Clinical Chemistry 43: 363-368, 1997;
(Clinical Chemistry. 1997;43:363-368.)
© 1997 American Association for Clinical Chemistry, Inc.
Competitive enzyme immunoassay with monoclonal antibody for homovanillic acid measurement in human urine samples
Frédéric Taran,
Yveline Frobert,
Christophe Créminon,
Jacques Grassi,
Didier Olichon1,
Charles Mioskowski and
Philippe Pradellesa
1
Laboratoire CERBA, 95066 Cergy-Pontoise, Cedex 9, France.
a Author for correspondence. Fax (33) 01 69 08 59 07; e-mail pradelles{at}dsvidf.cea.fr
 |
Abstract
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A fast competitive enzyme immunoassay (EIA) for measuring homovanillic
acid in human urine samples was developed with a monoclonal antibody
and acetylcholinesterase as enzyme label. Enzyme detection was
performed by an easy colorimetric assay. Monoclonal antibodies were
screened on the basis of sensitivity, specificity, and correlation
studies. EIA has a detection limit of 0.5 µmol/L, a CV <10% in the
1.25–10 µmol/L range, and intra- and interassay CVs of <10%.
Cross-reactivity with vanillylmandelic acid was 0.5% and <8% for
other structurally related catecholamine metabolites. Parallelism of
the EIA was shown in dilution studies and the correlation with routine
HPLC assay in 62 normal and pathologic samples was EIA = 1.492
(HPLC) - 3.46, Sy|x =
47.52, range = 4–1800 µmol/L, r2 =
0.977. Additional data concerning the validity of this assay were
provided by HPLC analysis of urinary immunoreactive material.
Key Words: indexing terms: catecholamines • neuroblastoma • vanillylmandelic acid
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Introduction
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Measurements of catecholamines and their metabolites remain an
important laboratory aid in the diagnosis of pheochromocytoma and
neuroblastoma (1)(2). Vanillylmandelic acid
(VMA) and homovanillic acid (HVA) correspond to the two major urinary
metabolites of both epinephrine (E) and norepinephrine (NE) for VMA
and dopamine for HVA, respectively.1
Several analytical methods have been described
(3), including gas chromatography–mass spectrometry
(4)(5) and HPLC with electrochemical detection
(ED) (6)(7), which is now routinely used for
the measurement of these metabolites. However, these methods are
generally time consuming, technically demanding, and often require
further sample purification before analysis, in contrast with
immunoassays, which are more suitable for highly sensitive and specific
screening of large numbers of samples.
Monoclonal antibodies for HVA and VMA have been previously described
(8) but have not been used to measure these metabolites in
urine, probably because of a lack of affinity and specificity. More
recently, a fluorescence polarization immunoassay of urinary VMA with
clinical applications and new strategies for polyclonal anti-HVA
antibody production have been described
(9)(10)(11)(12). Our laboratory, which is strongly
focused on the development of enzyme immunoassays (EIAs) with
acetylcholinesterase (AChE) as label, has produced monoclonal
antibodies for HVA. We describe here a competitive EIA for human
urinary HVA with one of these antibodies and AChE-HVA conjugate as
enzymatic tracer. Sensitivity, accuracy, specificity, and validity are
reported.
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Materials and Methods
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Apparatus.
Solid-phase EIA was performed with
specialized Titertek microtitration equipment (washer, dispenser, and
plate reader) from Labsystem (Helsinki, Finland). Microtiter plates
(Maxisorp) were from Nunc (Roskilde, Denmark). HPLC experiments were
performed with a Waters apparatus (St. Quentin en Yvelines, France),
including a 600 Controller, 996 photodiode array detector, 600 pump,
717 Autosampler, and Millennium chromatographic manager.
Chemicals.
HVA, VMA, 3-methoxy-4-hydroxyphenylethylene
glycol (MHPG), 3-methoxytyramine (MT), ethylene diamine,
3,4-dihydroxymandelic acid (DOMA), vanillin (VAN),
3,4-dihydroxyphenylacetic acid (DOPAC), normetanephrine (NM),
3-methoxytyrosine (3MTyr), dopamine (DA), E, NE, synephrine (Syn),
hydroxyphenylacetic acid (HPAA), phenylacetic acid (PAA), tyramine
(Tyr), N-hydroxysuccinimide (NHS),
1,3-dicyclohexylcarbodiimide (DCC),
N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate
(SMCC), and N-succinimidyl-S-acetylthioglycolic
acid (SATA) were purchased from Sigma (St. Louis, MO). Keyhole limpet
hemocyanin (KLH) and glutaraldehyde (25%) were from Merck (Darmstadt,
Germany). AChE was purified from electric eel (Electrophorus
electricus) by affinity chromatography as previously reported
(13).
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assay reagents
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H6 hapten synthesis (Fig. 1
).
To 25 mL of ethyl acetate were successively added HVA
(526 mg, 2.9 mmol), NHS (400 mg, 3.49 mmol), and DCC (600 mg, 2.9
mmol). The reaction was allowed to proceed for 12 h at room
temperature and then 2 mL of ethylene diamine (24.16 mmol) were added.
After 1 h, solvent was removed under reduced pressure and the
crude product was purified by flash chromatography on a silica columm
(3 x 10 cm) with dichloromethane:methanol:triethylamine (70:28:2
by vol) as eluting solvent.
Immunogen preparation (Fig. 1
).
To 1 mL (893 nmol) of H6
in 0.1 mol/L phosphate buffer (pH 7.4) were successively added 1 mL of
KLH (3 g/L) in 0.1 mol/L phosphate buffer (pH 7.4) and 8 µL of
glutaraldehyde. After a 12-h reaction at room temperature, the mixture
was dialyzed against 0.1 mol/L phosphate buffer (pH 7.4) at 4 °C.
Immunogen was stored at -20 °C until use.
Enzymatic tracer preparation (Fig. 1
).
H6 was covalently
coupled to AChE by using SMCC and SATA reagents as previously described
(14). This method involved the reaction of the thiol group
previously introduced by SATA into H6 with maleimido groups, previously
incorporated into the enzyme by the use of SMCC.
Monoclonal antibody production.
Monoclonal anti-H6
antibodies were produced according to the conventional hybridization
technique. Briefly, the immunogen (100 µg) was injected with
Freund's complete adjuvant into five Biozzi's mice. Boosters were
given at 2-week intervals. Anti-H6 polyclonal antibodies were screened
by competitive EIA (see below). The spleen cells of the mouse
presenting the highest affinity antiserum for HVA were fused with NS1
myeloma cells as previously described (15). After cloning,
mice were inoculated intraperitoneally with a selected clone and the
ascitic fluid was obtained 3 weeks after inoculation.
Competitive EIA.
Competitive EIA was performed as
previously described (16), with 0.1 mol/L phosphate buffer
(pH 7.4) containing 0.15 mol/L NaCl, 10-3 mol/L EDTA, 1
g/L bovine serum albumin, and 0.1 g/L sodium azide (EIA buffer).
Briefly, 96-well microtiter plates were coated with goat
affinity-purified antibodies specific for mouse IgGs (Jackson Labs, Bar
Harbor, ME). To each well were added 50 µL of calibrator, buffer, or
urine sample, 50 µL of enzymatic tracer [2000 Ellman's units
(EU)/L], and 50 µL of diluted monoclonal antibody. After a 2-h
incubation at room temperature, the wells were washed and Ellman's
reagent [7.5. 10-4 mol/L acetylthiocholine iodide (enzyme
substrate) and 5.10-4 mol/L 5,5'-dithiobis-(2-nitrobenzoic
acid) (chromogen) in 0.1 mol/L phosphate buffer, pH 7.4] (200 µL)
was dispensed into each well. After 1 h of enzymatic reaction, the
absorbance at 414 nm was measured in each well. AChE activity was
measured by Ellman's method (17). Enzymatic activity was
expressed in terms of EU corresponding to the quantity of enzyme
hydrolyzing 1 µmol/L of substrate during 1 min at 25 °C.
Calculations.
B and Bo represent the bound enzyme
activity measured in the presence or absence of competitor,
respectively. The results are expressed in terms of B/Bo as a function
of the logarithm of the dose. Fitting of calibration curve was
performed by using a linear log-logit transformation (18).
All measurements for calibrators or samples were made in duplicate, and
in quadruplicate for Bo values. Nonspecific binding was determined by
using an incubation mixture in which the specific monoclonal antibody
was replaced by 50 µL of EIA buffer. In all cases, nonspecific
binding was <0.15% of the total enzyme activity introduced in the
assay. The minimum detectable concentration (MDC) was taken as the
concentration of competitor inducing a significant decrease (3 SD) in
Bo. The precision profile of the calibration curve was established by
performing eightfold measurements of each concentration and was
expressed in terms of the CV of recalculated concentration vs HVA
concentration (19). Intra- and interassay (day-by-day)
repeatability was established by testing a urine sample eight times.
Specificity measurements.
The specificity of EIA was
checked by testing its capacity to detect compounds structurally
related to HVA by establishing for each of them the corresponding
calibration curves. Results were expressed in terms of percentage of
cross-reactivity, defined as the ratio (%) of the concentration of HVA
and HVA-related compounds at 50% B/Bo (20).
Biological samples.
Human urine samples were obtained
from 62 acidified 24-h collections, all of which had been previously
measured by HPLC in the CERBA Laboratory (Cergy-Pontoise, France)
following a routine procedure described elsewere (21).
Correlation studies.
Correlation studies were performed
by comparison of EIA results with the results of routine HPLC for HVA
determination in the CERBA Laboratory for 62 human urine samples. Eight
of these samples (three at low concentration, one at medium
concentration, and four at high concentration) were selected for
analysis of monoclonal antibody specificity.
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chromatographic characterization of immunoreactive material
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To validate the nature of the immunoreactivity present in urine
samples, HPLC was combined with competitive EIA. Ethyl acetate (1 mL)
was added to 1 mL of urine sample and, after shaking, the organic phase
was separated and the extraction was repeated three times. The organic
mixture was dried under reduced pressure and the extract was
resuspended in 200 µL of water:acetic acid mixture (98:2 by vol)
(mobile phase A). The solution (150 µL) was injected into a
reversed-phase C18 column (Nucleosil 300, 5 µm diameter, 250 x
4.6 mm). Chromatographic elution was achieved at a flow rate of 0.8
mL/min by using mobile phase A and mobile phase B
[water:methanol:acetic acid (68:30:2 by vol)] over successive runs
(gradient: 100% A to 20% A for 30 min; isocratic: 20% A for 20 min;
and gradient: 20% A to 100% A for 10 min, respectively). Fractions
(0.8 mL) were collected and a 50-µL aliquot of each fraction was
diluted in 450 µL of EIA buffer. Five urinary sample extracts were
prepared for EIA-HPLC validation studies.
|
Results
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Monoclonal antibody selection.
The monoclonal antibody
to be used in competitive EIA was selected on the basis of three
criteria: sensitivity, specificity, and correlation studies performed
as described above. Seven of 14 monoclonal antibodies were selected for
use in EIA on the basis of: (a) sensitivity (B/Bo 50% <10
µmol/L), (b) specificity for VMA (Table 1
) and other commercial catecholamine metabolites (results not
shown), (c) correlation with values obtained by HPLC
analysis with eight urine samples. Immunoassay with one of these
monoclonal antibodies (clone 127) showed the best sensitivity with the
highest coefficient of correlation (r2 = 0.99).
This antibody was chosen for further studies.
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Table 1. Sensitivity and specificity of EIAs with various monoclonal
anti-HVA antibodies and correlation studies with HPLC measurements for
eight urine samples.
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Sensitivity, accuracy, and specificity.
A routine EIA
calibration curve is presented in Fig. 2
. The MDC was 0.5 µmol/L and the CV was <10% between 1.25
and 10 µmol/L. The reference range (0–40 µmol/L) and limit of
detection (1 µmol/L) for the HPLC-ED method were similar to our assay
but 1 mL of urine sample was necessary to perform the assay compared
with 0.05 mL for the EIA method. The intra- and interassay
repeatability of HVA urinary content was <10%. EIA specificity is
detailed in Table 2
, showing low cross-reactivities with other catecholamine
urinary metabolites, particularly with VMA (cross-reactivity =
0.5%), which is present at high concentrations in normal urine samples
in comparison with other metabolites (1).
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Table 2. Structures and abbreviations of catecholamine urinary
metabolites, normal concentrations, and
cross-reactivity.
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Validation and correlation studies.
The dilution curve
for urine samples had a similar shape to the calibration curve (Fig. 2
). Using HPLC experiments coupled to EIA (Fig. 3
), we showed that immunoreactive material observed in a urine
sample containing a high concentration of VMA (68 µmol/L) was eluted
as a single homogeneous peak (Fig. 3B
) corresponding to the elution
profile of commercial HVA (Fig. 3A
). Similar results were obtained with
four other urinary extracts and only one experiment yielded an
unidentified significant cross-reactive product (retention time: 32
min) representing <10% of the total immunoreactive material (result
not shown). Good correlations were obtained for EIA and HPLC analysis
of HVA in 62 normal and pathologic samples. Linear regression analysis
gave the equation: EIA = 1.492 (±0.03) HPLC - 3.46 (±7),
Sy|x = 47.5, range = 4–1800 µmol/L,
r2 = 0.977, n = 62.
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Figure 3. HPLC fractionation of HVA immunoreactive material in
calibration solution (A) and urine samples (B).
The elution positions of other urinary catecholamine metabolites are
indicated by arrows.
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Discussion
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Immunoassays of catecholamine metabolites remain the preferred
methods for routine measurement of these molecules in biological fluids
(1). As catecholamines have closely related structures and
are chemically unstable, it is difficult to raise specific antibodies
against them (24). HVA and VMA molecules are chemically
stabler than molecules possessing a catechol ring, and the immunogen
synthesis therefore appears less problematic. However, their high
concentrations in normal or pathologic urine samples (1)
require very specific immunoassays. Thus, to direct the specificity of
antibodies toward R3 and R4 groups (Table 2
),
two different immunogens coupled via the phenolic group
(9)(10)(11)(12) or aromatic ring (8) were
prepared. We developed a different strategy in which the integrality of
the phenolic group was preserved by coupling via the R4
group. This leads to the production of monoclonal antibodies of various
specificities. We selected those monoclonal anti-HVA antibodies
allowing specific determination of HVA in human urine samples, as
estimated in comparison with HPLC measurement. The latter criterion
seems very important since poor correlation with HPLC values was seen
with three monoclonal antibodies (clones 7, 78, and 137) selected for
their high affinities for HVA and low cross-reactivity with VMA (Table 1
). We validated our EIA by chromatography studies on urine samples.
However, in the correlation study, the consistently higher
concentrations of HVA measured by EIA compared with those by HPLC could
be related to the measurement of an (or more) unidentified
cross-reacting substance(s) that could be present in normal or
pathologic urine following a parallel route of HVA biosynthesis. On the
other hand, the HPLC method could systematically underestimate the HVA
concentrations by loss of materials during the extraction and
chromatographic steps.
As already described for various haptens and antigens, the use of AChE
as enzyme label for competitive or immunometric EIAs allows an easy and
highly sensitive assay (25)(26)(27)(28). We report here
a routine EIA for HVA determination in urine that is particularly
suitable for neuroblastoma diagnosis in clinical analysis laboratories.
By modifying the experimental conditions (antibody concentration,
incubation, and visualization times), we have increased 35-fold the MDC
(0.013 µmol/L, results not shown), allowing us to assay HVA in human
plasma (0.05 to 0.15 µmol/L) and human cerebrospinal fluid (0.055 to
0.26 µmol/L) (3).
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Acknowledgments
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This study was conducted under the BIOAVENIR program financed by
Rhône-Poulenc and the Commissariat à L'Energie Atomique
(CEA) with the contribution of the "Ministère de
L'Éducation nationale, de L'Enseignement Supérieur et de
la Recherche." We are indebted to P. Lamourette and M. Plaisance for
their expert technical assistance in monoclonal antibody preparation.
We also thank D. Pétré (Rhône-Poulenc) for helpful
discussions.
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Footnotes
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CEA,1 Service des Molécules Marquées DBCM and 2 Service de Pharmacologie et d'Immunologie DRM, CE Saclay, 91191 Gif sur Yvette Cedex, France.
1 Nonstandard abbreviations: VMA, vanillylmandelic acid; HVA, homovanillic acid; E, epinephrine; NE, norepinephrine; ED, electrochemical detection; EIA, enzyme immunoassay; MHPG, 3-methoxy-4-hydroxyphenylethylene glycol; MT, 3-methoxytyramine; DOMA, 3,4-dihydroxymandelic acid; VAN, vanillin; DOPAC, 3,4-dihydroxyphenylacetic acid; NM, normetanephrine; 3MTyr, 3-methoxytyrosine; DA, dopamine; Syn, synephrine; HPAA, 4-hydroxyphenylacetic acid; PAA, phenylacetic acid; Tyr, tyramine; NHS, N-hydroxysuccinimide; DCC, 1,3-dicyclohexylcarbodiimide; SMCC, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate; SATA, N-succinimidyl-S-acetylthioglycolic acid; KLH, keyhole limpet hemocyanin; AChE, acetylcholinesterase; EU, Ellman's units; and MDC, minimal detectable concentration. 
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Top
Abstract
Introduction
) for HVA assayed with the
present EIA and typical dilution curve (
) for urine samples.