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Competitive enzyme immunoassay with monoclonal antibody for homovanillic acid measurement in human urine samples.

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. (4) 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 [9-12]. 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.

Materials and Methods

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-l-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].


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 [micro]L 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[degrees]C. Immunogen was stored at -20[degrees]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 [micro]g) 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.sup.-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 [micro]L of calibrator, buffer, or urine sample, 50 [micro]L of enzymatic tracer [2000 Ellmaris units (EU)/L], and 50 [micro]L of diluted monoclonal antibody. After a 2-h incubation at room temperature, the wells were washed and Ellmari s reagent [7.5. [10.sup.-4] mol/L acetylthiocholine iodide (enzyme substrate) and [5.10.sup.-4] mol/L 5,5'-dithiobis-(2-nitrobenzoic acid) (chromogen) in 0.1 mol/L phosphate buffer, pH 7.4] (200 [micro]L) 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 Ellmari s method [17]. Enzymatic activity was expressed in terms of EU corresponding to the quantity of enzyme hydrolyzing 1 [micro]mol/L of substrate during 1 min at 25[degrees]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 [micro]L 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.


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 [micro]L of water:acetic acid mixture (98:2 by vol) (mobile phase A). The solution (150 [micro]L) was injected into a reversed-phase C18 column (Nucleosil 300, 5 [micro]m 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-[micro]L aliquot of each fraction was diluted in 450 /.L of EIA buffer. Five urinary sample extracts were prepared for EIA-HPLC validation studies.


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 [micro]mol/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 ([r.sup.2] = 0.99). This antibody was chosen for further studies.

Sensitivity, accuracy, and specificity. A routine EIA calibration curve is presented in Fig. 2. The MDC was 0.5 [micro]mol/L and the CV was <10% between 1.25 and 10 [micro]mol/L. The reference range (0-40 [micro]mol/L) and limit of detection (1 [micro]mol/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].


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 [micro]mol/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 ([+ or -] 0.03) HPLC - 3.46 ( [+o r -] 7), [S.sub.y|x] = 47.5, range = 4-1800 [micro]mol/L, [r.sup.2] = 0.977, n = 62.


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 [9-12] 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 [R.sub.4] 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 [25-28]. 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 [micro]mol/L, results not shown), allowing us to assay HVA in human plasma (0.05 to 0.15 [micro]mol/L) and human cerebrospinal fluid (0.055 to 0.26 [micro]mol/L) [3].

This study was conducted under the BIOAVENIR program financed by Rh6ne-Poulenc and the Commissariat a L'Energie Atomi5ue (CEA) with the contribution of the "Ministere de L'Education nationale, de L'Enseignement Sup6rieur 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. Petre (Rhone-Poulenc) for helpful discussions.

Received August 7, 1996; revised October 3, 1996; accepted October 3, 1996.


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CEA, (1) Service des Molecules Marquees DBCM and (2) Service de Pharmacologie et d'Immunologie DRM, CE Saclay, 91191 Gif sur Yvette Cedex, France.

(3) Laboratoire CERBA, 95066 Cergy-Pontoise, Cedex 9, France.

(4) 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-l-carboxylate; SATA, N-succinimidyl-S-acetylthioglycolic acid; KLH, keyhole limpet hemocyanin; AChE, acetylcholinesterase; EU, Ellmari s units; and MDC, minimal detectable concentration.

* Author for correspondence. Fax (33) 01 69 08 59 07; e-mail
Table 1. Sensitivity and specificity of EIAs with various monoclonal
anti-HVA antibodies and correlation studies with HPLC measurements for
eight urine samples.

Clone # to B/[B.sub.0] (50%) VMA
HVA ([micro]mol/L HVA) (% cross-reactivity) [r.sup.2]

7 5 4 0.63
32 4 13 0.69
67 9 7 0.82
78 8 0.4 0.76
93 5 18 0.74
127 3 0.5 0.99
137 6 2 0.87

Clone # to
HVA Slope [gamma]-intercept x-intercept

7 8.50 -169.1 19.86
32 5.94 -94.1 15.85
67 5.16 -56.6 11
78 5.10 -65.2 12.8
93 6.25 -83.1 13.3
127 1.45 -1.1 0.76
137 1.67 -4 2.4

Table 2. Structures and abbreviations of catecholamine urinary
metabolites, normal concentrations, and cross-reactivity.

[R.sub.1] [R.sub.2] [R.sub.3] [R.sub.4]

OH [OCH.sub.3] H COOH
OH [OCH.sub.3] OH COOH
OH [OCH.sub.3] OH [CH.sub.2]OH
OH [OCH.sub.3] H [CH.sub.2][NH.sub.2]
OH [OCH.sub.3] OH [CH.sub.2][NH.sub.2]
OH [OCH.sub.3] H CH(COOH)[NH.sub.2]
OH [OCH.sub.3] =0
OH OH H [CH.sub.2][NH.sub.2]
OH OH OH [CH.sub.2]NH[CH.sub.3]
OH OH OH [CH.sub.2][NH.sub.2]
OH H OH [CH.sub.2]NH[CH.sub.3]
H H H [NH.sub.2]

 Compound Normal conc. in urine Cross-reactivity
[R.sub.1] (abbreviation) ([micro]moles/24 h) (%)

OH HVA 26 [3] 100
OH VMA 19 [3] 0.50
OH MHPG 7 [3] 0.50
OH MT 1.2 [23] 8
OH NM 1 [3] 0.80
OH 3M-Tyr 0.15 [22] 1
OH VAN <0.10
OH DOPAC 6 [3] <0.10
OH DOMA <0.10
OH DA 1.3 [3] <0.10
OH E 0.04 [3] <0.10
OH NE 0.2 [3] <0.10
OH Syn <0.10
OH HPAA 165 [5] <0.10
H PAA 12.5 [5] <0.10
H Tyr <0.10
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Article Details
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Title Annotation:Automation and Analytical Techniques
Author:Taran, Frederic; Frobert, Yveline; Creminon, Christophe; Grassi, Jacques; Olichon, Didier; Mioskowsk
Publication:Clinical Chemistry
Date:Feb 1, 1997
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