Anal. Chem. 1995, 67, 1617-1622
Two=Sitelmmunometric Assay for Substance P with increased Sensitivity and Specificity Christophe Cr6minon,*gt Olivier Wry,t Yveline Frobert,t Jean=YvesCouraud,tl* Philippe Pradelles,t and Jacques Grassit CEA, Service de Pharmacoiogie et dlmmunologie, DRIPP, CELSaclay, 91 191 Gif-sur-Yvette Cedex, France, and UFR de Biologie, Tour 54, Universite Paris 7,75251 Paris Cedex, France
A two-site immunometric assay of the undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specihdly directed against the N- and C-terminal parts of the peptide. Afiinity-purified polyclonal antibodies raised against the six amino-terminalresidues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy-terminalpart of substance P, covalently coupled to the enzyme acetylcholinesterase, was used as tracer antibody. The assay is very sensitive, with a detection limit close to 3 pg/mL. Precision is also very good, with a coefficient of variation of less than 10% in the 10-250 pg/mL range. More important, the assay is fulhl specific for SP since cross-reactivity coefficients below 0.01%were observed with other tachykinins, SP derivatives, and SP fragments. The assay was used to measure the SP content of rat brain extracts and was validated by HPLC experiments.
other peptides related to mammalian tachykinins which present N-terminal elongation, neuropeptide K and neuropeptide y, have also been isolated. All tachykinins exhibit similar spectra of biological actions even though their relative potencies may be quite different.’ To date, immunoassays are the best way to quantify the relative abundance of these molecules in tissue extracts or biological fluids. Different kinds of SP assay have been described in the literature, mainly radioimmun~assays~-~ and enzyme immunoassay^.^-^ All are based on a competitiveprocess using radiolabeled SP or SP enzyme conjugates as tracer. Most involve antibodies obtained from an immunogen where SP is linked to the carrier protein via its amino functions, i.e., mNH2 and E-NHz(lysine in position 3). The resulting polyclonal or monoclonal antibodies are mostly directed against the carboxyterminal part of the peptide which is common to the three tachykinins, and they generally cross-react significantlywith NKA and NKB. Few reports describe competitive assays which use antisera raised against the amino-terminal portion of SP and are therefore more specific with regard to NKA and NKB.8,9 However, these assays cannot discriminate SP from some of its fragments, which may also be present in the biological media. We have previously described the preparation and characterization of monoclonal antibodies (mAbs) raised against SP and their use in developing competitive immunoassays for SP.lo We used one of these mAbs (named SP31), which is specific for the carboxyterminal part of SP, and new polyclonal antibodies, directed against the amino-terminalmoiety of the molecule, and we describe here a two-site immunometric assay which proved to be sensitive, precise, and completely specific for the intact molecule.
During the last 30 years, immunoassays have proved to be very useful tools for quantifjmg every kind of organic substance in biological media. Although a large number of different procedures have been described, all immunoassays can be categorized as “competitive” or “immunometric”. It has been widely demonstrated that immunometric assays, because they use much more concentrated reagents, favor the formation of an antigen-antibody complex and potentially lead to much more sensitive measurements. As a consequence, immunometric assays (mainly two-site) have progressively replaced the originally developed conventional competitive immunoassays. This trend MATERIALS AND METHODS has been notable in the field of pituitary hormones (”ISH, LH, Reagents and Apparatus. Unless otherwise stated, all FSH, etc.), for instance, but has not been observed with low reagents were of analytical grade from Sigma (St Louis, MO). molecular weight haptens because it is a common belief that small molecules cannot simultaneously bind two different antibodies. (1) Otsuka, M.; Yoshioka, K Physioi. Rev. 1993, 73,229-308. In this paper we show that peptides as small as substance P (11 (2) Brodin, E.; Lindefors, N.; Dalsgaard, C. J.; Theodorsson-Norheim,E.;Rosell, amino acids, i.e., Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met- S. Regul. Pept. 1986, 13,253-272. (3) Toresson, G.; Brodin, E.; Wahlstrom, A; Bertilson, LJ Neurochem. 1988, NHz) can be efficiently sandwiched between two different antibod50, 1701-1707. ies. (4) Too, H. P.; Cordova, J.; Maggio, J. Peptides 1989, 10,25-30. (5) Stjemschantz,J.; Gregerson, D.; Bausher, L.; Sears, M.J.Neurochem. 1982, SP belongs to the tachykinin family, which comprises different 38, 1323-1328. naturally occumng peptides sharing a common carboxy-terminal (6) Couraud, J. Y.; Escher, E.; Regoli, D.; Imhoff, V.; Rossignol, B.; Pradelles, moiety, Phe-X-Gly-Leu-Met-NH2,where X is either an aromatic P. /. Biol. Chem. 1985,260,9461-9469. (7) Renzi, D.; Couraud, J. Y.;Frobert, Y.; Nevers, M. C.; Gepetti, P.; Pradelles, or a branched aliphatic amino acid. The mammalian tachykinin P.; Grassi, J. In Trends in Cluster Headnche; Sicuteri, F., Vecchiet, L., family comprises substance P (SP), neurokinin A (NKA, Le., HisFanciullacci, M., Eds.; Elsevier: Amsterdam, 1987; pp 125-134. Lys-Thr-AspSer-Phe-Val-Gly-Leu-Met-NHz), and neurokinin B (8) Lee, C.; Emson, P.; Iversen, L. Life Sci. 1 9 8 0 , 27, 535-543. (9) Toresson, G.; de las Carreras, C.; Bertilson, L.; Brodin, E. Regul. Pept. 1990, (NKB, Le., AspMet-His-AspPhe-PheVal-Gly-Leu-Met-NHz) . Two CEA Service de Pharamcologie et d‘Immunologie. * UFR de Biologie.
+
0003-2700/95/0367-1617$9.00/0 Q 1995 American Chemical Society
28, 39-46. (10) Couraud, J. Y.; Frobert, Y.; Conrath, M.; R e d , D.; Grassi, J.; Drapeau, G.; Regoli, D.; Pradelles, P. J Neurochem. 1987, 49, 1708-1719.
Analytical Chemistry, Vol. 67, No. 9, May 1, 1995 1617
(lZ5I-Tj+WPwas from DuPont (Wilmington, DE). The peptides (Alal)-SP, (Ala2)-SP, (Tfl-SP and SP-(2-11) were a gift from Dr Regoli (SherbrookeUniversity, Quebec, Canada). SP, NKA, and NKB were from Neosystem Laboratories (Strasbourg, France). Peptides SP-(1-8), i.e., R-P-K-P-Q-Q-F-FSP-(1-X), i.e., R-P-K-PQ-Q-GGC; (des-Glnq-SP, i.e., R-P-K-P-Q-F-F-GLM-NH2:(desPhe?-SP, Le., R-P-K-P-Q-Q-F-GLM-NHz;and (des-Gln5-GlnS-SP, Le., R-P-K-P-F-F-GLM-NH2,were synthesized in the laboratory using a Milligen 9050 (Waters, Milford, MI) with standard protocols. Punty was checked by high-pressure liquid chromatography (HPLC). Peptides were characterized by amino acid composition. Puflcation of acetylcholinesterase (AChE) from electric organs of the electric eel Electrophorus electricus (EC 3.1.1.7), preparation of the G4 form used, and measurement of its activity were performed as previously described.ll Solid-phase EIA was performed in 9Bwell microtiter plates QmmunoplateMaxisorp, Nunc, Denmark) using automatic Titertek microtitration equipment (washer, dispenser, and reader) from Labsystems @s Ulis, France). Antiserum Production. Peptide SP-(l-X) was covalently coupled via its thiol function to carrier protein bovine serum albumin (BSA) previously reacted with the heterobifunctional reagent N-succinimidyl maleimidocaproate (SMC) in order to introduce maleimido groups able to react speciticallywith the thiol function of the peptide. Briefly, to 15 mg of BSA (223 nmol) in 0.5 mL of 0.1 M phosphate buffer, pH 7.4, was added 50 pL of 0.1 M Nethyhaleimide (NEM) dissolved in the same buffer in order to ensure total blocking of every free thiol function. After 30 min of reaction at 20 "C, 6.87 mg (22.3 pmol) of SMC in 100 pL of DMF was added. After 1 h of reaction, maleimido-BSA was purified by molecular sieve chromatographythrough a Sephadex G25 (Pharmacia, Sweden) column (2.5 x 12 cm) eluted with 0.1 M phosphate buffer, pH 6, containing 0.005 M EDTA. The absorbance (280 nm) of each fraction was measured. In parallel, incorporation of maleimido groups into BSA was indirectly measured after reaction with /3-mercaptoethanolamine @-MEA). Excess unreacted b-MEA was quantified by reaction with 5 3 dithiobis(2-nitrobenzoicacid) OTNB). Briefly, 50 pL of 0.1 M phosphate buffer or 50 pL of each collected fraction was mixed on microtiter plate with 50 pL of 5 x M ,&MEA. After 30 min of reaction at 20 "C with gentle agitation, 100 ,uL of a M DTNB solution was added, and absorbance was measured at 414 nm after an additional 5 min of reaction. The decrease in coloration observed in fractions containing maleimido-BSAis due to reaction between P-MEA and maleimido groups and allows precise calculation of the maleimido content of these fractions. Fractions containing maleimido-BSA were pooled and revealed incorporation of about 13 mol of maleimido group/mol of BSA. Aliquots of maleimido-BSA were stored at -20 "C for months without a significant loss of reactivity. Immunogen was prepared by mixing maleimido-BSA and excess SP-(l-X) (SH/maleimido lO/l). The thiol content of the peptide was measured using DTNB immediately prior to coupling. The mixture was reacted for 3 h at 30 "C. Rabbits were immunized by intradermal multiple injections of 1 mg of immunogen as previously described.l2 They were bled twice after the first booster and after the following monthly boosters. Antisera (11) Grassi, J.; Frobert, Y.; Lamourette, P.; Lagoutte, B. Anal. Biochem. 1987, 168, 436-450. (12) Vaitukaitis, J. L Methods Enzymol. 1981, 73, 46-52.
1618 Analytical Chemistry, Vol. 67,No. 9,May 1, 1995
were kept at 4 "C after addition of sodium azide (0.01%final) and tested for titer and sensitivity using an enzymatic tracer. Preparation of Enzymatic Tracers. For competitive i"unoassays, peptide W(1-X)was covalently coupled via its thiol function to AChE using N-succinimidy14(N-maleimidomethyl)cyclohexane-lcarboxylate (SMCC) as previously described for other peptides and proteins.13-16 For immunometric assays, mAb SP31 was labeled by covalent coupling of its Fab' fragmentsto AChE using SMCC as previously described.16 Enzyme Immunoassays. Two different types of enzyme immunoassay (EN)for SP were developed. Polyclonal antibodies raised against the amino terminus of the peptide were first tested in competitive immunoassay using SP-(l-X)-AChEas tracer. Later, immunometric assay involving simultaneous use of polyclonal IgG coated on the solid phase and SP31-AChE as enzymatic conjugate was developed. Unless otherwise stated, all concentrations refer to the concentration of the reagents in the initial volume (50 pL for competitive assay and 100 pL for immunometric assay) before mixture with the other reagents. AU components of the immunoreaction were diluted in the following buffer (EIA buffer): 0.1 M phosphate buffer, pH 7.4, containing 0.15 M NaCl, 0.1%BSA, and 0.01%sodium azide. (i) Competitive Assay Using SP-(l-X)-AChEConjugate. Conventional competitive immunoassays using rabbit antisera as first antibodies and SP-(l-X)-AChE conjugate as tracer were performed under the conditions previously described for other haptens.13-15 These assays were performed in 9Bwell microtiter plates coated with monoclonal anti-rabbit IgG. The total reaction volume was 150pL, each component (tracer, antiserum dilutions, and standard) being added in a 50 pL volume. SP-(l-X)-AChE conjugate was used at a concentration of 2 Ellman units/mL (for the definition of an Ellman unit, see ref 16). The working dilutions of antisera were previously determined by performing serial dilution experiments. The sensitivity of the assay was characterized by the dose of SP inducing a 50%lowering of the binding observed in the absence of competitor (BIB0 = 50%),while the minimum detectable concentration (MDC) was estimated at 80% on the same curve (BIB0 = 80%). (ii) Immunometric Assay Using SP31-AChEConjugate. Immunometric assays were performed in 9Bwell microtiter plates (or in BreakApart modules (Nunc, Denmark) when (lZ5I-Tyr-8)SP was used as standard), using as capture antibodies either a total IgG fraction of the anti-SP-(l-X)polyclonal antiserum obtained by caprylic acid puri6cation17or specific IgG collected after afhity purification (see below). In most cases we used simultaneous reaction of SP solution (100 pL of SP standard or (1z51-Tfl-SPor sample) with SP31-AChE conjugate (100 pL, 10 Ellman units/ mL) for 18 h at 4 "C (onestep procedure). In a few experiments we used a two-step procedure. In this case, SP solution (standard radioiodinated or not, and sample) was reacted alone with solid(13) Caruelle, D.; Grassi, J.; Courty, J.; Grow-Muscatelli, B.; Pradelles, P.; Banitault, D.; Caruelle, J. P. Anal. Biochem. 1988, 173, 328-339. (14) Mor, A; Delfour, A;Amiche, M.; Sagan, S.; Nicolas, P.; Grassi, J.; Pradelles, P. Neuropeptides 1989, 13, 51-57. (15) Pradelles, P.; Frobert, Y.; Crhinon, C.; Liozon, E.; Masse, A; Frindel, E. Biochem. Biophys. Res. Commun. 1990, 170, 986-993. (16) Grassi, J.; Froberf Y.; Pradelles, P.; Chercuitte, F.; Gruaz, D.; Dayer, J. M.; Poubelle, P. J. Immunol. Methods 1989, 123, 193-210. (17) Reik, L;Maines, S.; Ryan, D.; Levin, W.; Banderia, S.; Thomas, P. j . Immunol. Methods 1987. 100, 123-130.
phase antibodies in a first step (18 h, 4 "C). After washing of the plate, solid-phase bound SP was reacted in a second step with SP31-AChE conjugate (18 h, 4 "C). Whatever the procedure used, solid-phase bound AChE activity was measured using Ellman's reagent18 after a final washing of the plate. When (lZ5I-Tyr4-SP was used as standard, after detection of enzyme activity, wells of the BreakApart modules were separated, and radioactivity was directly measured in a y counter (LKB, Sweden). Precision profiles of standard curves were established by performing all measurements (nonspecific binding and standard points) in eight replicates. For each concentration, the precision of the assay was expressed in terms of coefficient of variation (CV%). The sensitivity of the immunometric assay was defined as the MDC, which corresponds to the concentration of SP producing a statistically significant increase in the binding observed in the absence of SP, i.e., nonspecific plus 3 standard deviations (p = 0.005). Mouse IgGs from nonimmune serum (25 pg/mL) were routinely introduced into the assay to reduce nonspecific binding. Affinity Purification of Specific Polyclonal Anti-SP Antibodies. Anti-SP antibodies contained in anti-SP-(1-X) antisera were purifled by affinity chromatography using SP-(1-8) peptide linked to EAH Sepharose 4B (Pharmacia). Peptide was covalently coupled to amino groups of the matrix (6 pmol of NHdmL of gel) using an activated ester. To 20 pmol (21 mg) of SP-(l-8) dissolved in 2 mL of DMF were added 100 pmol of N-hydroxysuccinimide (11.5 mg/mL) and 20 pmol of dicyclohexylcarbodiimide (4.12 mg/mL), both in DMF. After 2 h of reaction at 20 "C, the mixture was added to 3 mL of EAH Sepharose 4B previously washed with 0.5 M NaCl and equilibrated in 0.1 M borate buffer, pH 8.5. After overnight reaction at 4 "C, the gel was extensively washed with 0.1 M HC1 and equilibrated with 0.1 M phosphate buffer, pH 7.4. Antiserum (4 mL) was diluted with 12 mL of equilibrating buffer and run three times through the column. The gel was washed with 20 mL of 0.1 M phosphate buffer, pH 7.4, containing 0.5 M NaC1. IgGs adsorbed on the gel were eluted by successive washings with 8 mL of 0.1 M acetate buffer, pH 4, containing 0.5 M NaC1, and then 8 mL of 0.1 M formate buffer, pH 3, and finally 8 mL of 0.03 M HCl /acetonitrile (80/20 v/v). The pH of all these acidic fractions was adjusted to 7 by addition of 1 M phosphate buffer, pH 8, immediately after collection. Serial dilutions of all the fractions and binding experiments with SP-(l-X)-AChEconjugate were performed in order to check both the depletion of anti-SP-(1-X) antibodies in the initial antiserum and the selective desorption of specific IgG under acidic conditions. The acidic fractions presenting a significant binding capacity were pooled and dialyzed for 18 h at 4 "C against 0.1 M phosphate buffer, pH 7.4, before being used to coat microtiter plates. Preparation of Biological Samples. Two Sprague-Dawley male rats were killed by inhalation of halothane. Brain minus cerebellum was homogenized (3 g in 12 mL of 2 M acetic acid) using an Ultra-Turrax apparatus. The brain extracts were heated to 95 "C for 10 min and centrifuged at 20000g for 45 min. Supernatant was kept at -20 "C before assay. Chromatographic Characterization of SP. The immunoreactive material detected in biological samples was characterized by HPLC fractionation using a Vydac C18 column (0.39 x 25 cm) (Shandon, France) and a Waters 600E system. Elution was (18) Ellman, G.; Courtney, K; Andres, V.; Featherstone, R Biochem. Pharmacol. 1961,7, 88-95.
achieved under isocratic conditions of 23%of solvent B for 30 min (solvent A, H20/0.1%TFA;solvent B, acetonitrile/0.04%TFA) and a flow rate of 1 mL/min. Fractions of 1mL were collected, dried under vacuum using a SpeedVac apparatus (Savant, Farmingdale, NY), and resuspended in 1 mL of EM buffer before SP assay. Under these conditions, recovery of known amounts of SP was estimated to be 80-90% of immunoreactive material. RESULTS AND DISCUSSION
Establishmentof Two-Site Immunometric Assay for SP. As specified in the introduction, the aim of this study was to develop a sandwich immunoassay for substance P using as tracer a monoclonal antibody (namely SP31) which has been previously characterized as recognizing the C-terminal part of the molecule, and more precisely the sequence F-F-GLM-NH2.1° In order to obtain complementary antibodies, we directed antiserum production against the amino-terminal part of the molecule using the synthetic nonapeptide SP-(l-X),Le., R-P-K-P-Q-Q-(GGC)possessing the six first amino acids of SP, two extra glycine residues acting as spacer arm,and a cysteine residue useful for specific covalent coupling with both immunogenic carrier and enzyme label via its thiol function. Antisera with high titers (ranging from 1/10 000 to WOO 000) were obtained at the first bleedings of the three immunized rabbits. With the antisera of two rabbits, there was a significant displacement of tracer SP-(l-X)-AChEby SP in competitive experiments,with poor sensitivity @/BO(50%) = 800 ng/mL and 1.2 pg/mL, respectively). Nevertheless, crossreactivity experiments performed with different SP-related peptides assessed the strict specificity of these antisera toward the amino terminus of the molecule. No significant cross-reactivity was observed with NKA, NKF3, or SP-(2-11) (CR < l%), while 2% cross-reactivity was seen with (Alal)-SP. To check the feasibility of performing a two-site immunometric assay of SP using these polyclonal antibodies, preliminary experiments were conducted with the total IgG fraction of the antisera as obtained by caprylic acid precipitation.17 This methodology yielded substantial quantities of IgG which were coated on microtiter plates and used as capture antibody (see Materials and Methods). These first trials revealed that it was actually possible to obtain a standard curve with a signal proportional to the SP concentration (see Figure 1). At this stage, we could verify that no signal was observed when either SP fragment (SP-(2-11)) or solid phase coated with unrelated antibodies was used (data not shown). This strongly suggests that two different antibodies, one on the solid phase and one as tracer, can simultaneously bind the peptide. In these experiments,however, the sensitivity of the assay was rather poor (MDC = 100 pg/mL) due to high nonspecific binding and low slope of the standard curve. This very likely reflects the poor binding capacity of the solid phase since anti-SP antibodies represent only a minor part of the total IgG fraction obtained by caprylic acid pudication. We therefore decided to aftinity-purify anti-SP antibodies in the anti-SP-(l-X) antisera, assuming this could signijicantly improve the performance of the immunometric assay. We designed for this purpose a gel derivatized with peptide SP-(1-8). SP-(l-X)was not used at this step in order to exclude antibodies directed against the three extra residues (GGC) added for immunogen preparation. Anti-SP IgGs were specifically retained on the corresponding column since binding of the SP(l-X)-AChE tracer was no longer observed in EIA experiments after the antisera were run through the column (see Materials Analytical Chemistry, Vol. 67,No. 9,May 1, 1995
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Figure I. Two-site immunometric assay for SP. Standard curves were established as described in Materials and Methods using simultaneous addition of SP standard and SP31-AChE conjugate. After ovemight incubation at 4 "C,plates were washed before addition of colorimetric substrate (Ellman's reagent). Absorbance was measured after 2 h of enzymatic reaction. 0, Standard curve obtained using total polyclonal IgG fraction (caprylic acid purification) as capture antibody; 0, correspond to a standard curve obtained using affinitypurified antibodies as capture antibodies. The inset shows the precision profile of the standard curve obtained using affinity-purified antibodies as capture antibodies.
and Methods). On the other hand, we observed anti-SP-(1-X) immunoreactivity in fractions corresponding to acidic elutions which provided good binding dilution curves with SP-(l-X)-AChE conjugate. Optimum recovery was observed in acetate and formate eluates (see Materials and Methods). These corresponding fractions were pooled and dialyzed against 0.1 M phosphate buffer, pH 7.4. IgG concentrationwas measured by absorbance at 280 nm and estimated to be 3-5% of the total IgG fraction. Use of solid phase coated with these specific IgGs substantially increased the standard curve slope of the immunometric assay and significantlylowered nonspecifk binding (see Figure 1). This resulted in increased sensitivity, with an MDC of 3 pg/mL. Precision of the assay was also very satisfying since CVs of less than 10%were obtained in the 10-250 pg/mL range (inset, Figure 1). It is worth noting that this immunometric assay is about 100fold more sensitive than the corresponding competitive immunoassay performed with mAb SP31 (MDC = 200 pg/mLIO) and about 10 times more sensitive than the corresponding competitive immunoassay performed with our best rabbit polyclonal antiserum (MDC = 20 pg/mLI9 ) (see Figure 2). In order to demonstrate further the existence of the sandwich, standard curves were also performed using radiolabeled CBI-Tyr4SP. In preliminary experiments using a competitive immunoassay, we observed that substitution of the Phes residue of SP by a tyrosine (iodinated or not) did not affect recognition of the molecule by mAb SP31. As seen in Figure 3, the use of radiolabeled SP as standard in the sandwich assay yielded a perfect parallelism between the dose of radioactivity bound to the solid phase and the binding of the SP31-AChE conjugate. No binding was obtained when control plates coated with unrelated antibodies were used (data not shown). Surprisingly, when the immuno(19) Couraud, J. Y.InAnti-ldiotypes, recepfon,andMolecular Mimicy, Linthicum, D.,Farid. N., Eds.; Springer-Verlag: New York, 1988; pp 45-59.
1620 Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
0
Substance P (pglml) Figure 2. Comparison of the standard curves obtained with the twosite immunometric assay (0) and two competitive immunoassays performed with a rabbit polyclonal antiserum (0)and mAb SP31 (a).
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Figure 3. Two-site immunometric assay performed using (1251-TyP)SP as standard. Comparison between radioactivity and enzyme activity bound to the solid phase. A standard curve was established as in Figure 1 using (lZ51-TyP)-SPas standard instead of SP. At the end of the assay, both radioactivity (0)and AChE activity (0)bound to the solid phase were measured as described in Materials and Methods.
metric assay was performed using a two-step procedure (see Materials and Methods),i.e., when C251-Tfl-SPwas reacted alone with the solid-phase, in a fist step, little radioactivity bound to the solid phase, and very low binding of the SP31-AChE conjugate was observed in the subsequent steps. In fact, in various experiments, we observed that either (lZ5I-Tfl-SPor SP is poorly recognized by anti-SP-(1-X)antibodies unless previously reacted with mAb SP31. These results strongly suggest that binding of mAb SP31 to SP favors recognition by complementary polyclonal anti-SP-(1-X)IgG. One attractive hypothesis would be that binding of mAb SP31 to SP induces a conformational change which enhances the aftinity of complementary antibodies by demasking the epitope they recognized. It acts as if the mAb SP31-SP complex mimicks the structure of the SP(l-X)-BSAimmunogen. This point is under examination in our laboratory and will be discussed in more detail in a forthcoming publication. These experiments provide further evidences that the enzymatic signal observed in standard curves is strictly related to the binding of SP to the solid phase. In order to define more clearly the epitope recognized by antiSP-(l-X)antibodies, three synthetic analogs lacking central amino acids were tested in the sandwich immunoassay. (des-Phe7-SP was not detected, which is not surprising since we have previously
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Substance P or related products (pglml) Figure 4. Interferences induced by an excess of different SP-related products in the SP assay. 0 , A typical SP immunometric assay. The other curves show the interference induced by increased doses of NKA (O),SP-(2-11) (H), or NKB (A)added to a constant dose of SP (180 pg/mL) during the assay. In these representative competitive curves, a 50% lowering of the signal was observed for 4 ng/mL of NKA, 3 ng/mL of SP-(2-1 I ) , and 50 ng/mL of NKB, respectively.
shown that this amino acid was essential for mAb SP31 recognition.l0 More interestingly, standard curves obtained with (desGlnG)-SP were identical to those given by SP, showing that one glutamine residue is not directly involved in the sandwich. By contrast, elimination of both glutamine residues in the (des-Gln5GlnG)-SPanalog resulted in a very faint signal, thus indicating that one glutamine is located in the epitope recognized by anti-SP-(1X) antibodies. The immunometric assay proved to be highly specific since neither other tachykinins (NKA and NKB) nor SP degradation products (SP-(1-8) or SP-(2-11)) were detected by this sandwich method. However, we observed that these SP-related substances could interfere with the assay by competing with either the solid phase or the tracer antibodies, thus lowering the signal. This point is illustrated in Figure 4,where it is shown that the signal induced by a low concentration of SP can be suppressed by an excess of NKA, NKB, or SP-(2-11). We previously reported the same kind of interference in developing an immunometric assay for endothelhZ0 In this case, interference could be totally suppressed by using a two-step assay in which the analyte is reacted alone with the solid phase, the tracer antibody being added in a second step after washing of the plates. Unfortunately, this cannot be done with this immunometric assay for SP since, as mentioned above, a two-step procedure yields standard curves with a very poor slope (less than 3%of the signal observed using the one-step procedure). It is noteworthy, however, from an analytical point of view, that interference due to SP-related peptides is not very problematic, since the presence of excess enzymatic conjugate means that interference will only be observed for samples in which NKA or NKB is highly concentrated (50% inhibition observed in the ng/mL range) and in large excess with regard to SP. To our knowledge, this situation is rarely encountered in the various biological media described so far. In fact, concentrations of NKA never exceed that of SP, while NKB is currently Dfold less concentrated in both central nervous system and peripheral tissues in normal as well as in pathological situations.' (20) CrCminon, C.; Frobert, Y.; Habib, A; Maclouf, J.; Patrono, C.; Pradelles, P.; Grassi, J. J. Immunol. Methods 1993,162,179-192.
0
5
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Fractions Figure 5. RP-HPLC of a mixture of NKA, SP, and SP-(4-11) (A) and of a rat brain extract (B). Elutions were performed as described in Materials and Methods. The immunoreactivityof each fraction was measured by a classical competitive EIA (open bars) and by the immunometric assay (full bars). Under the isocratic conditions used, NKA eluted at fractions 7-9, SP at fractions 18-19, and SP-(4-11) at fractions 21 -23.
Measurement of SP Content in Biological Samples. The performance of the present two-site immunometric assay using rat brain extracts was compared with that of a conventional competitive enzyme immunoassay which uses polyclonal anti-SP antibodies directed against the C-terminal part of the mole~ule.'~ Rat brain extracts were prepared and directly assayed by the two methods. The competitive immunoassaygave a higher concentration (21.8 ng/mL) than obtained with the immunometric assay (12.8 ng/mL). We thought that this discrepancy could result from cross-reactivityof the polyclonal antiserum used in the competitive assay with different SP-relatedpeptides, and therefore we checked this hypothesis using HPLC experiments (see Materials and Methods). Equal amounts (300 pg) of SP, NKA, and SP-(4-11) were coinjected and assayed by both the competitive and immunometric assays (Figure 5A). As expected, three immunoreactive peaks corresponding to SP, cross-reactive SP44-111, and NKA were revealed by competitive immunoassay,while immunometric assay detected a single peak corresponding to SP. Rat brain extract was chromatographed under the same conditions (Figure 5B). Accordingly, competitive immunoassay revealed at least three different immunoreactive entities, while two-site immunometric assay detected a single peak. Interestingly, the quantities of immunoreactive material eluted at the position of SP were identical when measured with the two immunoassays. These results explain the difference observed in the initial quantification of SP-like immunoreactivityin the whole extract and demonstrate the remarkable selectivity of the present immunometric assay. CONCLUSIONS Using a synthetic peptide corresponding to the amino-terminal portion of SP, we obtained polyclonal antibodies and purified them by aftinity chromatography. The concomitant use of these speci6c IgGs as capture antibodies, and of an mAb specific to the carboxyterminal moiety as enzymatic conjugate, allowed us to set up a two-site immunometric assay for SP. Considering the small size of SP (11amino acids), development of a sandwich immunoassay was not straightforward, and, as far as we know, SP is the smallest molecule for which a two-site immunometric assay has been Analytical Chemistty, Vol. 67, No. 9, May 1, 1995
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described. In fact, it is clear that it is possible to make a sandwich with a 10 amino acid peptide since the (des-G1nG)-SPanalog gave identical standard curves. In addition, we have been able to establish a similar immunometric assay for NKA (10 amino acids) using a comparable approach.21The lowest limit for a “sandwichable” peptide is unknown, but we believe it to be close to 10 amino acids since it is well established (and contirmed in this study taking into account the negative result observed with the (desGlnj-Glnq-SP analog) that most peptide epitopes involve at least four or five amino acid residues. Interestingly, we observed that the binding of anti-N-terminal antibodies was dependent on the binding of mAb SP31 to the C-terminal part of SP. This result is not surprising, considering the proximity of the two epitopes recognized by the two different antibodies in the sandwich. In fact, such synergistic antibody interactions were previously observed with antigens, such as class I major histocompatibility and human growth h0rmone.2~ This phenomenon can also be encountered with small peptides, since we have made similar observations for our endothelin sandwich assay.24 The two-site immunometric assay described here is both precise (CV < 10%for SP concentration of 10-250 pg/mL) and sensitive (MDC = 3 pg/mL). In a previous report,25we have (21) Creminon, C.; Dery, 0.;Frobert, Y.; Couraud, J. Y.; Pradelles, P.; Grassi, J. Neuropepeptides 1994, 26 (Suppl l), 10. (22) Diamond, A; Butcher, G.; Howard,J.J. Immunol. 1984, 132,1169-1175. (23) Mazza, M.; Retegui, L. Immunology 1989, 67, 148-153. (24) Grassi, J.; Creminon, C.; Frobert, Y.; Habib, A; Maclouf, J.; Pradelles, P. Abstract of 111 Forum Peptides et Proteines, Biarritz 1993, France. (25) Pradelles, P.; Grassi, J.; Creminon, C.; Boutten, B.; Mamas, S. Anal. Chem. 1994, 66, 16-22.
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described an immunometric assay for substance P using the new SPIEIA (solid-phase immobilized epitope immunoassay) methodology. This approach is based on the use of a single mAb for capturing and detecting solid-phase bound SP. The sensitivity achieved with SPIEIA is similar (MDC * 6 pg/mL) to the one obtained with the current two-site immunometric assay, but important cross-reactivities are observed with NKA, NKJ3, and Cterminal fragments. This is not surprising, since this assay is performed with mAb SP31. Very clearly, above all, the main advantage of this new two-site immunometric assay over all published immunoassays of SP lies in its remarkable specificity. This is simply explained by the concomitant use of two antibodies whose binding sites probably cover almost the entire SP molecule, while other immunoassays are performed using antibodies (polyclonal or monoclonal) directed only to a restricted portion of SP, either the C-terminal or less frequently the N-terminal part of SP. Hence, this two-site immunometric assay is an efficient and useful tool for determining SP concentrations in biological samples. ACKNOWLEDGMENT This work was supported by Le Commissariat a l’Energie Atomique (CEA, France). The expert technical assistance of P. Lamourette, M. C. Nevers, and M. Plaisance throughout this work was greatly appreciated. Received for review October 24, 1994. Accepted February 9, 1995.@ AC941043Z e.
Abstract published in Advance ACS Abstracts, March 15, 1995.
