Proc. Natl. Acad. Sci. USAVol. 83, pp. 3972-3976, June 1986Immunology

Antibodies of predetermined specificity to the major chargedspecies of human interleukin 1

(peptide/growth factor/monocyte interleukin 1)

GUADALUPE LImIJUCO*, STEFAN GALUSKAt, JAYNE CHIN*, PATRICIA CAMERON*, JOSHUA BOGERt,AND JOHN A. SCHMIDT**The Department of Biochemistry and Molecular Biology, tThe Department of Basic Animal Sciences Research, and tThe Department of MedicinalChemistry, Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065-0900

Communicated by P. Roy Vagelos, January 21, 1986

ABSTRACT The development of highly specific antisera tohuman interleukin 1 (IL-1) has been an elusive goal hamperedmainly by the availability of only limited amounts of pureimmunogen. To surmount this difficulty, three peptides of themajor charged species of IL-1 (pI 6.8) were synthesized andcovalently coupled to keyhole limpet hemocyanin (KLH). Allthree peptide-KLH conjugates raised rabbit heterologousantisera that bound intact pure IL-1 in a dose-dependent anddomain-specific manner. Immunoblot analysis of crude con-centrated culture supernatants with these antisera showed eachof them to be highly specific for mature 18-kDa IL-1. Im-munoblot analysis of monocyte lysates revealed a single 33-kDaband consistent with the size of the IL-1 precursor moleculededuced from cloned cDNA. These reagents should prove to bevaluable tools in the localization and measurement of IL-1 incells and fluids and may permit the separate study of individualIL-1 species as well as discrete domains of intact IL-1 mole-cules.

Antiserum to human interleukin 1 (IL-1) has been typicallyraised in rabbits immunized with partially purified prepara-tions derived from supernatants of cultured peripheral bloodmononuclear cells (1). The resultant antisera have beendemonstrated to be nonspecific in that they recognize avariety of serum and cell-derived proteins unrelated to IL-1(2). In addition, with the recent discovery of two distinctcomplementary DNA molecules (cDNA) coding for humanIL-1, termed IL-i-a and IL-1-, (3), it is not clear whetherthese antisera recognize one or both of these IL-1 species.Attempts to use hybridoma technology to obtain monospe-cific antibodies to human IL-1 have thus far been unsuccess-ful in producing useful antibody reagents.To surmount the difficulties imposed by limited amounts of

purified IL-1 protein and to avoid the circular logic thatsometimes arises when characterizing antisera with the sameantigens to which they were raised, we have immunizedrabbits with the keyhole limpet hemocyanin (KLH) conju-gates of three synthetic peptides ofthe major charged species(pI 6.8) of human IL-1 [IL-l-,8 (3)]. The current reportdemonstrates that the resultant anti-peptide antisera bind tointact pure IL-1 derived from monocytes in a dose-dependentand domain-specific manner. Moreover, immunoblot analy-sis reveals that the antisera specifically recognize mature18-kDa IL-1 in crude culture supernatants and 33-kDa IL-1precursor in crude cell lysates. In all, the results suggest thatthe anti-peptide antisera are monospecific reagents for thedetection of IL-1.

METHODS

Synthesis of Peptides and Preparation of Peptide-KLHConjugates. Three peptides of the pI 6.8 species of humanIL-1 (see Fig. 1) were synthesized by using Merrifieldsolid-phase peptide synthesis chemistry (4). The syntheses ofthe amino-terminal and internal peptides (Fig. 1) were per-formed on 4-methylbenzhydrylamine resins in place of thestandard Merrifield resin in order to obtain the COOH-terminal amide. The peptides were cleaved from the resinswith hydrofluoric acid, purified by preparative reverse-phaseHPLC, and lyophilized. Purity was established by analyticalreverse-phase HPLC, and composition was confirmed byamino acid analysis. Coupling to KLH (Calbiochem) wasperformed by first derivatizing the carrier with m-maleimido-benzoyl N-hydroxysuccinimide ester (MBS; ref. 5) andsubsequently reacting the derivatized carrier with peptide ata molar ratio of 1:10. Those peptides that did not have acysteine as part of the naturally occurring sequence had acysteine (internal peptide) or arginine and cysteine (carboxylpeptide) added to the NH2 terminus during solid-phasesynthesis to make coupling with MBS possible. The conju-gates were lyophilized and stored in an evacuated dessicatorat 4°C.

Production of Heterologous Antiserum. New Zealand Whiterabbits (1.5 kg) were bled and then immunized with 200 ,tg ofpeptide-KLH conjugate in complete Freund's adjuvant atmultiple intradermal sites. Repeat injections of conjugate inincomplete Freund's adjuvant were given 3 and 5 weeks laterat multiple subcutaneous sites. Ten days after the lastimmunization, the rabbits were bled and the serum wasaliquoted and frozen at -200C.

Leukotriene C4 was conjugated to KLH with MBS asdescribed (6). Hyperimmune antiserum to the conjugate wasraised in rabbits as described (6).

Solid-Phase Radioimmunoassay (SPRIA). The titer of theantisera raised to the peptide-KLH conjugates was deter-mined by using a solid-phase binding assay with eitherimmobilized peptide or pure IL-1 (pI 6.8). Peptide (50 ng perwell) or pure IL-1 (50 ng per well) was allowed to bind toflexible polyvinylchloride microtiter wells (Dynatech, Alex-andria, VA) in 140 mM NaCl/10 mM Na2HPO4 7H2O (PBS)(pH 7.2) overnight at 4°C. The following day, the wells werewashed in PBS containing 1.5% horse serum and thenblocked with PBS containing 10% horse serum for 2 hr atroom temperature. Antiserum diluted in PBS/1.5% horseserum was then applied in 100-,l volumes and allowed tostand for 2 hr at room temperature. After washing three timeswith PBS/1.5% horse serum, 100 IlI of 125I-labeled protein A(100,000 cpm per well; 5 x 107 cpm/,ug) was added to each

Abbreviations: IL-1, interleukin 1; KLH, keyhole limpet hemocya-nin; SPRIA, solid-phase radioimmunoassay; MBS, m-maleimido-benzoyl N-hydroxysuccinimide ester.

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well and incubated for 2 hr at room temperature. Afterwashing five times, the plates were dried. The bottoms of thewells were removed with a hot wire and counted in a ycounter.

In some experiments, inhibition of antibody binding toimmobilized peptide or IL-1 was attempted by preincubatinga constant amount of antiserum with serial logarithmicdilutions of relevant or irrelevant peptide for 2 hr at roomtemperature in polypropylene tubes. The mixtures were thentested for binding to immobilized antigen as described above.ELISA. Polystyrene microtiter wells (Dynatech) were

coated with various amounts of IL-1 in 100 pl of 0.015 Msodium carbonate buffer (pH 9.6) overnight at 40C. Thefollowing day, the wells were washed three times withPBS/0.05% Tween-20 and then blocked with 1% gelatin/PBS/Tween for 1 hr at room temperature. Antiserum dilutedin PBS/Tween was applied in 100-/.l volumes to the wells andincubated at room temperature for 1 hr. After washing threetimes, 100 41. of affinity-purified goat anti-rabbit IgG-horseradish peroxidase conjugate (Bio-Rad), diluted 1:1000in PBS/Tween, was added to the wells and incubated for 1 hrat room temperature. After washing three times, 100 A.l ofsubstrate [2,2'-azino-bis(3-ethylbenzthiazolinesulfonate)](Kirkegaard and Perry, Gaithersburg, MD) was added, incu-bated 10 min, and quenched with 100 /.l of 5% NaDodSO4.Optical density was then read with a multiscan plate reader(Titertek) at 405 nm.Immunoblot Analysis. To test the specificity of the anti-

peptide antisera for intact extracellular IL-1 and IL-1 pre-cursor, the antisera were used in immunoblot analysis ofcrude concentrated culture supernatant and mononuclear celllysates. The supernatants were harvested from human mono-nuclear cells that had been stimulated with both phytohemag-glutinin and lipopolysaccharide for 5 min or 96 hr (7). Thesupernatants were then concentrated 100-fold by ultrafiltra-tion (7) and 50-,ul aliquots were analyzed by NaDodSO4/PAGE in 15% homogeneous gels under reducing conditions.Mononuclear cell lysates were prepared by washing 1 x 107mononuclear cells at specified times three times with Hanks'balanced salt solution and scraping the adherent cells intoHanks' balanced salt solution containing 5 mM N-ethyl-maleimide, 2 mM phenylmethylsulfonyl fluoride, and 2 mMEDTA. The cells were pelleted, resuspended, and boiled inLaemmli sample buffer for 5 min, and analyzed byNaDodSO4/PAGE as described above. After electrophore-sis, the gels were preequilibrated in transfer buffer (25 mMTris/192 mM glycine/20% methanol/0. 1% NaDodSO4, pH8.3) for 20 min at room temperature and then rested on a sheetof presoaked filter paper (3 mm; Whatman). The sandwichwas completed by applying presoaked sheets of nitrocellu-lose and filter paper to the exposed surface of the gel andplacing the assembly between two sheets of Scotch Brite (3MCo.). Electrophoretic transfer to the nitrocellulose was per-formed in a Bio-Rad transblot apparatus at 4°C overnight ata constant voltage of 30 V. Completeness of transfer wasdemonstrated afterwards by silver staining of the polyacryl-

amide gel. The nitrocellulose sheet was washed three timesin PBS, blocked in 0.5% gelatin/PBS for 1 hr, and washedwith TEN buffer (0.05 M Tris'HCl/0.15 M NaCl/0.005 MEDTA/0.02% sodium azide/0.25% gelatin/0.05% TritonX-100, pH 7.4). The nitrocellulose strips were then incubatedin a 1:100 dilution of antiserum in TEN buffer for 1 hr withgentle agitation. Afterwards, the strips were washed threetimes in TEN buffer (10 min each wash) and then incubatedin 1251I-labeled protein A (200,000 cpm/ml; 25 ml; 5 X 107cpm/,ug) for 1 hr. After washing six times in TEN buffer, thesheets were air-dried and exposed to Kodak X-O-MAT filmovernight.

Purification of IL-1. The pI 6.8 species of IL-1 was purifiedas described by a protocol (7) consisting of anion exchangeand reverse-phase HPLC. The resulting material was pure byNaDodSO4/PAGE (Mr, 18,000), analytical isoelectric focus-ing (pI 6.8; ref. 8), and amino-terminal sequence analysis (7).The amount of IL-1 was determined by amino acid analysisas described (7).

RESULTS

The amino acid sequence of the pI 6.8 species of human IL-1has been deduced from cloned cDNA (2, 3) and confirmed byamino acid sequence analysis of purified protein (7). Begin-ning at alanine-117, the amino terminus of the matureextracellular protein (7), and ending at serine-269, the likelycarboxyl terminus (7), the primary amino acid sequence wasexamined for antigenic segments as described (9). Thisanalysis resulted in a list of peptides ranked in order of theirpotential antigenicity. It was decided to synthesize the first ofthese (Fig. 1; internal peptide) as well as peptides comprisingthe amino and carboxyl termini of the mature molecule (Fig.1). The carboxyl termini of the amino-terminal and internalpeptides were synthesized as amides in order to emulate theirinterior position within the native sequence. Cysteines wereadded to the amino termini of the internal and carboxyl-terminal peptides during synthesis to provide free sulfhydrylgroups for coupling to KLH. All three peptides were cova-lently coupled to KLH, a highly immunogenic carrier protein,using the heterobifunctional cross-linker MBS.The preimmune and immune sera of rabbits immunized

with the peptide-KLH conjugates were tested for their abilityto bind in SPRIA to immobilized peptide and pure intact IL-1(see Methods). As shown in Fig. 2, the preimmune serumfrom a rabbit subsequently immunized with the KLH con-jugate of the amino-terminal peptide did not bind to either therelevant peptide or intact IL-1 (pI 6.8). Similarly, rabbitantiserum to the KLH conjugate of leukotriene C4 (6) did notbind to either peptide or intact IL-1 (data not shown). Theimmune antiserum bound not only the peptide to which it wasraised but also to intact IL-1 in a dose-dependent manner.Identical results were obtained with antisera obtained fromrabbits immunized with the KLH conjugates of the internaland carboxyl-terminal peptides and with IgG isolated fromthese antisera by protein A affinity chromatography (data not

ResidueDesignation Number Sequence

amino terminal 117-128 H-ALA-PRO-VAL-ARG-SER-LEU-ASN-CYS-THR-LEU-ARG-ASP-NH2peptide

internal peptide 197-215 H-CYS-GLN-LEU-GLU-SER-VAL-ASP-PRO-LYS-ASN-TYR-PRO-LYS-LYS-LYS-MET-GLU-LYS -ARG-PHE-NH2

carboxyl terminal 258-269 H-CYS-ARG-ASP-ILE-THR-ASP-PHE-THR-MET-GLN-PHE-VAL-SER-SER-COOHpeptide

FIG. 1. The amino acid sequences of three synthetic IL-1 (pI 6.8) peptides. The residue number indicates the position of the peptides in thecomplete amino acid sequence deduced from a cDNA (2, 3) for this species of IL-1. The bold portions are modifications or additions to the nativesequence (see text).

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3974 Immunology: Limjuco et al.

30rA

25F

0

x

Ea

20F

151

10

5

B

x ~ ~ x

x~~~~~

o~~~~~

, O- _9_ 9~~~101 102 103 104 101 102 103 104

1 /serum dilution

FIG. 2. SPRIA of preimmune serum (o) and antiserum (x) raisedto the KLH conjugate of the amino-terminal peptide on wells coatedwith amino-terminal peptide (A) or intact pure IL-1 (pI 6.8) (B). Theaverage of duplicate determinations is plotted.

shown). The titers of the antisera shown in Fig. 2 arerepresentative of those obtained in rabbits immunized withthe various peptide-KLH conjugates once in completeFreund's adjuvant and twice in incomplete Freund's adjuvant(see Methods). Considerably higher half-maximal titers wereobtained (serum dilution = 1 x 10-5 to 1 x 10-6) with one ortwo additional immunizations in incomplete adjuvant.

Competitive solid-phase binding experiments were per-formed to demonstrate that the various anti-peptide antiserarecognized the appropriate domain of the intact IL-1 mole-cule. In Fig. 3, a single subsaturating dilution of antiserumraised to the KLH conjugate of the amino-terminal peptidewas preincubated with increasing amounts of relevant pep-tide or irrelevant peptide (internal peptide) prior to assay onwells coated with relevant peptide or pure IL-1. As shown,binding of the immune antiserum to peptide and IL-1 wasblocked in a dose-dependent manner by relevant peptide,whereas irrelevant peptide did not block in either case.Similar results were obtained with antisera to the KLHconjugates of the internal and carboxyl-terminal peptides.The amount of binding in solid-phase binding assays was

proportional to the amount of antiserum used (as shown in

\E

10 5-

0 101 102 103 104 O 101 102 103 104

Concentration of soluble peptide, ng/ml

FIG. 3. SPRIA of antiserum raised to the KLH conjugate ofamino-terminal peptide on wells coated with amino-terminal peptide(A) or intact pure IL-1 (B) after preincubation with serial logarithmicdilutions of relevant peptide (x) (amino-terminal peptide) or irrele-vant peptide (e) (internal peptide).

Fig. 2) and also to the amount of IL-1 used to coat the wells.In Fig. 4, wells were coated with pure IL-1 at concentrationsranging from 0.4 to 50 ng per well before incubation withserial logarithmic dilutions of antiserum raised to the KLHconjugate of the internal peptide. Subsequently, the wellswere reacted with a fixed concentration of peroxidase-conjugated goat anti-rabbit IgG. After addition of substrateand an appropriate incubation period, the absorbance ofeachwell was read by an automated spectrophotometer (seeMethods). As shown, the amount of chromophore obtainedwith each concentration of immune antiserum increased asthe amount of IL-1 used to coat the wells was increased. Noappreciable absorbance was obtained with preimmune serumeven when the highest concentrations of IL-1 and serum wereused (Fig. 4).The specificity of the anti-peptide antisera for intact IL-1

was tested by using them for immunoblot analysis of crudeconcentrated supernatants derived from cultures of stimu-lated human peripheral blood mononuclear cells (see Meth-ods). The crude concentrated culture supernatants were runon 15% NaDodSO4/polyacrylamide gels under reducingconditions and were transferred to nitrocellulose paper, asdetailed in Methods. The paper was then reacted withanti-peptide antiserum and 1251-labeled protein A and exam-ined by autoradiography. As shown in Fig. 5, antiserum to theKLH conjugate of the amino-terminal peptide recognized asingle band in crude concentrated culture supernatants (Fig.5A, lane 2). Silver staining of another lane loaded with crudeconcentrated culture supernatant showed it to be very com-plex with many different protein species (lane 1). Immunoblotanalysis of another lane loaded with 100 ng of pure IL-1showed that the band identified in the crude material had thesame mobility as IL-1 (lane 3). Culture medium in whichperipheral blood mononuclear cells were only briefly sus-pended for 5 min was negative by this analysis (data notshown). Fig. 5B (lane 1) shows a silver-stained NaDodSO4 gelof 100 ng of the IL-1 preparation used in Fig. 5A (lane 3). Asingle 18-kDa band is observed. Lane 2 in Fig. SB is anadjacent lane that was loaded with sample buffer not con-taining IL-1.The specificity of the anti-peptide antisera in immunoblot

analyses of crude concentrated culture supernatant wasfurther examined (Fig. 6). As shown in Fig. 6A (lanes 2-4),all three of the antisera bound pure IL-1. No bands wereobserved with preimmune serum or with 125I-labeled proteinA alone (lanes 1 and 5, respectively). As shown in Fig. 6B

E U.70 0.6

0.5-

0.4

0.2

0.1

10 1o2 1 V 104 105 106

1/serum dilution

FIG. 4. ELISA of preimmune serum (open symbols) and antise-rum (solid symbols) raised to the KLH conjugate of internal peptideon wells coated with the following concentrations (ng per well) ofpure intact IL-1: & and A, 50; m and o, 10; v and v, 2;e ando, 0.4.

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A1 2 3

kDa

94- U 467-

43 -

30 -

20 -14-

14-

BkDa94-6743-

30-

2 Time, hr0 4 24 48

kDa

33-

20-

14-

FIG. 5. Immunoblot analysis ofconcentrated culture supernatantand pure IL-1 using antiserum to the KLH conjugate of amino-terminal peptide. (A) Lanes: 1, silver stain of 50 1.l of crudeconcentrated (x 100) culture supernatant; 2, immunoblot of crudesupernatant; 3, immunoblot ofpure IL-1 (100 ng). (B) Lanes: 1, silverstain of 100 ng of pure IL-1 preparation used in lane 3 ofA; 2, silverstain of lane adjacent to lane 1, which did not contain IL-1.

(lanes 2-4), all three antisera recognized a major band incrude concentrated culture supernatant having a mobilityidentical to pure IL-1 with a molecular mass of 18 kDa. Minorbands were occasionally seen (lanes 3 and 4), but these werenot reproducible findings (compare with Fig. 5). Once again,no bands were seen with either pooled preimmune serum orwith "25I-labeled protein A alone (lanes 1 and 5, respectively).The specificity of the antisera was also tested by perform-

ing immunoblot analyses ofmononuclear cell lysates (Fig. 7).The adherent fraction of mononuclear cells stimulated withlipopolysaccharide and phytohemagglutinin was solubilizedin Laemmli sample buffer at 0, 4, 24, 48, 72 and 96 hr afterculture initiation (see Methods). After NaDodSO4/PAGEand transfer to nitrocellulose, the blot was treated with theantiserum to the KLH conjugate of the amino-terminalpeptide and 125I-labeled protein A. A single species of 33 kDawas not initially present but then increased to a maximum

A B1 2 3 4 5

kDa94 -a

67-

43-

30-

1 2 3 4 5kDa

-94

-67

-43

-30

20

14- -14

FIG. 6. Immunoblot analysis of pure IL-1 (100 ng; A) and crudeconcentrated (x 100) culture supernatant (50 ,ul; B) using preimmuneserum and protein A (lanes 1), protein A alone (lanes 5), or antiserato the KLH conjugates of carboxyl-terminal peptide (lanes 2),internal peptide (lanes 3), or amino-terminal peptide (lanes 4) andprotein A.

FIG. 7. Immunoblot analysis oflysates of the adherent fraction ofmononuclear cells using antiserum to the KLH conjugate of theamino-terminal peptide. Mononuclear cells (1 x 10') were culturedin the presence oflipopolysaccharide and phytohemagglutinin for theindicated times prior to washing and solubilization in Laemmlisample buffer.

level by 48 hr. Lower amounts were present at subsequenttimes but fewer adherent cells were recovered at these latertime points. Identical results were obtained at 48 hr with theantisera to the internal and carboxyl-terminal peptides (datanot shown).

DISCUSSIONWe have described three peptides of the major chargedspecies (pI 6.8) of human IL-1 that, when coupled to KLH,raise heterologous antisera to intact IL-1. The antisera boundIL-1 immobilized on plastic in a dose-dependent (Figs. 2 and4)- and domain-specific (Fig. 3) manner. Furthermore, theantisera were highly specific for IL-1 when used in im-munoblot analysis of crude concentrated culture superna-tants in that only the same single 18-kDa band having thesame mobility as pure IL-1 was consistently visualized (Figs.5 and 6). Immunoblot analysis of cell lysates of stimulatedcells (Fig. 7) showed the time-dependent increase of a single33-kDa band consistent with the size of the IL-1 precursormolecule deduced from cDNA (2, 3). Control supernatantscontaining no IL-1 activity and wells coated with serumproteins were nonreactive.The results obtained during immunoblot analysis of culture

supernatants with the three antisera gave no evidence of IL-1species of <18 kDa (Figs. 5 and 6). Small species (3-6 kDa)having IL-1-like activity and presumably derived from intactIL-1 have been reported by others in urine (10), plasma (11),and upon storage of partially purified material (12). Im-munoblot analysis of the cell lysates revealed only material ofhigh molecular mass and gave no evidence of intracellularprocessing to smaller forms (Fig. 7). A murine IL-1 precursorof similar size has recently been identified in the lysates ofperitoneal macrophages and the murine macrophage lineP388D1 (13). These findings, taken together, suggest that theIL-1 precursor is cleaved either during or shortly after releaseand raise the possibility that cleavage of IL-1 precursormolecules at a single site is linked to externalization.

72 96

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Recently, it has been discovered that there are at least twodistinct human IL-1 molecules encoded by different genes,termed IL-1-a and IL-1-,8 (3). The peptides described in thisreport were deduced from the pI 6.8 species of IL-1, whichis encoded by the IL-i-,3 gene (3, 7). The finding that theantisera to the amino-terminal peptide and carboxyl-terminalpeptide reacted with mature extracellular IL-1 on im-munoblot analysis confirms that these sequences lie withinmature extracellular IL-1 as predicted by amino acid se-quence analysis of the purified protein (7). Whether theseantisera cross-react with other anionic IL-1 species having pIvalues of 5.2 and 5.4 (14), which are presumably encoded bythe IL-1-a gene, is not yet known. These may not be resolvedfrom the pI 6.8 species by NaDodSO4/PAGE, given theremarkable conservation of length for all IL-1 moleculescloned to date (2, 3, 15). In addition, the other IL-1 speciesare considerably less abundant than the pI 6.8 species (3, 14,16). Thus, definitive testing for cross-reactivity awaits thefinal purification of these anionic species, a project nearcompletion in this laboratory. However, the limited amountof sequence homology between the synthetic peptides andtheir corresponding regions in the IL-1 encoded by the IL-1-acDNA (2/12, 4/19, and 3/12 residues for the amino-terminal,internal, and carboxyl-terminal peptides, respectively) sug-gests that cross-reactivity is an unlikely possibility.The utility of these antisera has only been partially ex-

plored. As demonstrated, all are useful reagents for im-munoblot analysis and solid-phase binding assays. Immuno-cytochemical analysis of cultured cells and tissues with thesereagents has also been successful (17). The availability oflarge amounts of pure relevant and irrelevant peptides forblocking studies makes them particularly well-suited for thislatter application. The study of these antisera as antagonistsof IL-1 biological activity has recently been initiated by firststudying their ability to bind soluble IL-1 as measured inimmunoprecipitation reactions. One of the antisera, theantiserum to the internal peptide, has already been identifiedas an excellent reagent for immunoprecipitation, but it blocksIL-1 activity weakly in the murine thymocyte assay (unpub-lished observations). This preliminary result suggests that thedomain of IL-1 included in the synthetic internal peptide maynot be directly involved in target-cell activation. Furtherexperiments of this kind with these and other anti-peptideantisera may be successful in identifying those domains oftheIL-1 molecule responsible for its many diverse bioactivities

(18) and, in conjunction with a receptor binding assay, inmapping its receptor recognition site(s).

Note Added in Proof. We have recently tested the three antiseradescribed in this paper in ELISA against pure monocyte-derivedhuman pI 5.2 IL-1 and pI 5.4 IL-1 (19). No cross-reactivity wasobserved.

We thank Dr. E. Hayes for several helpful discussions and Drs. E.Bayne, E. Cordes, P. Davies, E. Emini, A. Rosenthal, and M. Toccifor careful review of the manuscript.

1. Dinarello, C. A., Renfer, L. & Wolff, S. M. (1977) Proc. Natl.Acad. Sci. USA 74, 4624-4627.

2. Auron, P. E., Webb, A. C., Rosenwasser, L. J., Mucci, S. F.,Rich, A., Wolff, S. M. & Dinarello, C. A. (1984) Proc. Nati.Acad. Sci. USA 81, 7907-7911.

3. March, C. J., Mosley, B., Larsen, A., Cerretti, D. P., Braedt,G., Price, V., Gillis, S., Henney, C. J., Kronheim, S. R.,Grabstein, K., Conlon, P. J., Hopp, T. P. & Cosman, D.(1985) Nature (London) 315, 641-647.

4. Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154.5. Kitagawa, T. & Aikawa, T. (1976) J. Biochem. 79, 233-236.6. Hayes, E. C., Lombardo, D. L., Girard, Y., Maycock, A. L.,

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8. Schmidt, J. A. (1984) J. Exp. Med. 160, 772-787.9. Emini, E. A., Hughes, J. V., Perlow, D. S. & Boger, J. (1985)

J. Virol. 55, 836-839.10. Kimball, E. S., Pickeral, S. F., Oppenheim, J. J. & Rossio,

J. L. (1984) J. Immunol. 133, 256-260.11. Clowes, G. H. A., George, B. C., Villee, C. A. & Saravis,

C. A. (1983) N. Engl. J. Med. 308, 545-552.12. Dinarello, C. A., Clowes, G. H. A., Gordon, A. H., Saravis,

C. A. & Wolff, S. M. (1984) J. Immunol. 133, 1332-1338.13. Giri, J. G., Lomedico, P. T. & Mizel, S. B. (1985) J. Immunol.

134, 343-349.14. Schmidt, J. A., Oliver, C. N., Lepe-Zuniga, J. L., Green, I. &

Gery, I. (1984) J. Clin. Invest. 73, 1462-1472.15. Lomedico, P. T., Gubler, U., Hellman, C. P., Dukovich, M.,

Giri, J. G., Pan, Y. E., Collier, K., Semionow, R., Chua,A. 0. & Mizel, S. B. (1984) Nature (London) 312, 458-462.

16. Schmidt, J. A., Mizel, S. B., Cohen, D. & Green, I. (1982) J.Immunol. 128, 2177-2182.

17. Bayne, E. K., Rupp, E. A., Limjuco, G., Chin, J. & Schmidt,J. A. (1986) J. Exp. Med., in press.

18. Durum, S. K., Schmidt, J. A. & Oppenheim, J. J. (1985)Annu. Rev. Immunol. 3, 263-287.

19. Cameron, P. M., Limjuco, G. A., Chin, J., Silberstein, L. &Schmidt, J. A. (1986) J. Exp. Med., in press.

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