Anal. Chem. 1994,66, 16-22
Immunometric Assay of Low Molecular Weight Haptens Containing Primary Amino Groups Phlllppe Pradelles,’ Jacques Grassl, and Chrlstophe Crhlnon Laboratoire d’Etudes Radioimmunologiques, Service de Pharmacologie et d’lmmunologie, DRIPP, Commissariat 3 I’Energie Atomique, CE/Saclay, 9 1 19 1 Gif sur Yvette Caex, France Bruno Boutten and Suzanne Mamas Laboratoire de Pr6dhveloppement des Rhactifs Immunologiques, Institut Pasteur, 28, rue du Docteur Roux, 750 15 Paris, France
A new enzyme immunometric assay of small haptenscontaining primary aminogroup (thyroxine, MW 777; substance P, MW 1347; endothelin, MW 2492) is described. The procedure involves different sequential steps: (1) immunocapture of the haptens (standard or sample) by monoclonal anti-hapten antibodiescoatedon 96-well microtiter plates; (2) cross-linking of haptens via their amino groups to the wells using homobifuncdional reagents (glutaraldehyde or disuccinimidyl suberate); (3) denaturing treatments (HCI or methanol); (4) measurement of linked epitopeusing the same monoclonalantihapten antibodieslabeled with acetylcholinesterase. A minimal detectable concentration in the 4-10 fmoL/mL range was observed. Each assay appeared to be 70-200 times more sensitive than conventional competitive enzyme immunoassay using the same monoclonal antibody-coated plate technology and acetylcholinesterasehapten conjugates as enzymatic tracers. Precision and specificity were very satisfying. Good correlation was noted between this assay and the competitive assays performed for different biological samples (plasma, tissues, or supernatant cell culture).
Since the 1960s, when pioneering work established the basic rules of competitive analysis using specific antibodies’ or binding proteins,2 various technologies have appeared in the field of immunoassays. Variety is seen in the choice of label (radioactive, enzymatic, fluorescent, etc.), separation methods (precipitation of antigen-antibody complexes, coated solidphase antibody technology, etc.), and procedures. Nevertheless, two general principles have emerged: the competitive and the noncompetitive immunoassay. The competitive immunoassay uses a labeled antigen or hapten and a limiting concentration of the corresponding specific antibodies. Various noncompetitive immunoassays have been described, but the more widespread method is the two-site immunometric assay which uses two different antibodies to link the antigen. One is immobilized into a solid phase, while the other is labeled and used as tracer antibody. In this kind of assay, both antibodies are used in excess. This technology leads to increased sensitivity compared to a competitive assay using the same antib~dies.~ Unfortunately, low molecular weight (1) Yalow, R. S.; Berson, S. A. J. Clin.Inuesr. 1960, 39, 1157-1175. (2) Ekins, R. P. Clin.Chim. Aero 1960, 5, 453-459. (3) Ekins, R. P. Radioimmunoassays and Relared Procedures in Medicine; IAEA Vienna, 1977; Vol. I, pp 241-268.
16
Ana&ticalChemlstry, Vol. 66,No. 1, January 1, 1994
haptens cannot be determined by two-site immunometric assays because these molecules are too small to allow simultaneous binding of two different antibodies. As a consequence, so far, most of immunoassays concerning small haptenic substances have a sensitivity currently inferior to those observed with larger molecules. Recent attempts have been made to develop two-site immunoassays for small molecules. Ishikawa’s group4has developed a new method of measuring haptens with amino groups at attomole concentrations. Briefly, in this new approach, hapten is biotinylated and, after removal of unreacted biotin and other biotinylated substances, is measured using anti-hapten Fab’horseradish peroxidase conjugate and streptavidin-coated polystyrene balls. We report here a new method for sensitive and reliable enzyme-immunometric assay of hapten with an amino group: solid-phase immobilized epitope immunoassay (SPIE-IA). Basic principles are as follows: a specific monoclonal antihapten antibody (mAb) is immobilized on a 96-well microtiter plate. After immunological reaction with hapten and washing, the trapped molecule is covalently linked to the plate by means of a homobifunctional cross-linking reagent. Ater the washing and denaturing treatment, the “released” epitope covalently coupled to the solid phase can react with the same acetylcholinesterase-labeled monoclonal antibody used as tracer. Using a neuropeptide (substance P, SP), a thyroid hormone (thyroxine, T4), and a larger peptide (human endothelin 1, ET), the sensitivity, specificity, precision, and validity of this procedure have been evaluated. Each step of the new procedure is discussed and its application to other molecules, including antigens, is considered.
EXPERIMENTAL SECTION Reagents. Unless otherwise stated, all reagents were from Sigma (St. Louis, MO). Glutaraldehyde (25%) was from Merck (Darmstadt, Germany), disuccinimidyl suberate (DSS) was from Pierce (Rockord, IL), and substance P (SP), human endothelin 1 (ET), big endothelin (big ET-l), VIC peptide, and (Ala1*3,11J5)-ET-1 were from Neosystem Laboratories (Strasbourg, France). [1Z51]Thyroxine(5550 kBq/pg) was from Dupont (Wilmington, DE). Endothelins (ET-2 and ET3) were from Novabiochem (Clery en Vexin, France). (4) Ishikawa, E.; Tanaka, K.; Hashida, S. Clin. Chim. Acta 1990, 194, 51-72. 0003-2700/94/036&0016$04.50/0
0 1993 American Chemical Soclety
Sarafotoxin 6b analog (Thr2, Ile19) was synthesized by Dr. Lam Thanh (DIEP, CE Saclay, France) and (L-Ala) SP analogues were a generous gift of Dr. Regoli (CHUS, Sherbrooke, Canada). Radioimmunologicalmeasurements of T4 were performed using a commercial kit from CIS international (Saclay, France) following the recommendations of the manufacturer. Monoclonal Antibodies (mAb). Monoclonal anti-SP antibody (SP3 1) was obtained as described previously.’ Monoclonal anti-T4 antibody (no. 24488) under ascitic fluid, purified by the caprylic acid method? was kindly supplied by the Institut Pasteur (Paris, France). Monoclonal anti-ET antibody (Endo 4) was obtained and purified as described previ~usly.~ Apparatus. Solid-phasecompetitiveenzyme immunoassay and SPIE-IA were performed using Titertek microtitration equipment from Labsystem (Helsinki, Finland), including an automatic plate washer (microplatewasher 120), an automatic plate dispenser (Autodrop), and an automatic plate reader (Multiskan MCC). Microplate (Maxisorp) and BreakApart modules for lZ5Iradioactivity measurements were from Nunc (Denmark). Enzyme Label. Acetylcholinesterase (AChE; EC 3.1.1.7) was purified from electric eel (Electrophorus electricus) by affinity chromatography.8 The tetrameric form of the enzyme (G4 form)9 was used for labeling of haptens and antibodies. Preparationof EnzymaticTracers. Enzymatic tracers were obtained by covalent linkage of haptens or Fab’ fragments of monoclonal antibodies to the G4 form of AChE. The preparation of SP and ET-AChE conjugates have been described p r e v i o ~ s l y . ~T4-AChE J~ conjugate was prepared as follows: 100-pL aliquots of N-hydroxysuccinimide (1 pmol) and N,N’-dicyclohexylcarbodiimide (1 pmol) in anhydrous dimethylformamide (DMF) were successively added to 100 pL (1 pmol) of tetraiodothyroacetic acid in DMF. After 18 h at room temperature, 43 pL (125 nmol) of N-hydroxysuccinimidyl ester was added to 300 pL of G4-AChE (100 pg) in 0.1 M phosphate buffer (pH 7). The reaction was allowed to proceed for 30 min at room temperature and was stopped by addition of 200 pL of EIA buffer. Purification and storage of the conjugate were performed as described el~ewhere.~ Purified monoclonal antibodies were labeled by covalent coupling of the Fab’ fragments to AChE using N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).I1 Competitive EIA Procedures. All assays were performed in the following buffers. For SP EIA: 0.1 M phosphate buffer, pH7.4,containing0.15 MNaCl, 10-3M EDTA,O.l%bovine serum albumin, and 0.01% sodium azide (EIA buffer). For ET EIA: the same buffer with 0.1% Tween 20. For T4 EIA: 0.05 M veronal buffer (pH 8.6), 0.1% bovine serum albumin, (5) Couraud, J. Y.; Frobert, Y.; Conrath, M.; Renzi, D.; Grassi, J.; Drapeau, G.; Rcgoli, D.; Pradelles, P. J. Neurochem. 1987, 49, 1708-1719. (6) Reik, L.;Maines, S.;Ryan, D.; Levin, W.; Bauderia,S.;Thomas,P. J . Immunol. Methods 1987, 100, 123-130. (7) Creminon, C.; Frobert, Y.; Habib, A,; Maclouf, J.; Patrono, C.; Pradellcs, P.; Grassi, J. J . Immunol.Methods 1993, 162, 179-192. (8) Massoulic, J.; Eon, S.Eur. J . Biochem. 1976, 68, 531-539. (9) Pradelles, P.; Grassi, J.; Chabardcs, D.; Guiso, N. Anal. Chem. 1989, 61, 447-453. (IO) Rcnzi, D.; Couraud, J. Y.; Frobert, Y.; Nevets, M. C.; Geppetti, P.; Pradellcs, P.; Grassi, J. In Trends in Cluster Heodache; Sicuteri, F., et al., Eds.; Elsevier Science Publishers: Amsterdam, 1987; pp 125-134. (1 1) Grassi, J.; Frobert, Y.; Pradelles, P.; Chercuittc, F.; Gruaz, D.; Dayer, J. M.; Poubelle, P. J . Immunol.Methods 1989, 123, 193-210.
monoclonal anti-hapten
LQLQw
0
Step 1: coating
BSA
rNH2
hapten
Step 2: immunological reaction
IdQQlQI Step 3: epitope immobilization
Step 4: euitoue release
Ea
+
3
k
Fab’ labeled with AChE
Step 5 : tracer addition
+ acetylthiocholine and Ellman reagent Stet, 6: Enzvmatic reaction and colorimetric assay Figure 1. Different steps of the SPIE-IA procedure.
and 0.01% sodium azide. Competitive EIAs were performed as describede l ~ e w h e r e .Briefly, ~ ~ ~ the 96-well microtiter plates were coated with affinity-purified goat anti-mouse IgG antibodies as described previo~sly.~ The total reactionvolume. was 150 pL (50 pL of each component: enzymatic tracer, mAb, standard). Hapten-AChE conjugates were used at a concentration of 1 Ellman unit (EU)/mL (for Ellman unit definition, see ref 9). The working dilutions for mAbs determined by antibody dilution experiments were 50, 250, and 100 ng/mL for SP, T4, and ET EIA, respectively. SPIE-IA Procedures. Each step (Figure 1) was preceded by a washing procedure9 using five cycles with washer 120 andO.O1 M phosphatebuffer (pH 7.4) containingO.O5%Tween 20 as washing buffer. Step I. Coating. The 96-well microtiter plates werecoated with mouse monoclonal anti-hapten IgG (200 pL, 10 pg/mL in 0.05 M phosphate buffer (pH 7)) for 18 h at 22 OC and saturated with 300 pL of EIA buffer. Once coated, plates can be kept for months at +4 OC. Step 2. Immunological reaction. (SP or ET assay): incubation for 18 h at +4 OC of standard or sample (100 pL) in EIA buffer. (T4 assay): incubation for 1 h at 22 ‘C of standard or sample in EIA buffer. In the case of T4 plasma assay, sample (10 pL) was added to 100 p L of EIA buffer containing 0.4% 8-anilino-1-naphthalene sulfonic acid (ANS). T4-free plasma was used for standard curve construction. Step 3. Epitope immobilization. (SP assay): addition of 100 p L of 0.1 M borate buffer (pH 9) and 10 p L of 25% Ana&tibalChemWy, Vol. 68, No. 1, January 1, 1994
17
glutaraldehyde. One-hour reaction with gentle stirring at 22 OC. Washing and addition of 250 pL of stopping solution (4 mg/mL NaBH4) for 1-h reaction at 22 OC. (T4 or ET assay): addition of 250 p L of 0.1 M borate (pH 9) and 10 pL of DSS (1 mg/mL in anhydrous DMF). Ten-minute reaction with gentle stirring at 22 OC. Step 4. Epitope release. (SP assay): addition of 250 p L of HCl (0.1 N) for 10-min reaction at 22 "C. (T4 or ET assay): addition of 250 p L of methanol for 10-min reaction at 22 OC. Step 5. Tracer addition. (SP or ET assay): addition of 100 pL of monoclonal anti-hapten labeled with acetylcholinesterase (5 EU/mL) diluted in EIA buffer. Incubation for 18 h at +4 OC. (T4 assay): addition of 100 pL of acetylcholinesterase-labeled monoclonal anti-T4 (5 EU/mL) diluted in EIA buffer. Incubation for 1 h at 22 OC. Step 6. Enzymatic reaction and colorimetric assay: addition of Ellman's reagentg (200 pL/well). Thirty minutes to 1 h of enzymatic reaction and reading at 414 nm. SPIE-IA Procedure for T4 Assay Using [1251]T4as Standard and BreakApart Modules as Solid Phase. The protocol for this assay was the same as that described above. After enzymatic staining, reading, and washing, each well was separated from the module and the radioactivity was measured in a y radioactivity counter. Calculation. In competitive assay, B and BOrepresent the bound enzyme activity measured in the presence or absence of competitor, respectively. The results are expressed in terms of BIB0 X 100 as a function of the logarithm of the concentration. Unless otherwise stated, all measurements for standards or samples were made in duplicate, and in quadruplicate for Bovalues. In SPIE-IA, the results are expressed in terms of absorbance units (AU) as a function of the dose. In each assay, nonspecific binding was determined by replacing the incubation mixture containing specific antibody (competitive assay), or either standard (SPIE-IA assay), by EIA buffer. The minimum detectable concentration (MDC) was taken as the concentration of standard inducing a significant decrease (3 standard deviations) in Bo (competitive assay) or increase in nonspecific binding (SPIE-IA or two-site immunoassay). Precision profiles of standard curves were established by performing all measurements (nonspecific binding and standard points) eight times and were expressed in terms of coefficient of variation (% CV). Specificity Measurements. The specificity of each assay was checked by testing its capacity todetect structurallyrelated compounds. This was achieved by establishing for each of these compounds the corresponding standard curve. Results were expressed in terms of percentage cross-reactivity (% CR). For competitive assays, this corresponds to the ratio (in percentage) of standard and analog concentrations at BIB0 = 50%. For SPIE-IA, CR 7%were arbitrarily defined as the ratio (in percentage) of the dose of standard and analogue producing a 1 AU signal. Preparation of Biological Samples. Brain and spinal cord samples of mouse or rat were extracted in 0.1 N HCl and treated by heat deproteinization (95 OC, 15min). The buffered supernatants were tested (SPassay). Normal human plasma (10 pL) was directly assayed (T4 assay). Human umbilical vein endothelial cells were prepared and stimulated by 18 Ana!vticalChemisW, Vol. 66, No. 1, January 1, 1994
"i I
0.9-0.8-0.7-
0.8--
A. U. 0.5-
0
100
200
300
460
500
860
Substance P (pg/mL) Flgurr 2. Standard curve for substance P SPIE-IA. Inset: precision proflle of substance P SPIE-IA (0)and competitive enzyme Immunoassay (+).
thrombin as described previ~usly.~ Cell supernatants were directly assayed for ET.
RESULTS SPJE-IA for Substance P. A routine standard curve for SP measurement is presented in Figure 2. MDC was 6 pg/mL compared to 900 pg/mL for a competitive assay using the same monoclonal antibody (data not shown). Comparison of the precision profiles (see inset in Figure 2) obtained with the two assays revealed increased precision with SPIE-IA (CV less than 5% in 30-1000 pg/mL range). Standard curves established with different SP analogues (Table 1) revealed that SPIE-IA and thecompetitive assay present very similar specificities. In the same way, there was a good correlation between the two types of measurements performed in rat and mouse tissues extracts (Table 2). The effects of each chemical agent added during the different steps of SPIE-IA are shown in Figure 3. Both the action of glutaraldehyde and HCl treatment appeared essential to obtain a significant immunoreactivity in subsequent steps. The use of NaBH4 after glutaraldehyde cross-linking allows us to lower the nonspecific binding of tracer antibody by reduction of remaining aldehyde group. SPIE-IAfor Thyroxine. The sensitivity of T4 assay is presented in Figure 4. The MDC (14 pg/mL) was by far lower than that obtained with the competitiveassay using the same monoclonal antibody (1000 pg/mL) (data not shown). CV was less than 15% in the 20-1000 pg/mL range (Figure 4). The specificity of this assay was checked by the use of different related compounds: D-thyroxine, L-triiodothyronine, L-diiodothyronine, and 3,3',5,5'tetraiodothyroacetic acid (Tetrac). Respective CR values of 100% 356,096, and 0%were observed. The same values were obtained with the competitive assay, with the exception of Tetrac, which cross-reacted in this assay (1 5%) but not in SPIE-IA due to the lack of an amino group involved in the covalent linkage. When 30 normal human plasma samples
okmd WM COmpdUVO EIUYIM 1-y
TI#r 1. compul#n d AnrkgurrorDulv~
O1 SPI-EIA
for
PT a d
w (~-Ala)gP CR (%)
1 Arg
Ala Arg
Arg Arg Arg
Arg Arg Arg Arg Arg
2
3 Lys Lys Lys Ala Lys Lys Lys Lye Lys Lys
Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Pro
Lys
4
6
6
7
8
9
10
11
Pro Pro Pro Pro Ala Pro Pro Pro Pro Pro Pro
Gln
Gln Gln Gln Gln Gln Ala Gln Gln Gln Gln Gln
Phe Phe Phe Phe Phe Phe Ala Phe Phe Phe Phe
Phe Phe Phe Phe Phe Phe Phe Ala Phe Phe Phe
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Leu Leu Leu Leu Leu Leu Leu Leu Ala Leu Leu
Met Met Met Met Met Met Met Met Met Ala Met
Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln
compet away
SPI-EIA
100 86 100 68 85 89 0.2 102 19 22 0
100 67 56 46 66 46 4.6 137 27 34
NH2(SP) NH2 NH2 NH2 NH2
NH2 NH2 NH2 NH2 NH2 COOH
0
1.4-
0 1.2-
samples
competitive assay
SPI-EIA
rat brain rat spinal cord mouse brain mouse spinal cord
76.6 61.3 70 32.1
70 61.4 76.6 33.4
/
1--
0.8--
A.U. 0.6
1
0.2
Thyroxine (pg/mL) O!
A.U.
0
200
400
800
800
1000
1200
1400
Thyroxine (pg/mL) Flgure 4. Standard curve for thyroxine SPIE-IA. Inset: precision profile.
0
Substance P (pg/mL) Flgure 3. Effect of sequential additkn of chemlcei agents during substance P SPIE-IA (0) SlutaraMyde N a w HCI; (*) glutaraldehyde Hci (withoutNaBH,); (+)HCialone; ()ogiutaraldehyde NaBH, without HCI.
+
+
+
were assayed by this procedure, a mean value of 64.8 f 22.1 ng/mL was found, in agreement with values obtained using radioimmunological measurements (60.2 15.4 ng/mL, see methods) and in accordance with values determined by other techniques.12 The effects of various denaturating agents were tested during the epitope release step (step 4). The results presented in Figure 5 show that methanol and 10%formic acid were the most effective in this case. The use of I2SI radioiodinated thyroxine in this experiment allowed quantification of T4 (12) Bartalena, L.;Marioti, S.;Pinchera, A. In Radioimmunwssuy in Bosic u d CItnicul Phwmucology; F’atrono, C., et al., Eds.; Springcr-VerlagPublishers: Berlin, 1987; pp 401-431.
binding to the well at each step of the procedure. We observed that more than 70% of the radioactivity was immobilized during the immunological capture and conserved in the wells throughout the different steps (DSS treatment, epitoperelease with methanol, immunological reaction with tracer antibody). It is worth noting that if DSS was omitted at step 3, radioactivity bound during step 2 was fully removed by methanol treatment. In addition, as observed with SP,no significant immunoreactivity was observed in subsequent steps (results not shown). These data strongly suggest that the role of DSS is to covalently bind T4 onto the solid phase. SPIE-IAfor Endothelin. The MDC of SPIE-IA was 20 pg/mL, illustrating 250-fold greater sensitivity than the corresponding competitive enzyme immunoassay using the same monoclonal antibody.7 It is worth noting that a MDC of 1 pg/mL was determined for a two-site enzyme immunometric assay using the same tracer anti bod^.^ Specificity studies using ET-related peptides are shown in Table 3 and compared with those of the two-site enzyme immunometric assay.7 Logically, high cross-reactivity (63%) was observed with big ET- 1, a putative ET- 1 precursor known to be present in many biological samples. In this case, SPIE-IA appeared less specific than the two-site assay, which does not crossreact with big ET-1 since this molecule is not recognized by Ana!~ticalChemkby, Vol. 66, No. 1, Januery 1, 1994
19
0.1N BCI
L -t
+
40
0.1
0.2
0.3
0.4
0.5
I 0.5
A. U. Figure 5. Effect of denaturing agents during epkope release step of SPIE-IA for thyroxine.
the capture a n t i b ~ d y .Taking ~ into account this point, it is not surprising that SPIE-IA led to overestimation of ET-like material in the biological samples tested (Figure 6).
DISCUSSION The new procedure for immunometric assay of hapten with an amino group described here13 is based on two general findings. The first is that specific anti-hapten antibody coated on microtiter plates ensures an efficient capture of the hapten through a specific immunological reaction. The second is that a cross-linking agent permits covalent linkage of the hapten to the solid phase and that subsequent denaturing treatments allow epitope recognition by the same antibody used as tracer and added in subsequent steps. The first finding is now well established in the field of immunoassay, the principle of capture being widely used in two-site immunometric assays for instance. The second finding is more dependent on hapten structure. Each step of the SPIE-IA procedure (Figure 1) has to be discussed in light of knowledge acquired during development of this assay. Step 1. Coating. Passive immobilization of antibody on polystyrene microtiter plates is communly used in immun o a s s a y ~and , ~ ~the experimental conditions are well-known to users. Step 2. Immunological Reaction. As our aim was to develop highly sensitive assays, high-affinity antibodies were thus required. The Kd values of monoclonal antibodies used in this study were 25,0.25, and 0.07 nM for anti-ET, anti-T4, and anti-SP antibodies, respectively. This represents rather low affinities whencompared to the highest affinities measured for antibodies (Kd in the picomolar range). It is well-known that such high-affinity antibodies are currently found in good antisera. For instance, we have produced in our laboratory an anti-SP rabbit polyclonal antiserum allowing MDC close to 10 pg/mL when used in competitive enzyme immunoassay (13) Pradelles, P. Fr. Patent 93, 00869, 1993. (14) Butler,J.E.;Ni,L.;Ntssler,R.;Joshi,K.S.;Suter,M.;Rosenbcrg,B.;Chang, J.; Brown, W. R.; Cantarero, L. A. J. Immunol.Methods 1992,150, 77-90.
20
AnaMimlChemistry, Vol. 66, No. 1, January 1, 1994
(compared to 6 pg/mL for SPIE-IA and 900 pg/mL for competitive EIA using the same monoclonal antibodylo). It would thus be tempting to use affinity-purified polyclonal antibodies as both capture and tracer antibodies. This possibility, which could significantly increase the sensitivity of SPIE-IA, is presently under examination in our laboratory. This approach, however, presents some drawback linked to the difficulty of getting high amounts of specific antibodies by the use of immunoaffinity chromatography. Finally, it is clear that the availability of high-affinity monoclonal antibodies is the best way to take full advantage of the SPIE-IA technique. Step 3. Epitope Immobilization. Antibody interaction plays an essential role in this step. Indeed, the reaction conditions (medium, ionic strength, pH, time, etc.) must not disrupt the immunological complex during the cross-linking step. In addition, the chemical function (amino group in this study) borne by the hapten and reacting with the cross-linking agent must not be implicated in the epitope structure. Currently, this criterion is met if the chemical function is the same as that used during coupling to antigenic carrier to prepare the immunogen. For instance, the immunogen for SP was prepared using the homobifunctional reagent difluorodinitrobenzene (DFDNB),whereas glutaraldehyde was used for T4 or ET immunogen preparation. It is very likely that the role of the cross-linking reagent used at this step is to covalently immobilize the hapten onto the solid phase. This is clearly demonstrated by experiments performed with radioidinated T4 since we observed that the hormone is fully removed from the solid phase in step 4 when DSS is omitted in step 3. In addition, no significant immunoreactivity is observed in subsequent steps under these conditions (results not shown). The same conclusion can be indirectly derived from experiments presented in Figure 3 for SP. Accordingly, the reagents used in this step (glutaraldehyde and DSS) are known to react with primary amino groups and have been described previously as efficient peptide and protein crosslinkers. *,16 During SPIE-IA, the hapten is probably cross-linked with proteins surrounding the antigen-antibody complex formed during the capture step, i.e., paratope of the antibody, whole antibody, or protein of saturation (BSA). So far, we have no direct experimental evidence allowing us to discriminate between these different possibilities. This point is under examination in our laboratory. Even if glutaraldehyde has also been described as an efficient reagent for direct coupling of hapten to polystyrene microtiter plates,” it is very unlikely that haptens could be directly coupled to polystyrene since it is well-known that, under the conditions used here, the plastic is fully covered with proteins.14 Finally, in the case of a hapten with more than one amino group, if one of them is located on the epitope structure, it will be interesting to investigate whether the immunocomplex is able to protect this amino group from the action of crosslinking agents. (15) Richards, F. M.;Knowlcs, J. R. J. Mol. Biol. 1968, 37, 231-233. (16) Montcsano, L.; Cawley, D.; Herschman, H. R. Biochem. Biophys. Res. Commun. 1982,109.7-13. (17) Ordronneau, P.; Abdullah, L.H.; Petrusz, P. J. Immunol. Merhods 1991,142, 169-176.
T1#r 9. C r o r r - R ~ ~ ~ t lOf~ H EndothOUn-ROktOd y Plpt#.r
Ww T W M O
I"n0nnM0
CSCSSLMDKECVYFCHLDIIW ET-1
100
100
100
97
66
6
100
100
100
a2
co.01 co.01
63
I
CTCFTYKDKECWYCHLDIIW ET-3 I
I
CSCNS WLDKECVYFCHLDIIWVIC peptide I
I
CTCKDMTDKECLYFCHQDIIWsarafotoxin 6-banalogue I
I
ASASSLMDKEAWFAHLDIIW Ala (1,3,11,15)-ET-l CSCSSLMDKECVYF'CHLDIIW VNTPEHVVPYGLGSPRS big ET-1 I
SPI-EL4
I
CSCSS WLDKECWFCHLDIIW ET-2 I
~ andy 8PIE-IA
CR (%) two-site immunoassay
products (aminoacid sequence and name) 1
h
38
I
I 7
Alternatively, the critical role of this step could be attributable to other phenomena such as the cleaning or remodelingof the protein layers which have been disorganized during the cross-linking treatment, thus confining the hapten in a protein network and making it unaccessible for tracer antibody. Step 5 and Step 6. Incubation with Labeled Antibody, Enzymatic Reaction, and Colorimetric Assay. No special observations have to be made concerning these steps since they follow the classical procedure used in enzyme immunometric assays. Various antibody concentrations must be tested to ensure full immunoreactionwith immobilized hapten.
1 i, 10
1
12
14 15 16
Samples
Fburr 6. Correlation studies of endothelln content of endothelial cell supernatant assayed by SPIE-IA (open bar) and two-slte enzyme immunoassay (closed bar).
this step is strictly necessary to obtain a significant binding of tracer antibody in the following steps. We call it the "epitope release step" because we assume that its role is to dissociate the interaction between hapten and capture antibody and to present the hapten to the tracer antibody. In addition, it is very likely that such denaturating treatments can inactivate capture antibody,thus preventing competition between capture and tracer antibodies in subsequent steps. The choice of denaturing agent for more efficient release may depend on the characteristics of hapten-antibody interaction and thus vary depending on the antibody used. This is the reason why various denaturing agents have been tested, such as acids, bases, organic solvents, and detergents. In fact, variations have been observed since for SP optimal results were obtained using 0.1 N HCl while methanol proved to be more efficient for T4 and ET assays (see methods and Figure 5 ) . In order to support the hypothesis of epitope release, we are now investigating the correlation between the ability of various agents for releasing antibody from an affinity column in correlation with their efficiency in SPIE-IA.
CONCLUSIONS We have described a new enzyme immunometric assay for small haptens possessing a primary amino group. The three applications presented here clearly demonstrate the main characteristics of this assay, which appears sensitive, precise, and specific. The main advantage of this technique is that it involves the same antibody for the capture and revelation steps, thus avoiding the binding compementarity essential for two-site immunometric assays. When SPIE-IA and conventional competitive immunoassay are compared using the same monoclonal antibody, the former exhibits much increased sensitivity, mainly due to the immunometric format of the test. It is worth noting however that, in some cases, similar sensitivity can be obtained with competitive immunoassays using high-affinity antisera. Clearly, the use of affinitypurified polyclonal antibodiesor the availabilityof high-affinity monoclonal antibodies could further increase the sensitivity of SPIE-IA. In addition, some metabolites or hapten derivatives lacking the amine function that is essential for linkage in the epitope immobilization step and which easily cross-react in the competitive assay were not assayed in SPIE-IA. From this point of view, the SPIE-IA would be more specific. In other situations, as exemplified with the ET1 assay (see above), SPIE-IA leads to a loss in specificity. Extension of this methodology to antigens seems possible since preliminary encouraging results have been obtained with human recombinant interleukin-4 and Ras-p21 oncogen (MWs = 20 000) (preliminary results). In this context, it is very likely that SPIE-IA would not provide more sensitive immunoassaysthan the conventionaltwo-site immunometric assay (see the results AnaWcal Chemistry, Vol. 66, No. I,
1, lQ94
21
of the ET1 assay for instance). Here, the advantage of SPIEIA would be to allow the establishment of sensitive immunometric assays using a single monoclonal antibody. This is important from a practical point of view because, in many situations, it is very difficult to obtain two complementary monoclonal antibodies providing a good immunometric assay while one individual mAb can be available from a commercial source or a public collection (American Type Cell Collection, for instance).
22
Ane!YtICal Chemlstty, Vol. 66, No. 1, January 1, 1994
ACKNOWLEDQMENT We thank Y. Frobert, P. Lamourette, and M. Plaisance for the monoclonal antihapten antibody preparation and for their technical assistance. This work was supported by grants from the Commissariat A 1'Energie Atomique, France. Received for
June Q, 19Q3, Accepted
8, 1QQ3.a
Abstract published in Aduoncs ACS Abstracts, November 15, 1993.
