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Anal. Chem. I%SS,67,1170-1173
Enzyme Immunoassays of Eicosanoids Using Acetylcholine Esterase as Label: An Alternative to Radioimmunoassay Philippe Pradelles,*’ Jacques Grassi,’ a n d J a c q u e s M a c l o u P Section de Pharmacologie et d’lmmunologie, Laboratoire d’Etudes Radioimmunologiques, Dgpartement de Biologie, Commissariat Ci 1’Energie Atomique, CENISaclay, 91191 Gif sur Yvette Cedex, France, a n d U.150 INSERM-LA 334 CNRS Hopital Lariboisigre, 6 rue Guy Patin, 75475 Paris, France
Pure acetylcholine esterase (EC 3.1.1.7) from electric eel (Electrophorus electricus ) has been covalently coupled to dlfferent elcosanolds lncludlng thromboxane B,, POD,-methoxamlne, 6-keto-PGF,,, and leukotrlene C,. The corresponding conjugates have been tested In second-antibody enzyme Immunoassay. I n the case of thromboxane B,, a radlolodlnated tracer. Our comparlson was made wlth a (lBI) results show that the separatlon method has a slgnlflcant Influence on the performance of the assay. the best results are obtained by uslng mlcrotlter plates wlth 96 wells coated wlth the second antibody, as compared wlth classical lmmunoprecipltatlon. In these condltlons, EIA appears to possess a sensltlvlty equivalent to, or better than, RIA. The mlnlmal detectable concentratlons were 10 pg/rnL for prostaglandlns or thromboxane B, and 40 pg/mL for leukotrlene C4. For the range 20-400 pg/mL thromboxane B,, the lntraassay varlatlon was IO5Ellman units/mg of protein and, when analyzed on SDS poly(acry1amide) gels (15), presented the characteristic pattern described for the pure enzyme (16). Preparation of Tracers. Radioactive Tracers. The preparation of the lZ5I-TXB, tracer was performed as described earlier (1). Enzymatic Tracers. Enzymatic tracers were obtained by covalent coupling of eicosanoids with pure enzyme. The preparation of PG or TX-AChE conjugates was done as follows: to 1pmol of PG or TXB, were added 1 pmol of N-hydroxysuccinimide and 1 pmol of N,N’-dicyclohexylcarbodiimide in a total reaction volume of 300 pL of anhydrous dimethylformamide (DMF). After overnight incubation at room temperature, in the dark, the reaction products were analyzed by thin-layer chromatography using ethyl acetate/hexane (8020 v/v) as the developing system and silica gel HPTLC plates (Merck Darmstadt, Germany). For example, TXB2-N-hydroxysuccinimide ester migrated at R, 0.70 vs. 0.19 for control TXB2 Under these conditions, more than 90% of T X or PG was converted into the ester. At this stage, the reaction products were stored at -30 OC until use. PG or T X esters (1-100 nmol) in 50 pL of DMF were added to AChE (50-250 pg) in 500 pL of 0.1 M borate buffer, pH 9. After 1 h at 4 “C, the reaction was stopped by addition of 1 mL of assay buffer (see the following section). Addition of this buffer also prevented nonspecific adsorption of the enzyme on the walls of the reaction vials. At this stage, no significant loss of enzyme activity was observed. Purification of the conjugates was performed with molecular sieve chromatography by using a Biogel A 15-m column (90 X 1.5 cm ) eluted with M Tris M MgC12,and 0.01% buffer, pH 7.4, containing 1 M NaC1, sodium azide. In these conditions, uncoupled products were recovered in the total volume of the column (Figure 1). Fractions of 1 mL were collected in polystyrene tubes containing 1 mL of assay buffer. The enzymatic activity of each fraction was determined, and the fractions containing aggregates, or a mixture of As and AI2 forms, were pooled and stored at -30 O C . In order to evaluate the efficiency of the coupling procedure, the PG or T X content of the conjugates was analyzed by using radioimmunological measurements. This determination, which is based on the hypothesis that the immunoreactivity of eicosanoids is not modified when they are conjugated to the enzyme, cannot be regarded as a quantitative determination. It gives, however, a relative measurement of incorporation of PG or T X and was very useful for the determination of the optimum conditions for tracer preparation. We never observed any inhibition of the enzymatic activity of T X or PG conjugates in the presence
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of an excess of specific PG or T X antibodies. The preparation of the LTC,-AChE conjugate was carried out according to a different procedure (17). Thirty microliters of 1,5-difluoro-2,4-dinitrobenzene(100 nmol) in methanol was added to 10 nmol of LTC, in 50 pL of 0.1 M, pH 7,phosphate buffer. After 30 min at room temperature, the methanol was removed under a stream of Nz and then under vacuum. The unreacted reagent was removed by extraction with 2 x 500 pL of diethyl ether. AChE (200 pg) in 500 pL of borate buffer (0.1 M pH 9) was added to the reaction vial, which was left overnight at room temperature before the addition of 1mL of assay buffer. Subsequent purification and storage were performed as described above. Immunoassay Procedures. All assays were performed in the following assay buffer: phosphate buffer (0.1 M, pH 7.4) containing 0.4 M NaCl, M EDTA, 0.1% bovine serum albumin, and 0.01 % sodium azide. Radioimmunological measurements were obtained by using second-antibody immunoprecipitation, and EIAs were performed by using either second-antibody immunoprecipitation or the second-antibody solid-phase method. Second-Antibody Immunoprecipitation (RIA/EIA) with Swine Antirabbit IgG Antiserum. These experiments were performed in (13 X 75 mm) polystyrene tubes in a total volume of 300 pL composed of 100 pL of standard eicosanoid or biological sample, 100 pL of tracer (iodinated or an enzyme conjugate) and 100 pL of an apropriate dilution of specific antiserum. Nonspecific binding was determined by using an incubation mixture in which the specific antibody is replaced by 100 p L of assay buffer. The amount of tracer bound in the absence of eicosanoid competitors (Bo)was determined in separate tubes containing 100 pL of assay buffer instead of standard eicosanoid or sample. The final mixture was incubated overnight at 4 “C. All the concentrations mentioned in this paper refer to the initial 100 pL (RIA) or 50 pL (EIA) volumes. When radioactive tracers were used, antigen-antibody complexes were immunoprecipitated by adding the swine second antibody together with a 1/100 dilution of nonimmune rabbit serum in the presence of 2% poly(ethy1ene glycol 6000)(PEG) as previously described (18). This procedure appeared unsuitable when enzymatic tracers were used because both rabbit and swine sera contain choline esterase and acetylcholine esterase (IO),which induce an undesirable increase of nonspecific binding. We thus developed an alternative procedure involving the use of a preformed precipitate composed of both nonimmune rabbit IgG and swine second antibody. This “preprecipitate” was obtained by mixing a large volume (50-200 mL) of nonimmune serum and second antibody in the presence of 2% PEG in the conditions described for radioimmunoassay. The corresponding precipitate was then washed 10 times with the initial volume of 2 % PEG in order to eliminate the choline esterase and acetylcholine esterase contained in the mixture. This reagent was resuspended before use, and 200 pL of the suspension was added to each tube. Whatever the type of tracer used, the final mixture was further incubated for 30 min at room temperature before adding 2 mL of assay buffer and centrifuging for 30 min at 2000g. The supernatants were then discarded. For RIA, the radioactivity of the pellet was determined by using a solid scintillation y counter (Multigamma, LKB, Sweden). For EIA, the enzymatic activity of the pellet was measured as previously described (18). Results are expressed in terms of BIBo X 100 where B and Bo represent the radioactivity or the absorbance measured on the bound fraction in the presence or absence of eicosanoid competitors, respectively. Fitting of the standard curves and calculations of the quantity of eicosanoids in biological samples were done with a microcomputer (MiniMinc, Digital) using a linear log-logit transformation (19). All measurements were made in duplicate. When the precision profile of the standard curve was established, all measurements were made 10 times, and results are expressed in terms of the coefficient of variation (CV, %) vs. the logarithm of the dose (20). Second-Antibody Solid-Phase EIA. The different steps of this assay are presented in Figure 2. Swine antirabbit IgG antibodies purified by affinity chromatography were immobilized on 96 well microtiter plates as follows: 300 p L of a 10 pg/mL M, pH 7.4, containing anti-IgG solution (phosphate buffer 5 X 2% glutaraldehyde) was automatically dispensed into each well
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985
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Step 1 : Simultaneous i n c u b a t i o n of Ag, AgE and Abl
E--Ag
E--Ag
-
.
i n 96 w e l l m i c r o t i t e r
p l a t e s coeted w i t h AbZ. I n c u b s t l o n o v e r n i g h t a t 4 O C .
,Or
Abl
- Ab
Step 2 : Washing o f m i c r o t i t e r p l a t e Step 3 : A d d i t i o n o f E l l m a n ’ s reagent
E --Ag
-
Abl
-
(14 seconds)
PICOGRAM (TXB,)
Figure 4. Precision profiles of the different TXB, assays. (A)E I A (second-antibodyimmunoprecipitation),(0)RIA (second-antibody immunoprecipitation), (a)EIA (second-antibody solid phase).
Ab2
Step 4 : C o l o r i m e t r i c measurement (412 nm) (14 seconds)
Flgure 2. Experlmental procedure used In solid-phase EIA. Ag = antigen (PGs, Tx, or LTC); Ab, = speclfic antibody for POS, TX, or LTC; E = enzyme; Ab, = swine antirabbit IgG antibodies; S = substrate; P = enzymatic reaction product.
Table I. Comparative Specificity of RIA and EIA for TXB,
eichosanoid TXBz TXBl PGEz PGDZ PGFZ, PGBz 6-keto-PGF1,
90. 80
-x
-
‘..
1
70-
60.
40.
30. 20.. 10. I
1
I
, ,l.ll,l 10
,
I 1 1 1 1 1 1 1
,
I
a , ,
100
PICOGRAM
Flgure 3. Standard curves for R I A and EIA of TXB,. (A)EIA (second-antibody immunoprecipitation),( 0 )R I A (second-antibody Immunoprecipitation), (a)E I A (second-antibody solid phase).
by using the Autodrop apparatus. After overnight incubation at room temperature, the plates were extensively washed with M phosphate buffer, pH 7.4, containing 0.05% Tween 20 by using the Multiwash apparatus. Assay buffer (300 pL) was then added to each well, and the plates were stored at 4 “C for 24 h prior to use. The plates were washed again before use. The assay was performed in a total volume of 150 pL, each component being added in a 50 pL volume. The concentrations of each reagent were identical with those used in the second-antibody immunoprecipitation EIA. The final mixture was incubated overnight at +4 “C. The plates were then washed again as described above, and 200 p L of Ellman’s reagent was automatically dispensed into each well by using the Autodrop apparatus. After 30 min to 2 h, the absorbance at 414 nm of each well was measured by using the Multiskan MC spectrophotometer. Nonspecific binding and Bo were determined as described above for second-antibody immunoprecipitation, and the results were expressed in the same way. RESULTS AND DISCUSSION In order to evaluate the possibilities offered by the use of AChE as an enzymatic label in immunoassay, we first made comparisons between the performance obtained with enzyme conjugates and iodinated tracers. Two standard curves for TXB2 established with either lZ5I-TXB2or AChE-TXB2 (A8 AI, forms) are presented in Figure 3. Both curves were obtained by using second-antibody immunoprecipitation for the same dilution of specific TXB2 antiserum (1/30000). The two curves are quite superimposable, yet an analysis of the corresponding precision profiles presented in Figure 4 reveals that the precision for EIA is significantly lower than that obtained with RIA. As a consequence EIA has a lower sensitivity (detection limit = 40 pg/mL as compared to 15 pg/mL
+
EIA, % 100 17
0.02 0.39
0.27
