RR, relative reactivity.

samples were not detected. This discrepancy can be attributed to the maximum sensitivity of 3 to 5 pg/ml for TLC analysis of cocaine metabolites, while EMIT can detect concentrations as low as 1 pg/mP. False negatives (1.9Vo) in the Bastos5'j study were attributed to techni- cian error in performing either method of analysis. However, the specificity of the EMIT cocaine metabolite assay was demonstrated to be excellent and no cross-reactivity (see Table 5 ) with other commonly abused drugs was found. Cross-reactivity may occur with ecgonine, which also occurs as a metabolite of ergot al- kaloids and may be found in such prescription drugs as CafergotB, Bellergala, and Mether- gine@ (Sandoz).

Table 6 shows the lower percentage of false positives (<19'0) and false negatives (4 to 6016) by immunoassay for cocaine and its metabolites, which reflects the superiority of this irn- munoassay in comparison to TLC (2%) and GLC (49'0). The high percentages of false negatives when using TLC (16%) and GLC (63%) is due to the inability of these systems to detect cocaine metabolites, primarily because of low concentration and extraction problems. Ra- dioimmunoassay was evaluated by Cleeland et al.so who demonstrated detectability of less than 0.1 pg/ml in comparison to 1.0 pg/ml with enzyme immunoassay. An equal specificity was demonstrated.

TABLE 6

Center for Disease Control Survey of Cocaine and Metab- olites

TLC GLC

Method

Immunoassay

False False positive negative

(average To) (average To)

2 16.-14 4 63+-17

<1 6+-4

Note: Those marked + contained only benzoylecgonine; the others contained cocaine and/or benzoylecgonine.

Methadone The methadone enzyme immunoassay is sen-

sitive and specific for methadone alone and not its metabolites, 2-ethylidene-l , 5-dimethyl-3, 3- diphenylpyrrolidine and 2-ethyl-5-methyl-3, 3- diphenyl-1-pyrroline. Because this assay is specific (Table 7) only to free methadone in the urine, this fact becomes important in the detection of a missing dose in methadone maintenance programs, since free methadone is found in-the urine soon after administration. The metabolites of methadone may be secreted for several days following a single dose. Free methadone in a urine specimen can be detected as low as 0.5 pg/mf. Radioimmunoassay is about five times more sensitive than enzyme im-

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TABLE 7

Cross-reactivity of Methadone Assay

Concentration Drug W m l ) RR Ref.

Methadone Meperidine 2-Ethylidine-1 ,5-dimethyl-3,3- diphenylpyrrolidine

2-Ethyl-S-methy l -3.3-dipheny 1 - I-pyrroline

Promethasine Dextromethorphan Chlorpromazine Morphine dpropoxyphene

0.5 310 NR

NR

109 475 166 NR

600

1 .O 34 0.003 34 - 34

- 34

0.009 93 0.002 93 0.006 93

34 0.002 93

Note: NR, no cross-reactivity detected at concentration indicated; RR. relative reactivity.

TABLE 8

Center for Disease Control Survey - Methodology Correlation for Methadone

Concentration (pg/mf) False positives

Method I 1.2and 1.5 2 3 4 (average %)

False negatives (average %)

TLC 3 4 5 3 4 2 GLC 2 4 6 3 0 2 lmmunoassay 2 1 1 0 2 < I

munoassay, and the specificity is similar. Sen- sitivity of TLC is about 2 pg/ml. Immunoas- says have a distinct advantage over TLC in being rapid and able to detect lower concentrations. However, GLC is more sensitive and specific than TLC and equal to or better than the immunoassay technique. Unlike immunoassays, GLC does require sample preparation, and this may be a disadvantage in screening large numbers of samples.

In the CDC Surveys for 1973 to 1976 (see Ta- ble 8), 2 to 3% of the laboratories using EMIT, TLC, and GLC failed to detect methadone at the 1 pg/ml level. At higher concentrations, detectability increased by all methods. Overall, 2% of the laboratories using TLC or GLC alone reported false positives. Immunoassays had the lowest (<lVo) incidence of reported false positives. Chlorpromazine, which is exten- sively prescribed in high doses as a psychother-

apeutic agent, does give false positives for methadone with the EMIT system.

opiates The EMIT assay for opiates is designed to de-

tect free morphine, as well as morphine glucuronide, and therefore it is not necessary to hy- drolyze the sample prior to analysis as in T'LC and GC. There is a cross-reactivity associated with the EMIT opiate assay which is particularly more sensitive to codeine than to morphine itself (See Table 9). Codeine and destro- methorphan. which frequently are constituents of nonprescription cold remedies, will manifest false positives for morphine. None of the immunoassays can distinguish between morphine and codeine. Therefore, when the presence of opiates is indicated, an alternative nonimmunoassay is necessary to identify the specific opiate. Gas chromatography, TLC, fluorometric

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Deceiiiber 1978 31 I

TABLE 9

Cross-reactivity Opiate Assay

Drug Concentration

( rg /ml) tRR Ref.

Morphine Codeine Morphine glucuronide Diacetyl morphine Nalorphine Meperidine Dextromethorphan Chlorpromazine Diphenoxylate Cocaine Methadone Amphetamine Secobarbital Phenobarbital Dihydromorphinone Dih ydromorphine Levorphanol

0.5 0.78 2.6 2.4

15.8

268.0

I .o I .28 0.38 0.42 0.006 0.025 0.006 0.006

<0.006 <o. 00 I

- O.OOO1 <O.OOOI <O.OOOI <O.OOOI

2.6 0.38 1.8 0.56 6.2 0.16

57 34 34 34 34 34 34 34 34 34 34 34 34 34 34 34 34

Note: RR is relative reactivity,

TABLE 10

Center for Disease Control Survey for Morphine

False negative (average S) for morphine and morphine glucuronide

False positive

Concentration (pg/mf) 0.5 0.8 1.0 1.5 1.7 2.0 2.5 3.0 4 6 (average%)

Method TLC GLC FLUOROMETRIC FRAT RIA HI EMIT

17 4 17 13 29 9 6 8 5 2 2 29 10 22 16 37 16 19 7 5 5 5 4 0 5 8 1 1 7 0 4 0 0 4 9 9 7 5 14 0 0 0 0 0 1 8 0 3 1 1 3 0 0 I 5 4 3' 6 6 9 4 2 0 0 2 2 1 2 6 0 3 2 3 2 2 2 2 1 3'

. One of the test samples contained codeine instead of morphine. The CDC survey clearly indicated that the fluorometric method of detection for morphine is superior to other methods in distinguishing morphine from codeine. Immunoassays scored well in not reporting false positives in comparison to TLC and GLC, except when codeine was substituted for morphine. False negatives by immunoassay were also low. At lower concentrations, TLC and GLC had a high percentage of reported false negatives.

detection, or combinations thereof, must be used for positive identification. Codeine can be easily confirmed by GC or TLC. However, the best method for morphine identification is the fluorometric assay5* (See Table 10).

Mule, Bastos, and Jukofsky3' studied 422 human urine samples for the detection of morphine by TLC, EMIT, HI, and RIA. Using

TLC, 94.3% of these samples were negative and 5.7% were positive. The percentage of positive samples were 13.7, 14.0, and 25.5% with EMIT, HI, and RIA assays, respectively, using a concentration limit of 0.5 pg/ml. By lowering the concentration limit to 30 ng of morphine per milliliter, the percentage of positive samples for the HI and RIA assays was increased to 32.7

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and 36.4Q0, respectively. The variation in the detectability of positive samples was attributed to the varying level of sensitivity for each assay. The maximum practical levels of sensitivity for morphine by the individual technique are: TLC, 1 to 2 pg/ml; EMIT, 0.5 pg/ml; HI, 30 to 60 ng/ml ; and RIA, 30 ng/ml.

Mule, Whitlock, and Jukofsky." evaluated the performance of the morphine-RIA, the Mor-Barb brand of radioimmunoassay, differential elution TLC, EMIT, and routine TLC.* Twenty-two urine samples found positive by Mor-Barb RIA assay were tested for morphine by RIA, EMIT, differential elution TLC, and routine TLC. Of samples containing 0.5 pg/ml or less of morphine, 3 1.9% were found to be positive by RIA and Mor-Barb RIA, but were negative by EMIT, differential TLC, and routine TLC, due to a concentration below the sensitivity detection level. The false negative values by EMlT and TLC were 2.7% and 0%. respectively; the false positive values obtained were 0070 and 9.1 Qo, respectively.

Vander Slooten and Vander Helms9 reported poor correlation between the EMIT opiate assay and the GLC mass spectrometric assay for morphine and codeine in urine. For within-run precision, the coefficient of variation (CV) of the EMIT assay was 7% (N = 38), and with day- to-day precision the C V was 21 Qo (N = 29) (determined in the range 0.5 pg/mf .) The gas chromatographic mass spectrometric assay had within-run precision (CV) of 5% (N=25), and a day-to-day precision of 7% (N=21). The EMIT assay showed 4% false positives and 5.6 pg/ml of morphine as a cutoff concentration. Lowering the cutoff concentration of 0.1 pg/ ml for the GC mass spectrometric assay resulted in a decrease in false positives (l.6Q0) and an increase in false negatives (6.4%). The poor correlation between the two methods was due to:

I . Determination of morphine and codeine by GLC-mass spectrometry after complete hydrolysis Lack of absolute specificity, cross-reactivity, and reaction with other substances in the EMIT assay

2.

3. Loss of morphine (6 to 15%) and codeine (4 to 13%) due to hydrolysis and extraction for GC-mass spectrometry Lysozyme activity in urine specimens 4.

Vander Slooten and Vander Helm concluded that for practical purposes the EMIT assay can be useful for surveillance of drugs of abuse.

Propoxyphene The EMIT propoxyphene assay is sensitive to

propoxyphene and its major metabolite, mde- methyl dextropropoxyphene. There is no cross- reactivity observed from other commonly abused drugs. Occasionally a false positive may result due to high concentration of chlorpromazine. (Table 11). There are other sensitive methods for determining the presence of propoxyphene and its metabolite, such as the spectrophotometric,60*6' GC,61.63 and spectrofluo- r~metr ic .~ ' In addition, the preceeding methods also make use of serum and urine specimens. For drug abuse screening programs, the EMIT assay is most convenient and expedient. The minimum detectable level for this assay is 1 pg/ m l .

QUANTITATIVE EMIT ASSAYS

Anticonvulsant Drugs To obtain satisfactory control of seizures

with one or a combination of anticonvulsant drugs, monitoring may become necessary to achieve the optimum therapeutic level of drugs and, in many cases, may be essential for correct clinical management of the patient. Most of the analytical methods used for monitoring anticonvulsants, such as phenytoin, phenobarbital, primidone, ethosuximide, and carbamazepine, involve ~pectr~photornetric~~~~* or GLC69-7i systems. These methods are specific and accurate but relatively long, tedious, and subject to error, due to solvent extraction and derivatization steps. I r n m u n o a ~ s a y ~ ~ ~ ~ ~ and high pressure liquid chr~rnatography'~~~' have simplified determinations of anticonvulsants considerably, and it is possible to provide them on an emergent basis.

All EMIT quantitative anticonvulsant drug

Minimum detectable concentrarions: differential elution TLC, Ipcp/ml: TLC. I to 2 pg/ml; EMIT, 0 . 5 pg/mf; inorphiire- RIA, 5 nglmf: and Mor-Barb RIA. 5 0 ng/mf.

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TABLE I 1

Cross-reac tivit y

Drug

Propoxyphene Kdemethyl-dcxtropropoxyphene Codeine Chlorpromazine Morphine Methadone Barbiturates Amphetamines Benzoylecgonine Oxazepam Dextromethorphan

Note: R R is relative reactivity.

assays utilize the same principle as the qualitative assay for drugs of abuse. However, enzyme and substrate differ because all anticonvulsant assays are designed to detect levels of drug in plasma or serum rather than urine. The basic methodology is identical, and for a limited number of samples, one can make use of the same spectrophotometer and pipetteridiluter with a few minor instrument and reagent ad- justments such as wavelength, reaction time, temperature, and buffer. To achieve both effi- ciency and the ability to provide results.quickly for a large number of samples, it is essential to have two separate analytical systems, one for drugs of abuse and the other for quantitation of anticonvulsants. An automated system is preferable for the latter.

The enzyme, glucose-6-phosphate dehydrogenase, is coupled to the antiepileptic drug to be measured. The basic principle of reaction is similar to that which occurs in the analysis of drugs of abuse, as previously explained. How- ever, the basic components of the reaction differ in that the enzyme is glucosed-phosphate dehydrogenase instead of lysozyme; nicotina- mide adenine denucleotide (NAD) is used in place of the bacterial substrate. The buffer also differs. The active enzyme converts NAD to NADH, resulting in an absorbance change which is observed by a spectrophotometer at wavelength 340 nrn, 30°C.

Phenyroin In actual practice, 50 pl of serum or plasma

Concentration (&ml)

2.0 5.5

100.0 140.0

>200.0 370.0

>300.0 >300.0 >300.0 >300.0

550.0

RR

1 0. I 8 0.01 0.007 0.005 0.002 0.003 0.003 0.003 0.003 0.002

Ref.

93 93 93 93 93 93 93 93 93 93 93

are transferred to a 2-ml disposable plastic cup, along with 250 p l of Tris buffer. Subsequently, 50 p l of antibodylsubstrate and 250 p l of buffer are added to the 50 p l of diluted sample and 250 pf of the buffer. Next, 50 pl of enzyme-labeled drug and 250 p l of buffer are added, and the mixture is immediately aspir- ated into a flow-through thermoregulated cuvette. The rate of reaction is monitored by re- cording absorbances at 15 and 45 sec. The difference in optical density is plotted against concentration, and the concentration of the unknown sample is calculated from a calibration curve. The assay range for phenytoin is between 2.5 to 30 pg/ml and is specific for phenytoin. No significant cross-reactivity was observed from other antiepileptic drugs that may have been concomitantly a d m i n i ~ t e r e d ~ ’ - ~ ~ (See Ta- ble 12).

Correlation with Other MetJJods Booker and Darcy” reported correlation of

the EMIT phenytoin assay with a GC assay and found that the EMIT assay offered a higher de- gree of correlation, with a coefficient of correlation of 0.98. Legas and R a i s y ~ ’ ~ also found a similar correlation coefficient of 0.97 for phenytoin. Spiehler et correlated the EMIT assay with GC, radioimmunoassay, and spectrophotometry and found the following correlation coefficients: RIA vs. EMIT, 0.953; EMIT vs. GLC, 0.957; and EMIT vs. Spectro- photometry, 0.898.

Both the RIA and EMIT immunoassays cor-

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TABLE 12

Concentration Drug (8): Ref.

Diphenylhydantoin 5-Hydroxyphenyl-5- phenylhydantoin

Phenobarbital Primidone Methosuximide Ethosuximide Mephobarbital

100 78 0.5

78 0.5 78 0.5 78 0.5 78 0.5 18 0.5 78

relate well with each other and with the GLC method, but somewhat less with the spectrophotometric assay. The results obtained by using RIA, enzyme immunoassay, GLC and spectrophotometry did not differ sufficiently to change the clinical interpretation of the results. The enzyme immunoassay method for detecting phenytoin in serum or plasma is the most convenient with respect to sample requirement (50 p l ) , sample treatment, specificity, and rapidity of results. Small sample size, rapid results, sensitivity, and capability of analyzing five major antiepileptic drugs consecutively within a few minutes make the EMIT system the most desir- able of all methods for measuring anticonvulsants in a clinical laboratory. However, the cost of reagents is considerably higher than in other assay techniques. Each determination costs $1.75 plus the cost of reagents required for the calibration. A standard calibration curve re- quires five points with zero calibration determined in duplicate. Based on the authors’ ex- perience with 6 to 8 samples per day, a 100-test kit will yield sufficient reagents for 60 to 70 test samples; (higher, if a large number of samples per day are analyzed because the same calibration curves are used). The cost of reagents can be reduced considerably by adapting the EMIT assay procedure to a miniature centrifugal analyzer as reported by Brunk et a1.“ Reagent cost reduction was sixfold, and the correlation coefficient with GLC procedure for diphenylhydantoin was 0.91. The sample requirement for duplicate determinations is also reduced to only 3. p i . The coefficient of variation (CV) for the drug concentrations measured was large (15%) and was attributed to pipetting error, high blank reading, and the small slope of the log- log working curves.

TABLE 13

Crou-ructivity of Phenobarbital Assay .

Concentration Drug Olg/mO RR Ref.

Phenobarbital Mephobarbital Secobarbital Amobarbi ral Pen tobarbi tal Primidone Methosuximide Ethosuximide Carbamazepine Ethotoin Chlorpromazinc Diazepam Chlordiazepoxide

5 5

500 >700

>lo00 >lo00 >lo00 >lo00 >lo00 >lo00 >lo00 >lo00 >lo00

I 83 I 83 0.002 83 0.0014 83 0.001 83 0.001 83 0.001 83 0.001 83 0.001 83 0.001 83 0.001 83 0.001 83 0.001 83

Note: RR is relative reactivity.

Finley, Williams, and ByerP have also adapted the diphenylhydantoin EMIT assay to a centrifugal analyzer by diluting the reagents tenfold. No interference from phenobarbital, bilirubin (up to 150 pg/ml ), or lipemic sera was observed. However, there was significant suppression of absorbance by hemoglobin above lo00 mg/l. Therefore, a hemolyzed specimen may not be suitable for analysis using a centrifugal analyzer. The stability of the diluted reagents, if neither stored properly nor used within 5 days, or if analysis is not performed within 30 min after loading the centrifugal analyzer, may influence the accuracy of the results.

Phenobarbital The phenobarbital assay procedure is similar

to the diphenylhydantoin assay and is designed to detect concentrations between 5 to 80 pg/ml. Other barbiturates show cross-reactivity for the phenobarbital assay at higher concentrations. Mephobarbital reacts similarly to phenobarbital at equal concentrations. (Table 13).

Codation with Gas Ctrtoma tographic Deter- mination

Booker and Darcy” have reported that patients taking other anticonvulsants or other drugs showed no cross-reactivity. In their evaluation of the EMIT phenobarbital assay utilizing 202 patient samples and comparing it to the GC method for assaying phenobarbital, the

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correlation between two methods was good (r = 0.97). Reproducibility was good between duplicate determinations (r = 0.96). In their opinion, a single determination by EMIT assay was satisfactory in routine clinical practice. BastianieJ has also shown correlation between the two methods (r = 0.936). However, significant differences in the mean assay values by GLC were noted between the reporting laboratories, ranging from -5.70 pg/mP to + 4.37 p g / ml. Difficulties encountered in quantitative determination of barbiturates by GLC were attributed mainly to the instability of the methyl derivatives" and to other nonspecific interfer- ing substances.1s

Correlation of the EMIT phenobarbital assay with radioimmunoassay, GLC assay, and spectrophotometric assay methods has been reported by Spiehler et al.7e They found that patients receiving mephobarbital gave different apparent phenobarbital levels when the analysis was performed by different methods. Samples from patients receiving both phenobarbital and mephobarbital are reported as total phenobarbital by GC since the methyl derivatives of the two drugs cannot be separated. Likewise, the EMIT procedure for phenobarbital shows equal cross-reactivity with mephobarbital. Thus, an equal mixture of phenobarbital and mephobarbital in a sample will show the sum of the concentration of both drugs reported as phenobarbital. Similarly, the barbiturate ra- dioimmun~assay'~ shows reactivity with secobarbital to the extent of 100%, butabarbital 45%, amobarbital 35%, phenobarbital 25%, and barbital 10%. These data demonstrate that the enzyme immunoassay has much better specificity than the radioimmunoassay for phenobarbital determinations. The spectrophotometric assay procedure detects the sum of 5 , 5- substituted barbiturates (Amobarbital, Seco- barbital, Pentobarbital, and Phenobarbital). However, the 1, 5 , 5- substituted barbiturates such as hexobarbital. mephobarbital, and methabarbital do show an absorption shift between pH 14 and pH 9.0.B6 This latter fact makes the spectrophotometric phenobarbital procedure least affected by mephobarbital interference if the two drugs have been adminis- tered simultaneously. In the absence of other 5 , 5- substituted barbiturates, the spectrophotometric procedure for phenobarbitaI may provide accurate levels.

Primidone Like phenytoin and phenobarbital, the prim-

idone EMIT assay is specific. The operating procedure with respect to the sample size, diluting buffer, and instrumental parameters is similar. Cross-reactivity of the primidone assay with other anticonvulsant drugs is not clinically significant, except when 2 phenyl-2-ethylma- londiamide, a metabolite of primidone, is present in a concentration greater than 500 pg/ml of plasma or serum.

Conelation with Other Methods Sun and Walwicke7 reported on the correla-

tion of the EMIT primidone assay with the GLC method which is commonly used for the analysis of primidone in most laboratories. Of 94 specimens studied, the correlation coefficient between EMIT primidone and the GLC method was 0.976 (slope 0.97, intercept 0.51 pg/mf), suggesting that both methods could be used interchangeably .

Ethosuximide The EMIT ethosuximide assay is similar to

the other EMIT antiepileptic assays in its specificity, thereby permitting determination of ethosuximide along with the other EMIT anticonvulsants in the initial dilution of the same sample. Ethosuximide cross-reacts with glutethimide in concentrations greater than 100 pg/ mlB8 and cross-reacts equally with desmethyl- methsuximide (Cel~nt inB) . '~ The response is linear, thus enabling a quantitative determination of methosuximide after a calibration curve is established using the appropriate standard serum calibrators. At higher concentrations of serum ethosuximide leveb, a slight variation in enzyme activity may result in significant changes in apparent drug concentrations obtained from the calibration curve. Specimens with high ethosuximide concentrations can be accurately determined after appropriate dilution of the sample with serum or plasma.

The correlation of the EMIT ethosuximide assay with the GLC method has been very good (correlation coefficient 0.95).88 However, precision of this assay is less than that of other EMIT anticonvulsant assays with a coefficient of variation higher than 15%. However, when duplicate determinations are averaged, clinically acceptable quantitative levels result.

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Carbamazepine The introduction of the EMIT carbamaze-

pine assay has complemented other antiepileptic drug assays offered to laboratories using the EMIT system, and the methodology is identical to that of the other antiepileptic assays. The only cross-reactivity exhibited by this assay is from its own metabolite, carbamazepine-l0,ll- epoxide, a t a concentration level greater than 10 pg/mf. The coefficient of variation fur EMIT carbamazepine assay is less than 15% for day-to-day analysis and sample-to-sample analysis. The correlation coefficient between the GLC and EMIT carbamazepine assay is 0.95.8* The GLC procedure for carbamazepine is prone to error due to the alkaline methylating agent The formation of the methylated derivatives results from the injection technique and the geometry of the injection port of the gas chromatograph, since the length of time in which the carbamazepine remains in the injection port alters its decomposition profile.

Dig oxin Until the introduction of EMIT digoxin as-

say, radioimmunoassay was the only method available to clinical laboratories to provide accurate measurement of this widely used cardiac glycoside in biological specimens. The EMIT assay for digoxin has provided a choice to those who prefer methods involving nonradioactive reagents. The EMIT assay for digoxin employs the same principle as all other EMIT serum drug assays, but does require some additional inexpensive equipment, such as a constant temperature block or bath, a timer, a micropipette, and a vortex mixer, as well as additional labor per sample. Rosenthal, Vargas, and Klassso have compared a modified EMIT digoxin assay with a RIA digoxin assay. A correlation coefficient of 0.979 between the methods and excellent day-to-day reproducibility for commercial control sera containing digoxin were reported. These findings also point out the fact that slight hemolysis which ordinarily occurs in clinical specimens has no appreciable effect on the results. However, gross hemolysis of the spiked serum results in low recoveries for digoxin, rather than producing the falsely high values one might expect with this method. Modifica- tion of the EMIT procedure was necessary to minimize errors due to the following:

1.

2.

3.

4.

Inadequate mixing of some compounds of the reaction mixture Uneven temperature of the heating block- type incubator Malfunction of diluter/pippetter (inaccurate delivery and dilution of the reagents used) Light scattering errors in the spectrophotometer due to minute particles of fibrin, dissolved gases, o r extraneous particulate matter

The following modifications in the procedure were made:

1. Careful centrifugation of all serum specimens just before analysis

2. Use of deionized, degassed distilled water to reconstitute reagents

3. Filtering of all reagents through membrane filter after reconstitution

4. Use of lens paper rather than tissue paper as wipes

5 . Cleaning of cuvette twice a week by flush- ing with dilute surfactant and filtered, degassed, deionized water

Rosenthal, Vargas, and KlassP0 found that the above modifications were necessary to obtain accurate, precise, and reproducible digoxin levels which were comparable to the digoxin radioimmunoassay .

CONCLUSION

The introduction of the enzyme multiplied immunoassay technique has given hospitals and clinics an additional rapid and reliable method of analysis which is of particular value in the rapid screening of urine for drugs of abuse and for quantitation of serum concentrations of several important drugs. Rapid quantitation is of particular value in adjusting the dosage reg- imen of the difficult-to-manage patient, particularly on an outpatient basis. In the case of anticonvulsants, this type of therapeutic monitoring permits experimentation with var- ious combinations of these drugs and the rapidity of the laboratory results gives immediate information to the clinician, thus permitting dosage adjustment on the same visit and facili- tating quicker management of the patients. The future availability of such important drug as-

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says for theophylline, lidocaine, procainamide, and propranolol will do much to enhance the quality of care of patients taking these drugs and may reduce or even prevent hospitalization of these patients. The distinct advantages and disadvantages of the EMIT system can be sum- marized as follows.

Advantages

I . Minimal or no sample preparation 2. Small sample size (50~1) is sufficient, particularly for pe-

diatric patients 3. Excellent correlation with drug levels determined by

other methods, such as GLC 4. Rapid quantitation, once calibration of the standard

curve has been established 5. Objective interpretation of the results 6. Same parameters for five anticonvulsant drugs; all can

be determined from the same dilution

7. Most of the assays are specific for specific drugs

Disadvantages

I . Cost of reagents, ranging from $1 .SO to $2.75 per determination depending on the number of assays performed

2. Need to assay one drug at a time; simultaneous determination of multiple drugs is not possible as in GLC or high pressure liquid chromatography’0~76~~’~9z

3. Determined concentrations above and below calibra- tions are not accurate (high concentrations need to be diluted)

4. Lipemic or severely hemolyzed specimens may give er- roneous results due to increased light scattering

5. Urine specimens containing lysozymes for drugs of abuse screening cannot be analyzed by the EMIT system because of high background values caused by the activity of lysozyme on the substrate

6. Cross reactivity associated with some assays, particularly the amphetamine EMIT assay, results in false positives, especially in the absence of an alternative method of confirmation

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Enzyme Multiplied Immunoassay Technique: A Review