Anti-Immune Complex Antibodies Enhance Affinity and Specificity of Primary Antibodies (Affinity Labefing/Immunoassay/Molecular Recognition/Anti-Idiotype) EDWIN F

Proc. Nati. Acad. Sci. USA Vol. 90, pp. 1184-1189, February 1993 Colloquium Paper

This paper was presented at a coUoquium entitled "Molecular Recognition," organized by Ronald Breslow, held September 10 and 11, 1992, at the National Academy of Sciences, Washington, DC. Anti-immune complex antibodies enhance affinity and specificity of primary antibodies (affinity labefing/immunoassay/molecular recognition/anti-idiotype) EDWIN F. ULLMAN*, GARY MILBURNt, JOHN JELESKOt, KESAVAN RADIKA, MARCEL PIRIO, THOMAS KEMPE, AND CARL SKOLD Research Department, Syva Co., Palo Alto, CA 94303

ABSTRACT Antibodies have previously been described with the bound antigen, there should be an added recognition that enhance the binding of a second antibody to its antigen. element that could provide increased binding specificity. In The origin of this effect has been variously ascribed to binding this report we describe an anti-IC antibody, referred to as to a neodeterminant on the Fc region, to a combined determi- Ab2, which permits realization of these opportunities. nant representing portions of the second antibody and the immunogen, and to a ligand-induced conformation of the Fab MATERIALS AND METHODS fragment. This paper describes an antibody that recognizes an immune complex of an antibody to tetrahydrocannabinol Chemicals. Certified high-purity samples oftetrahydrocan- (THC). The antibody binds the anti-THC antibody at an nabinol (THC) and its derivatives (Fig. 1) from Research epitope recognized by an anti-idiotype antibody that is capable Triangle Institute (Durham, NC) were provided by the Na- of blocking THC binding. The ability of various THC deriva- tional Institute on Drug Abuse (Rockville, MD). A9-[3H]THC tives to enhance or inhibit binding taken together with equi- (2.6 Ci/mmol; 1 Ci = 37 GBq) was prepared by New England libria and kinetic data support a model in which the anti- Nuclear by Rh-catalyzed exchange with [3H1H20, purified by immune complex antibody interacts through adventitious bind- silica-gel thin layer chromatography, and stored in ethanol at ing to pendant groups on the THC derivatives. This type of -200C. interaction offers the opportunity to increase the sensitivity and m-Phenoxybenzoylamino-2-ethylsulfonic acid (PBES) was specificity of immunoassays beyond the limits imposed by prepared from the acid chloride of m-phenoxybenzoic acid normal antibody binding. The implications of these rmdings and tauric acid. Conjugates of 11-nor-9-carboxymethyloxi- with regard to earlier observations of anti-immune complex mino-6a,7,8,9,10,10a-hexahydrocannabinol (CMO) with antibodies are discussed. Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (THC-G6PDH) and with keyhole limpet hemocyanin were prepared by reaction of these proteins with the N-hy- It has long been recognized that antibodies elicited in re- droxysuccinimide (NHS) ester of CMO (from Syva). A8- sponse to an immunogen can themselves serve as immuno- [14C]THC-CO-P-Ala-/3-Ala was prepared by reaction of the gens in the same animal (1). The anti-idiotypic antibodies NHS ester of 11-nor-A8-THC-9-carboxylic acid (A8-THC-9- arising from this secondary response have been postulated as COOH) with f8-[1-14C]alanine to give A8-[14C]THC-9-CO-f- a critical control mechanism of the immune response (2). Ala followed by coupling with j3-alanine and N,N'- More recently, it has become clear that several types of dicyclohexylcarbodiimide. The NHS ester of A8-THC-CO- anti-idiotypic antibodies can arise. Antibodies can be di- (-Ala was combined with horseradish peroxidase (HRP) to rected toward nonbinding portions of the hypervariable re- prepare the THC-HRP conjugate. gion (anti-idiotope) or toward the binding region (anti- Antibody Preparation. Four-week-old female BALB/c paratope) (3). Additionally, there are several reports of mice were initially immunized i.p. with 100-250 mg of antibodies that bind preferentially to complexes of an anti- immunogen in complete Freund's adjuvant and in subsequent body and its cognate antigen (4-8). In this paper we refer to injections with incomplete Freund's adjuvant. Several days these as anti-immune complex (anti-IC) antibodies. These prior to splenectomization, 200-250 mg of immunogen in include IgM antibodies that were suggested to recognize saline was given i.p. Splenocytes from mice sensitized with changes in conformation in the Fc region of an anti-idiotype keyhole limpet hemocyanin-conjugated THC immunogen antibody when bound to the idiotype (4, 5) and antibodies that were fused with Ag8.653 cells and splenocytes from mice display enhanced binding to the variable region when the sensitized with affinity-labeled anti-THC antibody Ab1 were latter is bound to an antigen (7-9). The latter have been fused with NS-1 cells. After cell culture in Dulbecco's suggested to bind simultaneously to portions of both the modified Eagle's medium (GIBCO) supplemented with 10%o antigen and the antibody or to bind a determinant, termed a metatype (9), that results from a conformational change Abbreviations: ABTS, 2,2'-azinobis(3-ethylbenzothiazoline-6- induced by the immunogen. Although the exact nature ofthis sulfonic acid); CMO, 11-nor-9-carboxymethyloximino-6a,7, binding is controversial, the existence of such anti-IC anti- 8,9,10,10a-hexahydrocannabinol; HRP, horseradish peroxidase; bodies suggested the possibility that these antibodies could IC, immune complex; NHS, N-hydroxysuccinimide; PBES, m-phe- be used to enhance the sensitivity of immunoassays. More- noxybenzoylamino-2-ethylsulfonic acid; THC, tetrahydrocannab- over, if the anti-IC binding depends on a direct interaction inol; G6PDH, glucose-6-phosphate dehydrogenase; BSA, bovine serum albumin. *To whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge tPresent address: Pel-Freeze, Inc., Rogers, AR 72757. payment. This article must therefore be hereby marked "advertisement" tPresent address: Department of Plant Biology, University of Cali- in accordance with 18 U.S.C. §1734 solely to indicate this fact. fornia, Berkeley, CA 94720. 1184 Downloaded by guest on September 25, 2021 Colloquium Paper: Uffman et al. Proc. Natl. Acad. Sci. USA 90 (1993) 1185 determined by Paragon gel electrophoresis ofa mixture ofthe conjugate and THC-G6PDH. ELISAs. Except where noted, incubations were carried out at room temperature in phosphate-buffered saline (PBS, pH 7.2). Costar microtiter plate wells were coated with affinity- purified rabbit anti-mouse immunoglobulin antibodies (1 mg/ ml) for 1 h at 37TC and treated with 0.25% gelatin by incubation at 4TC overnight. After washing with 0.05% Tween 20, the wells were coated with the test antibody by incubation overnight at 4TC, washed, and blocked with irrelevant mouse THC _o u CMO IgG by incubation for 15 min. HRP activity was determined as indicated above. G6PDH activity was determined by incubation with Tris-HCl, pH 8.0/33 mM glucose 6-phosphate/20 mM NAD+/6 mM p-iodonitrotetrazolium violet/ 0.56 international unit of diaphorase. The absorption at 492 nm was read after 30 min. The competition between pairs of anti-idiotype antibodies for binding to Ab1 was determined by using one of the anti-idiotype antibodies as the test antibody. After the blocking step, the wells were incubated for 15 min with Abl-HRP PBES with and without the other anti-idiotype antibody and washed five times, and the activity of the bound enzyme was mea- FIG. 1. Structures and numbering of A9-THC, immunogen CMO, sured. A similar procedure was used for testing the cross and PBES. reactivity of Ab2 to various isotypes. Ab2 was first bound to the wells followed by incubation with the test isotype and (vol/vol) fetal calf serum (SMEM), cloning of hybridomas Abl-HRP. The wells were then washed and the enzyme was carried out in the presence of nonimmune peritoneal activity was measured. macrophages. Screening was carried out for anti-THC anti- For determination of the Ab2 binding specificity to Ab1 in bodies by binding the culture supernatants to microtiter wells the presence of various THC derivatives, the appropriate precoated with rabbit anti-mouse IgG, blocking the surfaces THC derivative was incubated for 30 min with Ab1-HRP and with excess mouse IgG, and determining the binding of the immobilized Ab2. SMEM with or without 10%o (vol/vol) THC-HRP conjugate in the presence and absence ofA9-THC. ethanol was used as buffer. Although equilibrium was not Screening for anti-IC antibodies was carried out in the same reached in this time, independent measurements of the time way except that THC-HRP was replaced by Abl-HRP. course of the binding showed no change over 22 h in the Enzyme rates were determined in a solution of 9 mM H202, relative binding with various THC derivatives. After incu- 2 mM 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) bation, the enzyme rate was determined by washing the (ABTS), and 0.1 M citrate (pH 4.2) and read at 414 nm. wells, incubating with 9 mM H202/2 mM ABTS, pH 4.2, and Anti-THC antibody Ab1 and anti-IC antibody Ab2 were reading at 414 nm. prepared by culture in SMEM using a hollow-fiber device The ability of Ab1 and Ab2 to bind to immobilized THC (CD Medical, Miami Lakes, FL), and the antibodies were hapten was determined by using microtiter wells that were adsorbed on r-protein A-agarose (Repligen, Cambridge, coated with THC-HRP by a procedure similar to that used to MA), eluted with 0.1 M citrate (pH 6.0), and concentrated by coat the wells with anti-mouse immunoglobulin antibodies. centrifugation ultrafiltration. Antibody purity was checked The wells were then incubated for 15 min with the appropriate by SDS/PAGE, and the antibodies were isotyped with a kit antibody in PBS containing 10%6 ethanol with or without 1.6 ,uM A9-THC. Binding was measured by treating the wells from Southern Biotechnology Associates (Birmingham, AL). with anti-mouse phosphatase con- were with pepsin at pH immunoglobulin-alkaline F(ab')2 fragments of Ab2 prepared jugate and measuring the enzyme activity with 4.5 mM 4.3, followed by purification on DEAE-Sephacel. Ab2 Fab' p-nitrophenyl phosphate/2 mM MgCl2, pH 9.2, at 405 nm. fragments were prepared by treatment of Ab2 F(ab')2 in 0.15 The specificity ofAb1 was determined by incubating THC- M Tris-HCl (pH 8.0) with 5 mM'dithioerythritol followed by G6PDH and THC or THC-9-COOH derivatives in PBS quenching with 10 mM iodoacetamide. containing 10%6 ethanol for 1 h at 37°C in microtiter wells that Affinity Labeling of Ab1 (THC-Abl). The NHS ester of were coated as above with Abl. The wells were washed three [14C]THC-CO-f3-Ala-f3-Ala and Ab1 (3:1) were incubated in times, and the enzyme activity was measured as above. 14% (vol/vol) dimethylformamide/10 mM phosphate (pH Radioimmunoassays. The specificity ofAb1 in ethanol-free 8.5) for 16 h at 4°C, gel-filtered on Sephadex G-50, and then PBS was determined by incubating 40 nM A9-[3HJTHC, 10 alternately chromatographed first on CNBr-activated Seph- nM Abl-HRP, and the THC derivative for 2 h, adding arose 4B coupled to Ab1 and then on AH-Sepharose 4B anti-HRP-coated ImmunoBeads (Bio-Rad), and incubating coupled to A8-THC-9-COOH. After two rounds of chroma- again, followed by separation, washing, and determining the tography, the product had a THC/Ab1 molar ratio of 2.1:1 radioactivity on the ImmunoBeads. and THC-G6PDH did not affect its migration on a Beckman Binding Constants. Ab2 Fab' (0.36 umol) was tritiated in SPE Paragon agarose gel electrophoresis. PBS (pH 7.2) with 0.5 mCi (11 ,mol) of N-([2-3H]ethyl)ma- Abl-HRP Conjugate. HRP labeled with 1.2 sulfhydryl leimide for 2 h at 30°C followed by treatment with 10 mM groups was prepared by coupling with N-succinimidyl 3-(py- unlabeled N-ethylmaleimide. Binding constants were mea- ridyldithio)propionate followed by reduction with dithio- sured at 25°C in the presence of 1.0%6 bovine serum albumin threitol. Ab1 labeled with 7.8 maleimide groups was prepared (BSA). For determining binding of Ab1 to Ab2, 12.5 nM Ab1 with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1- was incubated overnight with various amounts of 3H-labeled carboxylate. Coupling of these preparations at pH 6.0 fol- Ab2 Fab' and then adsorbed with Fc-specific goat anti-mouse lowed by fractionation on Sephacryl S-300 yielded an anti- IgG bound to ImmunoBeads. For measurement of Ab2 bind- body conjugate having an average of -3.8 HRP molecules ing in the presence ofa ligand, Ab1 was preincubated with 3.2 per IgG. The preparation had 100%o immunological activity as mM 49-THC prior to initiating the preceding procedure. For Downloaded by guest on September 25, 2021 1186 Colloquium Paper: Ullman et al. Proc. Natl. Acad Sci. USA 90 (1993) determination of A9-THC binding to Abl, various amounts of A study was carried out on the binding of Ab1 and Ab2 to A9-[3H]THC were incubated overnight with 1 nM Ab1, the the immobilized THC derivative THC-HRP, which had a antibody was separated, and radioactivity was measured as hapten structure similar to that of the immunogen THC-Ab1 before. In all cases, measurements were made in duplicate used to prepare Ab2. The binding was evaluated in the with an irrelevant mouse IgG used in place of Ab1 as a presence and absence of A9-THC for each antibody sepa- control. rately and for a mixture ofAb1 and Ab2 (Table 1). Ab1 binding Kinetic Measurements. Rates ofassociation ofA9-[3H]THC to THC-HRP was, as expected, inhibited by A9-THC. Since with Ab1 (40 nM) were measured at room temperature with Ab2 also inhibited Ab1 binding and did not itself bind to the the same buffer as for the binding constant measurements but immobilized THC-HRP, it must bind directly to Abl. The with sufficient irrelevant mouse IgGl(A) to provide a total increased inhibition caused by adding A9-THC to wells con- immunoglobulin concentration of 122 nM. After mixing, taining Ab1 and Ab2 is consistent with A9-THC further aliquots were periodically withdrawn and added to rabbit enhancing binding of Ab1 and Ab2. However, the additional anti-mouse IgG-coated ImmunoBeads suspended in 1 ,uM inhibition can also be explained simply by A9-THC prevent- A9-THC. The beads were then washed and radioactivity was ing binding of Ab1 that is not associated with Ab2. measured. A similar procedure was used for measurement of Binding ofAb2 to irrelevant murine monoclonal antibodies the rates of binding of A9-[3H]THC to Ab1-Ab2 complexes. of isotypes IgGl(K), IgG2a(K), IgG2b(K), IgG3(K), IgGl(A), Mixtures of 20 nM Ab1 with either 0.1 or 1.2 mM intact Ab2 IgM(K), and IgM(A) or to other anti-THC monoclonal anti- IgG were preequilibrated overnight with irrelevant IgG added bodies was not observed. The absence of binding was estab- to equalize the total immunoglobulin concentrations. These lished by the failure of these antibodies to compete with mixtures were then diluted 1:10 into the assay solution. Since Abj-HRP for binding to immobilized Ab2. passage of a solution containing a 1:1 molar ratio of the The epitopes recognized by Ab2, anti-idiotope 1E2, and antibodies over a Sephacryl S-300 column gave a peak for an anti-paratope 4B12 on Ab1 were mapped by binding one Ab1-Ab2 complex with no detectible uncomplexed antibody, antibody to a well surface and causing it to complete with a the preincubation procedure assured that only the Ab1-Ab2 second antibody in solution for binding to HRP-Abl. By complex would be present during reaction with labeled THC. studying all six combinations, it was found that anti-idiotope The rates of dissociation of A9-[3H]THC from Ab1 or 1E2 only competes with itself for binding to HRP-Ab1; Ab1-Ab2 were measured by first preforming the A9-THC anti-paratope 4B12 competes with itself and with Ab2; and complexes by incubating 5 nM A9-[3H]THC with 2 nM Ab1 Ab2 competes with itself and with anti-paratope 4B12. Thus alone or combined with 200 nM Ab2 for 20 h. A9-THC at 1-2 Ab2 and anti-paratope 4B12 bind to overlapping epitopes and mM was then added and aliquots were periodically with- anti-idiotope 1E2 binds to an independent epitope on Ab1. drawn and added to rabbit anti-mouse IgG ImmunoBeads, Binding Constants and Kinetics. All measurements were which were then washed, and radioactivity was measured. made in the presence of 1% BSA, which was needed to prevent binding of A9-THC to surfaces. Since this protein is RESULTS reported to bind to A9-THC with a binding constant of2 x 106 M-1 (10), only -0.3% of the A9-THC was free. The A9-THC Characterization of Antibodies. Murine monoclonal anti- binding constants and on-rates given here are not corrected bodies were prepared using an affinity-labeled anti-THC for binding to BSA and are accordingly a factor of -330 too antibody IgGl(A) (THC-Ab1) as immunogen and were se- low but are correct relative to each other. Since measurement lected based on their ability to bind Abj-HRP in the presence of binding of Ab1 to Ab2 would be complicated by the or absence of A9-THC. In these determinations, the test possibility ofbivalent interactions, rate and equilibrium mea- antibody was bound to a microtiter well and challenged with surements were carried out with Ab2 Fab' fragments and the labeled Ab1. Out of five fusions, initial screening yielded intact Ab1 IgG. This had the added advantages that Ab2 Fab' 130 anti-idiotope antibodies (binding unaffected by A9-THC), could be radiolabeled at its sulfhydryl groups by N-[3Hleth- 67 anti-paratope antibodies (binding only in the absence of ylmaleimide and that Ab1-Ab2 Fab' complexes could be A9-THC), and one anti-IC antibody (binding enhanced by readily separated from the unbound Ab2 Fab' with beads A9-THC). The anti-IC antibody (Ab2) was shown to have an coated with anti-mouse Fc antibodies. IgGl(K) isotype. The A9-THC-dependent modulation ofbind- Binding constants for Ab2 Fab' binding to Ab1 that was free ing ofAbj-HRP to Ab2 and to anti-idiotope 1E2 [IgGl(K)] and of hapten or was saturated with excess A9-THC (T) were anti-paratope 4B12 [IgGl(K)] in an ELISA are given in Table determined as K1+2 = 4.5 x 106 M-1 and K1T+2 = 3.1 X 108 1. The data illustrate that A9-THC enhances the binding of M-1, respectively. The binding constant of A9-THC to Ab1 Ab1 to Ab2. was found to be K1+T = 4.5 x 109 M-1. Binding of A9-THC to the Ab1-Ab2 complex could not be measured directly but Table 1. Effect of 1.6 ,uM A9-THC on the binding of can be calculated to be K12+T = 3.1 x 1011 M-1 from the anti-idiotype antibodies relationship K12+T = K1+TKlT+2/K1+2. All measurements were made in duplicate and good linear Scatchard plots were Soluble Immobilized Enzyme activity all component component - A9-THC + A9-THC obtained in cases. Ab1+ Ab2+ T Ab1- T + Ab2 Ab1-HRP Ab2 0.30 0.90 K1+T Ab1-HRP Anti-idiotope 1E2 0.52 0.52 KI+21 1 K1T+2 Ab1-HRP Anti-paratope 4B12 0.24 0.14 K12+T Ab2 THC-HRP 0.00 0.01 Ab1-Ab2+ T _ Ab1 - Ab2 - T Ab1 THC-HRP 0.86 0.30 Ab1 + Ab2 THC-HRP 0.17 0.10 The rates of A9-THC binding to and dissociation from Ab1 = 2.2 x 105 M-1-s-1 and = 2.8 X 10-5 s-'] Antibodies were bound to microtiter wells that were precoated [kl+T(on) klT(oM with affinity-purified rabbit anti-mouse immunoglobulin. THC-HRP were determined by following A9-[3H]THC binding to Ab1. was bound to the wells by nonspecific adsorption. Incubations were K1+T = 7.9 X 109 M-1 derived from these data is within in PBS for 15 min at room temperature. Ab1-HRP binding was experimental error of the direct measurement for binding of determined from the change in A414 produced by HRP catalysis. Ab1 Ab1 and A9-THC. Determination of the rate of binding of and Ab2 binding was determined from the change in A405 produced A9-THC to Ab1-Ab2 Fab' was complicated by the need to by alkaline phosphatase catalysis (see text). assure that all the Ab1 was complexed with Ab2 during the Downloaded by guest on September 25, 2021 Colloquium Paper: Ullman et al. Proc. Natl. Acad. Sci. USA 90 (1993) 1187 binding reaction. This was accomplished by preparing an antibody complex from 20 nM Ab1 and 1.2 mM Ab2 where stronger binding is expected because of the likely bivalent binding of the intact IgGs and because size-exclusion gel chromatography suggested that a stable Ab1-Ab2 complex can be formed. The validity of this approach was supported ._ by the observation that the on-rate observed when the complex was diluted 1:10 [k12,T(on) = 2.7 x 104 M-1ls-1] was only slightly increased to 3.2 x 104 M-1lso1 when the complex was diluted 1:120. A~~~~~~~~~~~~~~~~~~~~1~~~~~1 Dissociation of A9-[3H]THC from its ternary complex with the two intact antibodies, Ab1 and Ab2, could not be detected even after incubation for 5 days in the presence of excess 1.1l_ A.; : 0I unlabeled A9-THC. The off-rate [k2+T(off)] is, therefore, <2 X 1' A 10-7 s-1. This is consistent with the calculated off-rate of k12+T(om = kl2+T(on)/Kl2+T = 8.7 x 10-8 so1, which is based on the reasonable assumption that the rate of binding of A9-THC to Ab1-Ab2 Fab' is the same as for binding to intact IgG Abj-Ab2. Anti-THC Antibody (Ab1). Binding specificities of Ab1 were measured with and without the addition of 10% ethanol as cosolvent. With ethanol present, specificities were studied :i ()Xf).xs()liT,!'( I by allowing THC-G6PDH to compete with various THC derivatives for binding to microtiter wells coated with Abl. Each of the compounds A9- and A8-THC and A9- and A8_ (1. . i .}.ii THC-9-COOH equally inhibited THC-G6PDH binding over a 1000-fold range of concentrations. In a related experiment in li;'11 1 _ t .___L 1 ~~~~~~~~~~~~~~~11Ah which THC-HRP was used in place of THC-G6PDH, the antibody was shown to have a detectable but weak cross reactivity to cannabinol and to PBES. In the absence of the cosolvent, dissolution of the THC analogs was dependent upon binding to added BSA, and the FIG. 2. Effect of THC derivatives on binding of Ab1-HRP to specificity of binding to Ab1 was determined by allowing microtiter wells coated with Ab2. Solutions containing 4 nM IgG as various compounds to compete with A9-[3H]THC for Ab1- Abj-HRP in 10% ethanol (A) and ethanol-free SMEM (B) were incubated 30 min, and the wells were washed, incubated with HRP in solution. Again A9-THC and A9-THC-9-COOH were H202/ABTS, and measured at 414 nm. equally effective competitors and an 3-fold higher concentration ofthese compounds was required for competition than ent (Fig. 2B). 8,11-Dihydroxy and 11-hydroxy THC deriva- for 11-OH-, 813-OH-, and 813,11-(OH)2-THC (Table 2). tives promoted 50% maximal binding at similar concentra- Anti-IC Antibody (Ab2). The specificity ofbinding ofAb2 to tions, which were -10-fold lower than the corresponding various Abl-antigen complexes was studied by measuring concentrations ofunsubstituted THC derivatives. In contrast binding ofthe complexes to microtiter wells coated with Ab2. the maximal binding produced by these compounds at high In these experiments, various concentrations of the antigen concentrations diminished in the following order: unsubsti- were mixed with a fixed concentration of Abj-HRP that was tuted > 11-hydroxylated > 8-hydroxylated > 9-carboxy selected such that, in the absence ofadded antigen, there was substituents. only 5-10% of maximal binding to the wells. In 10%o ethanol, various THC derivatives enhanced binding at different concentrations and were variable in their ability to affect max- DISCUSSION imal binding (Fig. 2A). Each ofthe compounds THC-G6PDH There have been several previous reports of enhanced bind- (data not shown), A9-THC-9-COOH, and PBES inhibited binding of Ab1 to Ab2- ing ofantibodies to immune complexes (4-9). Although it has In a similar study in which a larger set ofTHC analogs was been claimed (4, 5) that IgM antibodies bind to idiotype-anti- investigated in the absence of ethanol, A9-THC-9-COOH idiotype complexes on neodeterminants on the Fc fragment, changed from a weak inhibitor to a weak promoter and a clear enhancement may be due to the frequently observed coop- relation between structure and enhancement became appar- erative formation ofaggregates produced by multisite binding (11, 12). More recently, an anti-IC monoclonal antibody to a Table 2. Relative affinities of Ab1 for THC derivatives thyrotropin immune complex was shown to interact with Krel bound anti-thyrotropin Fab fragments (7). In a related paper, a polyclonal antibody is reported that binds preferentially to THC derivative(s) No EtOH 10% EtOH alprenolol antibodies complexed to iodocyanopindolol, a A9-80-OH-THC 3.4 hapten having a 2-cyano-4-indolinyl group in place of the A9-11-OH-THC 3.4 o-allylphenyl group of alprenolol (8). Although both studies A9-88,11-(OH)2-THC 2.8 demonstrated that the anti-IC bound to Fab fragments and A9,11-THC 1.7 that the binding was enhanced in the presence ofthe antigen, A8- and A9-THC 1.0 1.0 it could not be determined whether the binding was to an A8- and A9-THC-9-COOH 1.0 1.0 antigen-induced conformation of the primary antibody or to Relative reciprocal concentrations required for 50% inhibition of an epitope composed of portions of both the antigen and the binding are shown. For no ethanol, 40 nM A9-3H]THC, 10 nM Ab1, antibody. Additionally, Voss et al. (9) have described an PBS, and 0.1% BSA were incubated for 2 h at room temperature. For anti-IC polyclonal antibody that they suggest binds to a 10% ethanol, THC-G6PDH in PBS with Ab1 bound to microtiter ligand-induced conformational determinant of an anti- wells was used and incubations were for 1 h at 370C. fluorescein monoclonal antibody that they call a "meta- Downloaded by guest on September 25, 2021 1188 Colloquium Paper: Ullman et al. Proc. Natl. Acad Sci. USA 90 (1993) type." The complexes formed with fluorescein-bound Fab hydrophobic unoxygenated THC derivatives. Although the fragments involved several anti-IC antibodies binding simul- binding ofAb2 to ligand immune complexes was less solvent- taneously to the same Fab fragment (13). Different off-rates sensitive, the carboxylic acid derivatives shift from inhibiting were determined for various complexed fluorescein deriva- binding in 10% ethanol to causing a slight enhancement with tives but no binding constants were measured (14). no cosolvent added. This shift could be due either to de- In the present work, the affinity ofAb1 for anti-IC antibody creased binding of Ab1 to Ab2 in the absence of ligand or Ab2 was found to be enhanced in the presence of excess stronger binding to Ab2 in the presence of ligand and is A9-THC. Binding ofAb2 is specific to Ab1; no binding to other particularly noticeable only because there is change in bind- immunoglobulins having the same or different isotypes was ing relative to ligand-free Ab1. observed. Additionally, although Ab2 bound to its immuno- The observation that Ab2 produces only a small (8-fold) gen, affinity-labeled Abl, it did not bind to a THC conjugate of decrease in the rate of association of A9-THC with Ab1 an unrelated protein (HRP). The A9-THC-induced enhance- suggests that Ab2 only weakly obstructs the Ab, binding site. ment of binding of Ab1 to Ab2 Fab' fragments eliminates the The observed 70-fold enhancement ofbinding (2.5 kcal/mol; possibility that enhanced binding was associated with bridg- 1 cal = 4.184 J) is similar to the enhancements reported in ing. The observation that Ab2 competes with an anti-paratope early studies (7, 8) and is consistent with the formation of antibody for binding to Ab1 requires that Ab2 must bind to an little more than one additional hydrogen bond upon binding epitopic site on Ab1 that overlaps at least a portion of an Ab2. Since ligand structures that enhance binding appear epitope that encompasses the hapten binding site. unrelated to the structure of the THC-CO-f-Ala-p-Ala im- In the cross-reactivity experiments in which Abl-HRP is munogen used for preparing Ab2, the relationship between allowed to bind to Ab2 on a surface, it is safe to assume that structure and enhancement appears to stem from purely binding to the surface has a negligible effect on solution adventitious binding of groups projecting from the Ab1 bind- concentrations. When excess ligand is present, the Ab1-HRP ing site and interacting with a functionality on the bound Ab2. binding sites can be assumed to be fully occupied. The In this regard it is noteworthy that in some of the earlier amount of Abl-HRP bound to surface Ab2 under these studies, anti-IC antibodies were produced even when no conditions bm., therefore, is controlled only by the affinity antigen was associated with the primary antibody used as an constant of Ab2 for the Ab1-ligand complex; immunogen (4, 8). Further, with one exception (9), even when antigen was present, it was not covalently bound to the bmax = [Abl]o[Ab2]oKlT+2/(l + [Abl]oKlT+2). antibody and would, therefore, be unlikely to remain associated with the antibody during processing by an antigen- In the absence ofadded ligand the amount ofAbl-HRP bound presenting cell. bmin depends only on K1+2; These conclusions are inconsistent with the formation of a unique conformationally distinct metatype upon antigen bmn= [AbliO[Ab2ioK1+2/(l + [Abl]OK1+2). binding as was proposed by Voss et al. (9) to explain anti-IC The amount of ligand required to effect 50%o binding [TO]50 is polyclonal antibody binding to a fluorescein-anti-fluorescein independently controlled by the affinity constant of Ab1 for immune complex. Although the metatype hypothesis cannot the be excluded in the latter system, the current data raise the ligand; possibility of an alternative explanation. Thus it seems likely + 1/K1+T. that the observation of Voss and coworkers (13) of multi- [To1so= [Abl]0/2 meric binding to the anti-fluorescein immune complex is a Thus bm. and [TO]50 independently reflect the respective result of enhanced binding of one antibody through a weak values for K1T+2 and K1+T. adventitious binding interaction of the type described here Ab2 binding enhancement, bmax, appears to be related in part followed by binding of additional anti-idiotype antibodies, to the polarity of the THC derivatives. Significantly, the where each binding event is further enhanced by weak affinity of Ab2 for the immune complexes as reflected in bmax nonspecific interactions with the bound antibodies. is not correlated with the affinity ofthe various ligands for Ab1 The observed interaction of haptenic pendant groups and (see Fig. 2). For example, A9-THC and A9-THC-COOH bind anti-idiotypic antibodies is demonstrated by this study to identically to Ab1 (Table 1) but only A9-THC enhances Ab2 greatly increase the specificity of immunoassays. Thus es- binding (bx,,~); and A9-8,11-(OH)2-THC and cannabinol differ sentially complete specificity ofdetection ofA9-THC relative greatly in binding to Ab1 but provide similar enhancement of to A9-THC-COOH was achieved with an anti-IC antibody Ab2 binding. Where the structural variations in the ligand were when using an antibody that was otherwise completely non- at C-9, the attachment site for the hapten used toaprepare Ab1, specific with respect to these compounds. The additional binding of Ab2 (bma,) decreased in the order of polarity of the property of anti-IC antibodies to increase the affinity of the C-9 substituent, CH3 > CH20H > CO-(3-Ala-,3-Ala > COOH. primary antibody also offers the opportunity to significantly Additionally Ab2 binding was strongly affected by the intro- increase assay sensitivity. Although this was not realized in duction ofcharge. Thus, A9-THC-COOH was a weak inhibitor the present study because of the use of insufficiently radio- of binding and PBES, which is more acidic but only roughly active THC, the use of more readily detectable labels should conforms to the shape of the immunogenic hapten CMO (see make this possible. The present findings also offer the Fig. 1), is a stronger binding inhibitor. This dependence of possibility of engineering catalytic or additional recognition enhancement on the structure of the group at the attachment sites into anti-IC antibodies to take further advantage of the site ofthe hapten that must extend away from the binding site spatial organization of this system. implies direct contact ofAb2 and the ligand in the termolecular complex. The absence ofcorrelation between Ab2 binding and We are grateful to Ann Brady for preparation of the THC immu- the to Ab1 that variations in nogen, Dr. Mae Hue for preparation of PBES, Beth Hutchins for binding affinity requires confor- preparation of the antibody-peroxidase conjugate, and Dr. Sat Nam mation ofAbl associated with tightness offit ofthe ligand must for of the anti-THC have a minimal effect. Hanjan preparation antibody. The effect on ligand binding to Ab1 ([T501) when the carrier 1. Oudin, J. & Michel, M. (1%3) C. R. Hebd. Seances Acad. 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