Anal. Chem. W89, 61, 447-453
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Enzyme Immunoassays of Adenosine Cyclic 3’,5‘-Monophosphate and Guanosine Cyclic 3’,5’-Monophosphate Using Acetylcholinesterase Philippe Pradelles* and Jacques Grassi Laboratoire d’Etudes Radioimmunologiques, Section de Pharmacologie et d’lmmunologie, Dgpartement de Biologie, Commissariat ci 1’Energie Atomique, CENISaclay, 91191 Gif sur Yvette C&dex,France
Danielle Chabardes Laboratoire de Physiologie Cellulaire, Collsge de France, 11 place Marcellin Berthelot, 75231 Paris CQdex 05,France
Nicole Guiso Unit6 d’Ecologie Bactgrienne, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris C6dex 15,France
The pure tetramerk form of acetykhdlnesterase (EC 3.1.1.7) from the electrlc eel Electrophorus elecfrlcushas been covalently coupled to 2’-O-succlnyCcAMP tyroslne methyl ester and 2‘-O-succlnyl-cGMP. Both enzymatic conjugates have been used as tracers In a classical heterogeneous competltlve enzyme immunoassay allowlng the determlnatlon of cAMP and cGMP, respectively. The test was performed In 96-well mlcrotlter plates coated wlth a mouse monoclonal antl-rabblt Immunoglobulin antlbody In order to ensure separation between bound and free moletles of the tracer. Acetylchollnesterase actlvlty bound to the solld phase was measured by colorimetric assay. When standards or samples were flrst acetylated by treatment wlth acetlc anhydride, the sensltlvlty of both assays appeared very good slnce minimum detectable concentratlon close l o 0.04 pmol/mL (2 fmol/weil) could be calculated for each assay. Preclslon was also very satisfying since the coefficient of varlation was less than 5 % In the 0.2-10 pmWmL range. Good correlatlon was noted between enzymdmmunologkal and radlolnnnundogkal measurements of cAMP performed for dlfferent Mologlcal samples (urlne, serum, or tlssue extracts).
been made, particularly in the field of nonisotopic immunoassays. Some attempts have been made to develop an enzymoimmunoassay (EIA) for cAMP a t the picomole level ( mol) by using P-galactosidase as label (7) and a homogeneous immunoassay based on chemiluminescence energy transfer (8)allowing the determination of cAMP in biological samples with a detection limit of 25 fmol. In recent years, our laboratory has developed enzyme immunoassays based on the use of acetylcholinesterase (AChE) from Electrophorus electricus as label (9). We have shown that AChE conjugates of high specific activity can be used to quantify various substances such as eicosanoids (IO), substance P (ll),atriopeptin (12),or thymulin (13),at sensitivities equal to or greater than those of conventional RIAs using lZ6I-iodinated tracers. In addition, the combined use of enzymatic tracers and second antibody solid phase allows a high degree of automation of the method, thus rendering it very suitable for routine analysis. The present study reports the development of a simple, highly sensitive and reliable EIA for cAMP and cGMP using AChE conjugates, second antibody coated microtiter plates as solid phase, and acetylation procedures to increase the sensitivity of the assay. EIA-RIA correlation studies for biological samples are presented and the advantages of the technique are discussed.
INTRODUCTION The ubiquitous roles played by adenosine cyclic 3’,5‘phosphate (CAMP) and guanosine cyclic 3’,5’-phosphate (cGMP) as intracellular mediators have rendered increasingly desirable a precise measurement of the nucleotides in tissue and body fluids. These substances being present at very low concentrations in tissues the corresponding analytical methods must be both sensitive and specific. Three types of methods emerge among those described to assay these molecules: enzymatic methods (1)protein binding assay (2), and radioimmunoassay (RIA). Radioimmunoassays of cyclic nucleotides (mainly cAMP and cGMP) in biological fluids or tissue extracts currently involve the use of specific antibodies and lEI-iodinated tracers. The pioneering studies of Steiner et al. (3),Cailla et al. (4), Harper and Brooker (5),and Delaage et al. (6) have established the fundamental and practical basis for RIA or these substances, showing that femtomole levels mol) could be measured when 12SI-iodinatedtracers and 2’-O-derivatization procedures (succinylation or acetylation of the competitor) are used. Since these studies, few significant advances have
EXPERIMENTAL SECTION Reagents. Unless otherwise stated, all reagents were from Sigma (St Louis, MO). The cAMP antiserum used in the EIA studies was obtained by immunizing rabbits with an immunogen prepared by conjugating the 2’-O-succinyl derivative of cAMP with keyhole limpet hemocyanin according to a previously described method (3). Rabbit cGMP antiserum (code 79594) and the kit for CAMPradioimmunoassay (code 79830) were purchased from Institut Pasteur Production (Paris, France). Mouse monoclonal antibody (code 173) against rabbit immunoglobulins (IgG) was obtained in our laboratory by using standard hybridomas techniques (unpublished data) and was purified from ascitic fluids by Protein A affinity chromatography (14). The characteristics of these monoclonal antibodies have already been described (13). Acetylcholinesterase (AChE) (EC 3.1.1.7) was extracted from the electric organ of the electric eel Electrophorus electricus, and was purified by one-step affinity chromatography as described by Massoulig and Bon (15). The tetrametric form of AChE (the polymorphism of AChE is detailed elsewere (16))used in this study and named “G4-AChE”was prepared from the purified AChE preparation by treatment with trypsin (1pg/mL) for 18 h at 25 “C in 0.1 M phosphate buffer pH 7 (1/2000 trypsin-AChE ratio (w/w)). This mixture, which contained mainly G4-AChE, was
0003-2700/89/0361-0447$01.50/0
0 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989
t
t
t
minutes
Figure 1. Purification of SCAMP-TME-AChE(0,)conjugate: Vo, void
volume; Vt, total volume; G,, pooled fractions corresponding to SCAMP-TME-AChE (0, form) conjugate. used without further purification. Acetylcholinesterase activity was measured by the method of Ellman et al. (17). Ellman's medium comprises a mixture of 7.5 x lo4 M acetylthiocholine M 5,5'-dithiobis(2iodide (enzyme substrate) and 5 X nitrobenzoic acid) (reagent for thiol colorimetric measurement) in 0.1 M phosphate buffer, pH 7.4. Routinely, Ellman's reagent was lyophilized so that one vial was reconstituted with 100 mL of water. Enzymatic activity was expressed in terms of the Ellman unit (EU), which is defined as the amount of enzyme producing an absorbance increase of 1 during 1min in 1 mL of medium, for an optical path length of 1 cm: it corresponds to about 8 ng of enzyme and 7.35 X lo-* enzyme unit (one enzyme unit corresponds to the quantity of enzyme hydrolyzing 1pmol of substrate during 1 min at 25 "C). AChE concentrations were determined enzymatically on the basis of a turnover number of 4.4 x lo+' mo1.h-l per enzymatic site (18),and by assuming a molecular weight of 80000 for the catalytic subunit. When a detection limit is arbitrarily defined as the amount of AChE producing an absorbance increase of 0.01 during 1 h in 200 pL of Ellman medium, for a 0.5 cm path length, a value of 1.6 X lo-'* mol can be calculated for G4-AChE. Apparatus. Solid-phase EIA was performed by using specialized Titertek microtitration equipment from Flow Laboratories (Helsinki, Finland), including an automatic plate washer (Microplate Washer 120), an automatic plate dispenser (Autodrop), and an automatic plate reader (Multiskan MC). Microtiter plates (96 F immunoplate I with certificate) were from Nunc (Denmark). Enzyme activity measurements were made with a Stasar I11 spectrophotometer (Gilford, Oberlin OH). Molecular sieve chromatography was performed with the FPLC system of Pharmacia (Uppsala, Sweden) using a Superose 6 column HR 10/30. Preparation of Enzymatic Tracers. Enzymatic tracers were obtained by covalent linkage of succinyl-CAMP tyrosine methyl ester or succinyl-cGMP to G4-AChE. SCAMP-TMEAChEConjugatePreparation. A 50-pL (lo00 nmol) portion of DFDB (1-5-difluoro-2-4-dinitrobenzene) in anhydrous methanol was added to 100 pL (100 nmol) of ScAMPTME (2'-O-monosuccinyl adenosine cyclic 3',5'-phosphate tyrosyl methyl ester) in 0.1 M phosphate buffer pH 7.4. After 30 min at room temperature in the dark, an aliquot of the reaction mixture was analyzed by thin-layer chromatography (TLC) on a silica gel plate, with 1-butanol-acetic acid-water (75/10/25 (v/v/v)) as the developing solvent system. Under UV light, a newly formed product (Itr= 0.5), assumed to be the monofluoronitrobenzene (MFDB) derivative, was detected. The reaction mixture was dried under nitrogen and 100 pL of 0.1 M phosphate buffer, pH 7.4, was added. Excess DFDB was extracted with ether. After addition of 2 mL of ether and shaking, the upper phase (ether) was removed by suction and this operation was repeated 3 times. After the last extraction, the ether remaining with the aqueous phase was evaporated under nitrogen and the presence of the MFDB derivative was again checked by TLC. The MFDB derivative (100 pL) was added to 200 p L of GI-AChE preparation (0.63 mg/mL) in 0.1 M borate buffer, pH 8.5. After 18 h of incubation at room temperature, 200 pL of EIA buffer (see immunoassay procedures section) was added and 15 min later the enzyme activity was measured. At this stage, no significant loss of activity was noted. Purification of the enzymatic tracer was performed by molecular
sieve chromatography using the FPLC system with EIA buffer as eluting solvent (flow rate 0.4 mL/min). Fractions were collected each minute. The enzymatic profile was determined (Figure 1) and fractions containing GI-AChE were pooled and stored at -80 "C until use. The two peaks eluted before the G4 peak correspond to the high molecular weight asymmetric forms remaining after treatment of the AChE preparation with trypsin (10). For routine assay, the diluted tracer was lyophilized and stored under vacuum at +4 "C. Under these conditions, no significant loss of enzyme activity or immunoreactivity was observed, after 6 months of storage. ScGMP-AChEConjugate Preparation. A 20-pL portion of N-hydroxysuccinimide (NHS) (200 nmol) and 20 pL of N,N'dicyclohexylcarbodiimide (DCC) (200 nmol), both in anhydrous dimethylformamide (DMF),were successivelyadded to 20 pL (200 nmol) of ScGMP (2'-0-monosuccinylguanosine cyclic 3',5'phosphate) in DMF. After 4 h at room temperature, 30 pL of this mixture was added to 300 p L of G4-AChE (0.3 mg/mL) in 0.1 M borate buffer pH 8.5. The reaction was allowed to proceed for 30 min at room temperature and was stopped by the addition of 200 p L of EIA buffer. No significant loss of enzyme activity was observed. Purification and storage of the conjugate were performed as described above. Competitive Immunoassay Procedures. Both EIA and RIA for cyclic nucleotides were competitive immunoassays using AChE-cyclic nucleotides or lZ6Ilabeled cyclic nucleotides as tracers, respectively. EIA Procedures. All assays were performed in the following assay buffer (named "EIA buffer"): 0.1 M phosphate buffer, pH 7.4, containing 0.4 M NaCl, M EDTA, 0.1% bovine serum albumin, and 0.01% sodium azide. EIA was performed as described elsewhere for eicosanoids (10) and peptides (11-13). The 96-well microtiter plates were coated with mouse monoclonal antibodies specific for rabbit IgG, as previously described (13). Before use, the plates were extensively washed with lo-* M phosphate buffer pH 7.4 containing 0.05% Tween 20 (washing buffer) using the Multiwash apparatus (300 rL/well, five wash cycles). The assay was performed in a total volume of 150 pL, each component being added in a volume of 50 pL. In routine assay, reagents were dispensed as follows: 50 pL of cAMP or cGMP diluted antiserum (1/10000 and 1/20000 in EIA buffer so that the final dilutions were 1/3oooO and l/6oooO, respectively); 50 pL of cAMP or cGMP-AChE conjugate diluted in EIA buffer (3.7 and 5.3 EU/mL, respectively);50 pL of standard or biological sample (derivatized or not). The plates were then covered with a plastic adhesive sheet (from Flow Laboratories) and were left for 18 h at room temperature. The plates were washed again as described above and shaken to eliminate the remaining washing buffer and 200 pL of Ellman's medium were automatically dispensed into each well by using an Autodrop apparatus. During the enzymatic reaction the plates were gently agitated. At this stage strong illumination of the plate with natural or artificial light must be avoided. When the absorbance in the Bo well (see below) reached 0.2 (30-60 min), the absorbance at 414 nm as automatically measured in each well by a Multiskan MC spectrophotometer (reading time, 20 s for one plate). 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 BIB, X 100 as a function of the logarithm of the dose. Fitting of the standard curves was performed with an Apple 2e microcomputer using a linear log-logit transformation (19). Unless otherwise stated, all measurements for standards 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 antibody was replaced by 50 pL of EIA buffer. In all cases, nonspecific binding was less than 0.170of the total enzyme activity introduced in the assay. The minimum detectable concentration was taken as the concentration of competitor inducing a significant decrease (3 standard deviations) in Bo. All the concentrations or dilutions mentioned in this paper are expressed in terms of "final concentration or dilution" and refer to the concentration or dilution of the reagent in the total volume used in the assay.
ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989
Determination of the Balance Sheet for Enzymatic Activity. Two different antibody dilution curves were established simultaneously using microtiter plates coated with a second antibody as described in the Experimental Section. After immunoreaction (6 days at room temperature), the liquid contained in each well used for data collection for the f i t curve was carefully transferred to an uncoated microtiter plate before addition of 150 pL of Ellman’s medium (concentrated 2-fold) in order to measure unbound enzymatic activity. A 160-pLportion of washing solution was then added to each well and the plate was gently agitated for 5 min before measurement of enzymatic activity. Activity present in the first washing solution was measured as for unbound activity. After a further washing performed with the automatic washer, solid-phase bound activity was measured as usual after addition of 300 pL of Ellman’s medium. Activity in the liquid phase, solid-phase bound activity, and activity present in the first washing solution were expressed as a percentage of total activity introduced in the test. In order to check the completeness of immunoreaction, liquid-phasealiquots corresponding to the second antibody dilution curves were transferred to second antibody coated microtiter plates in the presence of an excess of first antibody (1/1000dilution of cAMP antiserum) and were allowed to react for 48 h. At the end of the second period no significant solid-phase bound activity could be measured. Specificity Measurements. The specificity of the assays was checked by testing their capacity to detect structurally related compounds (i.e. other cyclic or noncyclic nucleotides, acetylated or not). This was obtained by establishing for each of these compounds the corresponding standard curve (concentrationsup to micromoles per milliliter were tested if necessary). Results were expressed in terms of “cross-reactivitypercent”, taking the cAMP or cGMP standard curve as reference as defined by Abraham (20). This corresponds to the following ratio: (dose of cAMP or cGMP/dose of analogue) X 100. RIA Procedures. RIA measurements of cAMP content in microdissected segments of the rat nephron, using a commercial kit purchased from Institut Pasteur Production, with minor modifications, have been described in detail elsewhere (21,22). For human serum and urine, cAMP content was measured by the Service de Radioanalyse (Institut Pasteur Lyon, France) using the 1261-RIAcommercial kit. The cAMP and cGMP contents of enzymatic conjugates were measured by RIA as follows: 50 pL of cAMP or cGMP diluted antiserum, 50 pL of 1261-cAMPor cGMP (4000dpm), and 50 pL of either acetylated standards (CAMPor cGMP) or enzymatic conjugate were dispensed into the wells of microtiter plates as described in EIA Procedures. After 6 days of immunoreaction at room temperature, the plates were washed and 200 pL of 0.1 N HC1 was added to each well. Thirty minutes later, the contents counting. of each well were transferred into plastic tubes for 1261 Preparation of Biological Samples. Medullary cortical portions (MCT) of rat collecting tubules were microdissected and cAMP extracts were prepared as previously described (21). A 50-pL portion of 50 mM sodium phosphate buffer, pH 6.2, was added to the dry extracts in polypropylene tubes. In each EIA-RIA correlation study, samples containing 2 pL of incubation solution but no tubule fragment were processed in the same way and were used to determine nonspecific binding and Bo values. For the EIA-RIA correlation of cAMP content in human urine, the samples were directly assayed after dilution in EIA buffer. For the human serum, the extraction of CAMPwas performed by using ethanol as described in the RIA kit protocol: 2 mL of cold ethanol was added to 0.5 mL of serum, and after 5 min the tubes were centrifuged and the supernatant was collected. After the solution was dried under nitrogen, 0.5 mL of 50 mM phosphate buffer, pH 6.2, was added and the samples were treated for the acylation process (see below). Derivatization Procedures. Acetylation of cyclic nucleotides by acetic anhydride (AA)in aqueous medium (5) was performed as described elsewhere (6). Briefly, to 500 pL of standard cAMP or cGMP or biological extract in 50 mM phosphate buffer, pH 6.2, were successively and rapidly added 100 pL of 4 N KOH, 25 pL of AA, and, after 15 s of shaking, 25 pL of 4 N KOH. For the EIA-RIA correlation studies with microdissected tubule extracts some modifications were introduced. To 50 pL of biological extract were successively
449
and rapidly added 10 pL of 4 N KOH, 5 pL of AA, and, after a few seconds of shaking, 10 pL of 4 N KOH. After addition of each immunological component, 50 pL of 1 M phosphate buffer, pH 7.4,were added to each well. In each case, due to the interfering effects of acetate salt (6),phosphate buffer was treated in the same way and introduced in the assay for the nonspecific and Bo measurements. In the RIA experiments, acetylation was performed as described in the protocol of the commercial kit, using triethylamine (21) instead of KOH.
RESULTS AND DISCUSSION In previous papers (10-13) we have shown that primary amino groups of acetylcholinesterase could be efficiently used for coupling the enzyme with haptens, without altering the catalytic properties of the enzyme. For haptens containing a single carboxylic group (i.e. prostaglandins, thromboxane B2), an active NHS ester was prepared in anhydrous organic medium before reaction with the enzyme in weak alkaline aqueous medium (IO). A different approach was developed in the case of leukotriene C4 which contains three carboxylic groups. Here the coupling procedure involved the single primary amino group of the hapten which was cross-linked to AChE by DFDB (10). Altematively, another procedure was used for the coupling of small peptides such as substance P (11)or atriopeptin (12). Here the coupling was achieved with a heterobifunctional reagent (SMCC, succinimidyl I-(N-ma1eimidomethylcyclohexane)-1-carboxylate).In this case, the cross linking involved reaction of maleimido groups carried by AChE after reaction with SMCC and thiol groups previously introduced into the primary amino groups of the peptide. Whatever the coupling procedure used, the preparation of the hapten-enzyme conjugate was carried out in accordance with the following rules: (i) Avoid any loss of enzyme activity during the coupling with the hapten. This rule is currently obeyed with AChE providing only the primary amino groups of the enzyme are involved. (ii) Avoid polymerization of both enzyme and hapten since this could be detrimental to the immunoreactivity of the conjugate. In addition, polymerization is undesirable in that it renders precise control of enzyme-hapten stoichiometry problematical and has a negative influence on nonspecific binding of tracer. (iii) As far as possible the synthesis of the enzymatic conjugate is copied from that of the immunogen preparation in order to ensure full immunoreactivity for the tracer. All these considerations were taken into account when a strategy was chosen for the preparation of cAMP and cGMP conjugates for determination of cAMP and cGMP. ScGMP and SCAMP-TMEwere chosen as labeling precursors since both ScGMP and ScAMP were used for immunogen preparation (3). Coupling of ScGMP to enzyme was obtained through the intermediate formation of an NHS ester in DMF as for prostaglandins and thromboxane B2 (10). This procedure could not be applied to SCAMP,which was insoluble in anhydrous DMF. We thus turned to SCAMP-TME,which was successfully used as precursor in the preparation of a radioiodinated tracer for RIA. SCAMP-TMEwas coupled to AChE through the intermediary of DFDB. Here, however, the coupling did not involve primary amino groups as for leukotriene C4 (10)but the phenolic group of SCAMP-TME. This polyvalent property of DFDB has already been described for protein cross linking (for review see ref 23) and this homobifunctional reagent is known to react with amino, tyrosyl, sulfhydryl, or imidazole groups (24,25). The precise procedures used for the preparation of both tracers are detailed in the Experimental Section. The structural features of the corresponding enzymatic conjugates are presented in Figure 2. The presence of immunoreactive cyclic nucleotide in the AChE molecule after coupling could be demonstrated in two ways. First way is by performing radioimmunological measurements on the enzymatic conjugates (see Experimental
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989 una H.)(
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:'
~ I P V O N H
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Flgure 2. Structure of SCAMP-TME-AChE (G,) and cGMP-AChE (G,) conjugates.
Bound activity (f: o f toto. toto octlvlty)
P
A n t i s e r w dilutior
Flgure 3. Antibody dilution curve for anti-CAMP antiserum: (curve A) standard procedure; (curve B) sequential procedure.
Section). These tests revealed that 1.8 and 2 molecules of haptens were coupled per molecule of G4-AChE for cAMP and cGMP, respectively. However these measurements cannot be regarded as quantitative since it is very likely that cyclic nucleotides linked to the enzyme and acetylated cyclic nucleotides used for establishing the standard curves differ in their immunoreactivity. This hypothesis is supported by the fact that serial dilutions of enzymatic conjugates gave curves that were not parallel to the standard curve (data not shown). The effectiveness of the coupling was confirmed by the fact that the enzymatic conjugates could be immobilized on a second antibody solid phase in the presence of specific anti-cyclic nucleotide antibodies. A typical antibody- dilution curve obtained with a cAMP antiserum and ScAMP-TMEAChE conjugate is presented in Figure 3 (curve A). In this standard procedure, specific cAMP antiserum was diluted in EIA buffer and incubated with enzymatic tracer and solid phase for 18 h at room temperature as described in the Experimental Section. Similar curves were obtained with the cGMP system (data not shown). These curves differ markedly from classical antibody dilution curves in two ways. First, a marked reduction in bound enzyme activity is observed when high concentrations of antiserum are tested, and secondly, the solid-phase bound enzyme activity observed at the plateau does not exceed 8% of the total enzyme activity introduced in the assay. These two characteristic features were observed with all enzyme immunoassays developed in our laboratory and we should like to take this opportunity on this paper to clarify these points. The decreased binding observed in the presence of excess antibody (left part of the curve) is not due to inhibition of enzyme activity caused by reaction with antibodies since, in solution, prolonged incubation of enzymatic conjugate with
a high concentration of cyclic nucleotide antiserum does not induce any inhibition of enzyme activity (data not shown). It is worth noting that such inhibition has been observed with anti-acetylcholinesteraseantibodies (26). It is very likely that the decrease in bound enzyme activity is not due to the use of an enzymatic tracer but is rather connected to the limited binding capacity of the solid phase. What we observe here is a classical "hook effect" where an increasing proportion of tracer-antibody complexes fails to bind to the solid phase as the total immunoglobulin concentration exceeds the binding capacity of the second antibody solid phase. This hypothesis is supported by the fact that this decreased binding is suppressed when antibodies and enzyme conjugates are allowed to react sequentially on the solid phase (unbound antibodies being eliminated by washing in an intermediary step); Figure 3 shows the curve obtained (curve B) when specific cAMP antiserum was first incubated alone for 18 h at room temperature. In this case, enzymatic tracer was added in a second step (18 h at room temperature). In this curve the plateau is restored throughout the excess antibody zone. Different hypotheses can be put forward to explain the low binding value observed at the plateau, including (1)partial inhibition of enzyme activity due to the binding of antibodies to the enzymatic conjugate, (2) incomplete reaction of tracer with first antibodies and/or of first andibody with second antibody solid phase due to very slow kinetics, and (3) underestimation of bound enzyme activity which could be attributed to different factors, including poor accessibility of substrates to the solid-phase bound enzyme, lowering of substrate concentration at the liquid-solid interface, or accumulation of acetic acid (acetic acid is produced by hydrolysis of acetylthiocholine) at this interface. These last two possibilities actually exist since AChE activity is closely dependent on substrate concentration and because the turnover of the enzyme is significantly lowered in acidic medium (for a review see ref 27). Two additional hypotheses are (4) the loss of solid-phase bound enzyme activity during the washing of plates and (5)the poor effectiveness of the coupling procedure, plateau binding values below 100% being observed when a proportion of AChE molecules do not carry any hapten. As discussed above, the f i t hypothesis can be rejected since under homogeneous conditions reaction of first antibody with AChE conjugate is not accompanied by any inhibition of enzyme activity. On the other hand, it is clear that under our experimental conditions (overnight incubation at room temperature), binding equilibrium is not reached. This can be seen from Figure 4, which shows the binding kinetics obtained by using an enzymatic conjugate or radioactive tracer. These curves were established as follows: cAMP antiserum and radioiodinated or enzymatic tracers were incubated for various times at room temperature in second antibody coated plates. EIA measurements were performed as usual and for the RIA measurements, 200 I.LLof 0.1 N HC1 was added to each well after washing in order to remove material bound to the solid phase. Thirty minutes later the content of each well was transferred into plastic tubes for y scintillation counting. It is clear that when AChE conjugates are used, the binding equilibrium is only reached after 4 or 5 days of incubation. The marked difference between the binding kinetics of the radioiodinated tracer and those of the enzymatic tracer is probably due to differences in the coefficient of diffusion of each tracer as a function of size. However, this does not explain the low value of the plateau observed in antiserum dilution curves, since even after 6-day incubation, no more that 12% of enzyme activity was bound on the solid phase. In order to check hypotheses 3 and 4, we tried to establish a complete balance sheet of enzyme activity. For this purpose, enzyme present in the liquid phase, as well as any enzyme
ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989
Table I. Balance Sheet of Enzyme Activity after 6-Day Immunoreaction % total enzyme activity
Table 11. Effect of cAMP Antiserum Dilution of B o Values and Sensitivity in EIA Experiments Using the Acetylation Procedure BO
in the
antiserum dilution 1/3OOO l/6000 1/12000 1/24000 1/48000 1/96000 1/192000 1/384000 1/768000 1/1536000 1/3072000 1/614oooO
in the liquid
phase
bound to the solid phase
first washing solution
91.47 90.86 86.40 78.50 88.61 93.16 95.40 97.75 97.16 97.38 99.17 97.16
7.64 9.60 11.35 13.37 11.64 8.44 5.17 2.91 1.62 0.87 0.30 0.08
0.01 0.32 0.65 0.54 0.22 0.20 0.10 0.02 0 0.08 0 0
451
antise-
% of
(BIB0 50%),
rum
total
total
dilution
activity
absorbance unit
pmol/mL
99.21 100.78 98.40 92.44 100.47 101.80 100.67 100.68 98.78 98.33 99.47 97.24
1/12000 1/24000 1/ 48OOO 1/96OOO 1/192000
6.6 6.1 4.5 2.1 1.5
1.532 1.368 0.946 0.481 0.320
0.520 0.320 0.180 0.120 0.120
,
Table 111. Specificity of cAMP and cGMP Antisera in EIA % cross reactivity
competitor
cAMP
acetylated cAMP acetylated cGMP cAMP cGMP acetylated mixture'
100 co.001 0.300 0.003
