Clinical Chemistry 47: 540-547, 2001;
(Clinical Chemistry. 2001;47:540-547.)
© 2001 American Association for Clinical Chemistry, Inc.
Comparison of ELISAs for Opiates, Methamphetamine, Cocaine Metabolite, Benzodiazepines, Phencyclidine, and Cannabinoids in Whole Blood and Urine
Sarah Kerrigana,1,1 and
William H. Phillips Jr1
1
California Department of Justice, Bureau of Forensic Services, Toxicology Laboratory, 4949 Broadway, Sacramento, CA 95820.
a Author for correspondence. Fax 916-227-4751; e-mail
kerrigas{at}hdcdojnet.state.ca.us.
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Abstract
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Background: ELISAs are widely utilized in forensic drug analysis.
A comparative assessment of microtiter plate assays for the detection
of six common classes of drug in blood and urine is described.
Methods: ELISAs for opiates, methamphetamine, benzodiazepines,
cocaine metabolite, phencyclidine (PCP), and tetrahydrocannabinol (THC)
metabolite were evaluated in a side-by-side study. The analytical
performance of 12 commercially available ELISAs was determined in terms
of binding characteristics, dose–response curves, limits of detection,
sensitivity, intra- and interassay imprecision, and lot-to-lot
reproducibility. Assay performance was also compared using 855 forensic
casework samples.
Results: Detection limits in whole blood for morphine,
D-methamphetamine, nordiazepam, benzoylecgonine,
nordiazepam, PCP, and L-11-nor-9-carboxy-
9-THC
were 3, 2, <4, 5, 25, and 3 µg/L, respectively, for the STC ELISAs.
Corresponding detection limits for Immunalysis ELISAs were <1, <2,
<4, 5, <1, and 1 µg/L, respectively. Intraassay CVs (n = 8) at
the immunoassay cutoff concentrations were 4.1–5.6% and
3.5–11% for STC and Immunalysis ELISAs, respectively.
Corresponding interassay CVs were 3.1–10% and 6.5–20%. Of the 855
casework samples, there were a total of 92 discordant results (44
cannabinoid, 15 opiate, 15 methamphetamine, 11 benzodiazepine,
and 7 cocaine metabolite). Gas chromatography–mass spectrometry
analysis indicated a total of three unconfirmed positive results for
Immunalysis assays and one unconfirmed positive for STC assays.
Conclusions: A comparative assessment of drugs-of-abuse assays
from two manufacturers indicated some key differences in analytical
performance. Overall, Immunalysis assays offered superior binding
characteristics and detection limits, whereas STC assays offered
improved overall precision and lot-to-lot
reproducibility.
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Introduction
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ELISAs are becoming increasingly popular among the forensic
toxicology community because of their relative ease of use, growing
potential for automation, and their adaptability for use with blood and
urine samples (1). Unlike many of their homogeneous
immunoassay counterparts, ELISAs are amenable to whole-blood samples
without the need for sample pretreatment. Other advantages of enzyme
immunoassays include small sample volumes, high sample throughput,
rapid turnaround times, long shelf lives, and the lack of
radioisotopes.
Each month the California Department of Justice Toxicology Laboratory
receives 
1000 blood and urine samples from law enforcement agencies
across the state. These cases comprise health and safety violations,
driving under the influence, homicides, rapes, and other felonies.
Immediately after submission, samples are presumptively screened for
opiates, methamphetamine, benzodiazepines, cocaine metabolite,
phencyclidine
(PCP),2
and cannabinoids to qualitatively determine whether drugs are
present. The cutoff concentrations, above which a sample is considered
positive, are as follows: morphine, 10 µg/L;
D-methamphetamine, 100 µg/L; nordiazepam, 100 µg/L;
benzoylecgonine (BE), 150 µg/L; PCP, 10 µg/L; and
L-11-nor-9-carboxy-9-tetrahydrocannabinol
(THCA), 30 µg/L. BE and THCA are major metabolites of cocaine and
marijuana, respectively. These cutoff concentrations, which are
considerably lower than those used for federal workplace drug-testing
programs, were selected to reflect the performance of the immunoassays,
goals of the analyses, and organization and specialization of the
laboratory (2).
All presumptively positive immunoassay results are confirmed by gas
chromatography–mass spectrometry (GC-MS) before the appearance of a
toxicologist in court. With the exception of felony casework, which
undergoes confirmatory analysis regardless of the screening outcome,
the immunoassay results dictate whether subsequent confirmatory GC-MS
analyses will be performed. As a result, the reliability and
performance of the screening test are of paramount importance.
We describe a comparative assessment of drugs-of-abuse ELISAs purchased
from a prospective vendor (Immunalysis Corp., San Dimas, CA)
with our existing vendor (STC Diagnostics Inc., Bethlehem, PA). The
comparison consisted of two parts: (a) comparison of the
analytical performance of all 12 assays in terms of binding
characteristics, limit of detection (LOD), sensitivity, intra- and
interassay imprecision, and lot-to-lot reproducibility; and
(b) evaluation of casework samples over a 3-week period,
during which time 855 consecutive toxicological submissions of blood
and urine were assayed for drugs of abuse using both manufacturers’
assays.
Each test relies on the principle of direct ELISA. Antibodies raised
against the drug of interest are coated onto the surface of a
polystyrene microtiter plate. Blood or urine samples are added to the
plate, along with a drug-enzyme conjugate. The drug in the sample and
the drug-enzyme conjugate compete for antibody binding sites on the
surface of the well. After an appropriate incubation time, the unbound
drug is removed by washing. A colorimetric reaction is used to
determine how much drug-enzyme conjugate is bound to the microtiter
well. A spectrophotometer is then used to measure the absorbance, which
is inversely proportional to the concentration of drug in the sample.
Each of the assays utilizes horseradish peroxidase-labeled drug-enzyme
conjugates, tetramethylbenzidine enzyme substrate reagents, and acid
stop solutions (3)(4).
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Materials and Methods
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reagents
STC immunoassays, which have been used in our laboratory for
several years, were performed in accordance with standard operating
procedures. The new Immunalysis assays were performed in accordance
with the manufacturer’s instructions. With the exception of the
phosphate buffer, immunoassay reagents and plates were supplied by each
manufacturer: STC Diagnostics, cat. nos. 1150EB (opiates), 1104EB
(methamphetamine), 1110EB (benzodiazepines), 1122EB (cocaine
metabolite), 1154EB (PCP), and 1118EB (cannabinoids); Immunalysis
Corp., cat. nos. 207-0480 (opiates), 211-0480
(methamphetamine), 214-0480 (benzodiazepines), 206-0480 (cocaine, BE
specific), 208-0408 (PCP), and 205-0408 [tetrahydrocannabinol (THC)
metabolite].
stc elisas
Blood (500 µL) and urine (200 µL) samples were aliquoted into
polypropylene tubes. Urine samples were diluted with 300 µL of 100
mmol/L phosphate buffer, pH 7 (Sigma). Deionized water (100
µL) and the appropriate volume of blood or diluted urine (opiate, 25
µL; methamphetamine, 25 µL; benzodiazepine, 10 µL; BE, 25 µL;
PCP, 25 µL; THCA, 10 µL) were added to microtiter wells with a
microtiter pipettor (Hamilton Microlab AT). In accordance with the
manufacturer’s instructions, the STC PCP assay utilized a proprietary
buffer solution provided by the manufacturer instead of
deionized water. Drug-enzyme conjugate (100 µL) was added and allowed
to incubate for 30 min. Microtiter wells were washed with deionized
water six times, after which 100 µL of substrate solution was
added. After a 30-min incubation, 50 µL of acid stop
solution was added, and the absorbance (A450–620
nm) was measured (BioTek Omni Microtiter plate Processor; BioTek
Instruments).
immunalysis elisas
Immunalysis assays were performed similarly to those of the STC
assays, with the exception of sample volume and sample incubation time.
In accordance with the recommendations of the manufacturer, sample
volumes of blood and urine were 10 µL for each assay, and incubation
of the sample with the drug-enzyme conjugate was 60 min.
evaluation of analytical performance
The analytical performance of each of the assays was evaluated in
terms of binding characteristics, LOD, sensitivity, and precision
(5). Drug-free blood and urine were fortified with the
target drugs at the concentrations of interest. Target drugs that were
used to fortify drug-free matrix were
D-methamphetamine, nordiazepam, BE, PCP, and
THCA. Morphine base was the target drug in whole blood, and its
metabolite, morphine-3-glucuronide, was the target drug in urine
samples. Dose–response curves were generated for all 12 assays by
plotting (A/A0) x 100
against the log of the concentration in µg/L, where A is
the absorbance of the test sample and
A0 is the absorbance of drug-free
blood or urine. The concentration of drug at which 50% of the
drug-enzyme conjugate was bound to the antibody
(EC50) was determined for each assay. The LODs
for both blood and urine were estimated using drug-free matrix
(mean - 3 SD; n = 8) (6). The sensitivity,
i.e., slope of the calibration curve between the negative
control and the cutoff calibrator, was determined. Intra- and
interassay imprecision was estimated by replicate analysis of
blood and urine samples that had been fortified with a known quantity
of drug, and the lot-to-lot reproducibility was evaluated over a period
of 1 year.
comparison of casework samples
A parallel study was undertaken using 855 casework samples (550
blood, 305 urine) that were submitted to the laboratory over a 3-week
period. Samples were screened for opiates, methamphetamine,
benzodiazepines, cocaine metabolite, PCP, and cannabinoids using ELISAs
from each manufacturer. Toxicological evidence was refrigerated upon
arrival, and the screening test was performed within 24 h of
receipt. Tests using both manufacturers’ assays were performed
on the same day in accordance with the manufacturers’ recommendations
and the standard operating procedures indicated above. The following
controls were run routinely in each assay: cutoff calibrator (morphine,
10 µg/L; D-methamphetamine, 100 µg/L; nordiazepam, 100
µg/L; BE, 150 µg/L; PCP, 10 µg/L; THCA, 30 µg/L); negative
control (drug-free blood or urine); positive blood control (morphine,
100 µg/L; D-methamphetamine, 300 µg/L; nordiazepam, 300
µg/L; BE, 300 µg/L; PCP, 15 µg/L; THCA, 50 µg/L); and positive
urine control (morphine-3-glucuronide, 300 µg/L;
D-methamphetamine, 300 µg/L; nordiazepam, 500 µg/L; BE,
300 µg/L; PCP, 15 µg/L; THCA, 50 µg/L). After each set of 20 case
samples, a positive urine control was inserted to assess homogeneity
across the plate. A control fortified with drugs at 50% of the cutoff
concentrations was also included in each analytical run. The mean
response of the cutoff calibrator (n = 2) was used to determine
whether a sample screened positive or negative. Samples that produced
absorbance readings above the cutoff calibrator were considered
negative, whereas those below the cutoff calibrator were presumptively
positive. An immunoassay response factor (IRF) is routinely assigned to
casework samples as follows: IRF =
(A0 x C)/A,
where A is the absorbance reading of the sample,
A0 is the absorbance of the negative
control, and C is the concentration of drug (µg/L) in the
cutoff calibrator. The IRF is determined only for positive samples,
i.e., when the absorbance of the sample is less than the mean
absorbance of the cutoff calibrator. The immunoassay is used
qualitatively to identify samples that require GC-MS confirmation.
However, the IRFs are numerical estimates that are used to indicate
whether the sample contains a "high" or "low" concentration of
drug and/or metabolite. This information is useful during subsequent
confirmatory analyses to determine appropriate dilution of the sample.
The concordance of results obtained by the STC and Immunalysis ELISAs
were compared, as were the IRFs. Discordant results were further
investigated by confirmatory GC-MS analysis as outlined briefly below.
Basic drugs, including PCP and methamphetamine, were isolated from
blood and urine by liquid-liquid extraction. Extracts were derivatized
using acetic anhydride and analyzed by GC-MS using electron ionization.
This procedure identifies numerous sympathomimetic amines, including
methamphetamine and amphetamine, as well as synthetic designer
amphetamines such as methylenedioxymethamphetamine (ecstasy, Adam) and
methylenedioxyamphetamine (Eve). Opiates (morphine, codeine, and
6-monoacetylmorphine) were extracted using solid-phase extraction.
Trimethylsilyl derivatives were analyzed by either GC-MS or GC combined
with tandem MS. Synthetic opioids, including the keto-opioids
(e.g., hydrocodone, hydromorphone, oxycodone, and oxymorphone) were
identified using the basic drug extraction outlined above. Cocaine, BE,
and ecgonine methyl ester were isolated using solid-phase extraction.
Trimethylsilylation followed by either GC-MS using electron ionization
or GC-tandem MS using positive chemical ionization was used for
spectroscopic identification. Benzodiazepines and their metabolites
were identified using liquid-liquid extraction followed by GC-MS
analysis using negative chemical ionization and trimethylsilylation
when necessary. THC and THCA isolated by liquid-liquid extraction were
subsequently derivatized by trifluoroacetylation and methyl
esterification. Target drugs were identified using negative chemical
ionization GC-MS.
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Results and Discussion
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binding characteristics
Dose–response curves for morphine-3-glucuronide, methamphetamine,
nordiazepam, BE, PCP, and THCA in urine are shown in Fig. 1
. EC50s were used as a numerical
estimate of the binding efficiency (Table 1
). Immunalysis ELISAs produced lower EC50
values with opiate, methamphetamine, benzodiazepine, cocaine
metabolite, and PCP assays. It should be noted that STC and Immunalysis
sample volumes and incubation times, factors that are known to affect
binding characteristics, were different. Optimum sample volumes and
conditions used for STC assays were determined in our laboratory
several years ago during implementation of these methods. Conditions
used for the Immunalysis assays were in accordance with the
manufacturer’s recommendations. Although incubation times for
Immunalysis assays were longer, opiate, methamphetamine, BE, and PCP
assays used a reduced sample volume (10 µL instead of 25 µL), which
is advantageous in terms of reduced matrix effect.
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Figure 1. Dose–response curves of STC ( ) and Immunalysis (•)
ELISAs for morphine-3-glucuronide (A), methamphetamine
(B), nordiazepam (C), BE
(D), PCP (E), and THCA (F)
in urine.
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lod
LODs were measured in both blood and urine (Table 2
). Overall, the LODs for the Immunalysis assays were slightly
lower than those for the STC assays. This was probably attributable in
part to the improved binding characteristics and lower
EC50s for the Immunalysis assays. The LODs were
well below the cutoff concentrations for all drugs with the exception
of the STC PCP assay, which is no longer used in our laboratory. Since
the time of this study, a new and improved PCP assay has been
introduced (Brian Feeley, STC Diagnostics Inc., Bethlehem, PA, personal
communication).
intra- and interassay imprecision
The intraassay CVs for blood and urine samples are shown in Tables 3
and 4
. Both intra- and interassay CVs were consistently lower for STC
ELISAs and followed the same overall trend between assays. Interassay
CVs were collected over a period of 2 weeks, during which there were no
changes in lot numbers. Fig. 2
depicts the effects of lot-to-lot variations for each assay
over a period of 1 year. After each run, the absorbance values of the
immunoassay controls were entered into a database. Control charts were
used to assess the performance of each assay over time. Variations in
assay performance over time were evaluated by plotting
(A/A0) x 100 for the
cutoff calibrator. A change in this value between lots was indicative
of a change in the binding characteristics of the assay, e.g., a
displacement in the dose–response curve, and not absolute changes in
absorbance, which are to be expected. ELISAs were run each working day,
with each data point representing the monthly average (typically 20
assays per month; 240 runs per year). These results confirmed the
observation that Immunalysis reagents and assays were more susceptible
to lot-to-lot differences. The total CVs for the STC opiate,
methamphetamine, benzodiazepine, BE, PCP, and THC assays were 9.8%,
18%, 40%, 7.5%, 8.8%, and 27%. Data for the STC PCP assay
were collected over 6 months because of replacement of the method.
Corresponding CVs for the opiate, methamphetamine, benzodiazepine, BE,
PCP, and THC assays from Immunalysis were 36%, 18%, 30%, 23%, 26%,
and 26%, respectively.
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Figure 2. Lot-to-lot variations for STC (A) and
Immunalysis (B) assays.
Plots show the monthly average value of
(A/A0) x 100 for the
cutoff calibrator for the period of 1 year.
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analytical sensitivity
The sensitivity of the assay refers to the change in analytical
response for a given change in analyte concentration. One way to
compare the sensitivity is to measure the slope in the calibration
curve between the negative control and the cutoff calibrator, which is
the critical region in forensic testing (7). In this way,
the theoretical ability of the assay to distinguish positive samples
from negative samples can be determined. Absorbance values for negative
controls and cutoff calibrators were used to calculate the slope of the
calibration as follows: slope =
(A0 -
Acutoff)/C, where
A0 is the absorbance of the negative
control, Acutoff is the absorbance of
the cutoff calibrator, and C is the cutoff concentration
(Fig. 3
). Slopes for methamphetamine, nordiazepam, and BE assays were
comparable. Marked improvements in the slope were observed with the
Immunalysis morphine and PCP assays, and there was a slight improvement
with the STC cannabinoid assay. Although the slope, or analytical
sensitivity, can be used to predict the reliability of the assay at the
cutoff concentration, the true sensitivity and specificity must be
determined using real casework samples that contain not only one target
drug of interest, but also multiple drugs, metabolites, and other
cross-reacting species.
casework comparison using stc and immunalysis elisas
The concordance of STC and Immunalysis results is summarized in
Table 5
. The mean presumptively positive rates for all toxicological
submissions decreased in the order cannabinoids (36%) >
methamphetamine (33%) > opiates (21%) > cocaine
metabolite (15%) > benzodiazepines (8%). Casework comparisons
for PCP were not available because the STC PCP assay is no longer used
in our laboratory. The percentage of discordant immunoassay results was
<1% for cocaine metabolite; <2% for opiates, benzodiazepines, and
methamphetamine; and 5% for cannabinoids. Of the 855 samples analyzed,
there were a total of 92 discordant results (15 opiate, 15
methamphetamine, 11 benzodiazepine, 7 cocaine, and 44
marijuana), largely because of the differences in cross-reactivity
between manufacturers (Table 6
). The majority (96%) of the discordant results gave IRFs that
approached that of the cutoff calibrator. When samples that fell within
50% of the cutoff calibrator were eliminated, only four urine samples
remained (three cannabinoid and one opiate).
Confirmatory GC-MS analysis was used to establish whether these
discordant results were false-positive or false-negative immunoassay
responses. Opiates were not detected in the urine sample that screened
positive in the Immunalysis opiate assay, indicating one false-positive
result. Confirmatory analysis for cannabinoids indicated that the STC
assay produced one false-negative result and the Immunalysis assay
produced one false-negative and one false-positive result. Despite the
low number of unconfirmed positive results, these results reinforce the
fact that all positive drug findings by immunoassay must be confirmed
using a more rigorous confirmatory technique.
The IRF is a function of the difference in absorbance between the
negative control and the sample. The larger the difference in
absorbance, the larger the IRF, and vice versa. Indirectly, these
values are related to the dynamic range of the assay. Although the
units of these values were µg/L, they do not represent true
concentrations because the dose–response curves were nonlinear.
Statistical summaries of the IRFs for each assay are depicted in Table 7
. The opiates, methamphetamine, and cannabinoid assays gave very
similar results. Although the median IRFs for both benzodiazepine
assays were similar, the range of values was more than fivefold
wider in the Immunalysis method. The cocaine metabolite assays
also performed differently when casework samples were used. The median
and range of values increased more than twofold in the Immunalysis
assay, indicating a more than twofold increase in the range of
absorbance. The larger the difference in absorbance between the sample
and the negative control, the greater the confidence in the result.
Cross-reactivity also plays an important role in these values. An
explanation for the difference in immunoassay responses in the cocaine
metabolite assays could be the low cross-reactivity of the STC assay
toward cocaine (1%) compared with the Immunalysis assay (9%)
(3)(4). Likewise, measurable differences in
cross-reactivity toward different benzodiazepines (Table 6
) account for
the differences between these immunoassays.
In conclusion, our comparative assessment of ELISAs for six drugs
of abuse demonstrated some important differences in performance between
the assays. In general, binding characteristics and LODs were more
favorable for the Immunalysis assays. However, the STC assays offered
improved overall precision and were less susceptible to lot-to-lot
variations in assay performance.
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Footnotes
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1 Present address: New Mexico Department of Health, Scientific
Laboratory Division, Toxicology Bureau, PO Box 4700, Albuquerque, NM
87196-4700. 
2 Nonstandard abbreviations: PCP, phencyclidine; BE, benzoylecgonine; THCA, L-11-nor-9-carboxy-9-tetrahydrocannabinol; GC-MS, gas chromatography–mass spectrometry; LOD, limit of detection; THC, tetrahydrocannabinol; EC50, concentration of drug at which 50% of the drug-enzyme conjugate is bound to antibody; and IRF, immunoassay response factor.
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Abstract
Introduction
) and
Immunalysis (
) assays.