Journal oflmmunological Methods, 156 (1992) 163-170

163

© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00

JIM 06498

Development of a monoclonal antibody to the conserved region of p34 cdc2 protein kinase Kathryn K a m o a, R a m o n J o r d a n a, Hei-ti Hsu a and Derek H u d s o n b a Florist and Nursery Crops, U.S.D.A., Beltsville, MD 20705-2350, USA, and b Arris Pharmaceuticals, South San Francisco, CA 94080, USA

(Received 2 March 1992, revised received 27 May 1992, accepted 17 June 1992)

Mice and rabbits were injected with various forms of a 16 amino acid synthetic peptide representing PSTAIR, the evolutionarily conserved region of the protein kinase p34 cJc2, for polyclonal antisera and hybridoma-monoclonal antibody production. Antisera from mice injected with an unconjugated monomeric form of the peptide showed no reaction to the peptide. Of four animals injected with the monomeric form of the peptide conjugated to keyhole limpet hemocyanin via m-maleimidobenzoyl-N-hydroxysulfosuccinimide (MBS), antisera from only one mouse had a very low titer to the peptide, and all four animals produced antibody to the MBS bridge. Both mice injected with an octameric multiple antigen peptide (MAP) of PSTAIR produced antisera reactive to the octameric MAP form of the peptide in ELISA and also to the cdc2 protein expressed in bacteria in an immunoblotting assay. Splenocytes from one mouse injected with the octameric MAP form of the peptide were successfully used for hybridoma-monoclonal antibody production. A monoclonal antibody was produced that reacted with octamer, monomer and cdc2-expressed protein and specifically with the carboxyl terminus of the 16 amino acid peptide. Key words: p34cdc2; Multiple antigen peptide; Antipeptide antibody

Introduction Correspondence to: K. Kamo, Florist and Nursery Crops,

Bldg. 004, Rm. 208, BARC-West, U.S.D.A., Beltsville, MD 20705-2350, USA. Tel.: 301-504-5350; Fax: 302-504-5096. Abbreviations: KLH, keyhole limpet hemocyanin; MBS, m-maleimidobenzoyl-N-hydroxysuccinimideester; t-Boc, tertbutoxycarbonyl; BSA, bovine serum albumin; sulfo-MBS, rnmaleimidobenzoyl-N-hydroxysulfosuccinimidq ester; TBST, TBS containing 0.05% Tween 20; MAP, multiple antigen peptide; PBS, phosphate-buffered saline (50mM sodium phosphate, 150 mM NaCI, pH 7.5); SDS/PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Fmoc, 9-fluorenylmethyloxycarbonyl; PAL, peptide amide linker; BOP, Castro's reagent, 1-benzotriazolyloxy-tris(dimethylamino)phosphonium hexafluorophosphate; HOBt, 1-hydroxy-benzotriazole; OtBu, t-butyl ester; tBu, t-butyl ether; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl.

The protein kinase p34 cdc2, first discovered in yeast, is important in regulating cell division. In every eukaryote so far examined, p34 cdc2 contains an evolutionarily conserved 16 amino acid sequence called PSTAIR (EGVPSTAIREISLLKE) which distinguishes p34 c°c2 from other protein kinases. Although p34 c°ca is presently one of the most widely studied protein kinases, there are only two reports where antibody has been produced to the characteristic PSTAIR region. Rabbit polyclonal antisera has been produced to the PSTAIR sequence of p34 ~dc2 by using the 16

164

amino acid PSTAIR peptide coupled to a carrier protein (Lee and Nurse, 1987). There is only one recent report of a monoclonal antibody, but it does not describe production of the monoclonal antibody in detail, and the antibody was made using the conventional method of conjugating the synthetic peptide to either BSA or KLH (Yamashita et al., 1991). The difficulty in making antibody to conserved proteins has been well documented (Wallace and Cheung, 1979; Van Eldik and Watterson, 1981; Kitajima et al., 1983; Winkler et al., 1987; Harlow and Lane, 1988; Sacks et al., 1991) and may be the reason for only two reports of antibody made to the PSTAIR region of p34 cdc2. Even when one has obtained a suitable polyclonal antibody, the same animal may not always be used successfully for monoclonal antibody production because of difficulties encountered in growing antibody-producing hybridoma cell lines (Sacks et al., 1991). There are many advantages for using peptides representing specific regions of ~, protein rather than the entire protein as the antigen. Peptides are, however, typically relatively poor immunogens (Harlow and Lane, 1988). Tam (1988) created the multiple antigen peptide (MAP) system for improving the immunogenicity of a synthetic peptide and allowing sensitive detection by solidphase methods. The MAP was found to be a successful antigen for obtaining polyclonal antibodies to all six MAPs created, and titers were higher with the MAP rather than the KLH-conjugated form used as the antigen (Tam, 1988). Troalen et al. (1990) compared polyclonal antibodies made when either the MAP or conjugated form of a peptide representing human lutropin was used as the immunogen, and both forms of the peptide resulted in comparably high antibody titers. A few others have successfully used the MAP system for antibody production (Tam, 1989; Del-Giudice et al., 1990; Zavala and Chai, 1990). Use of a synthetic peptide representing an evolutionarily conserved sequence presents an additional potential disadvantage for its use as a suitable antigen. In this study we have evaluated the use of various forms of a synthetic peptide, including the octameric MAP form representing the PSTAIR sequence of p34 cdc2 for producing polyclonal and monoclonal antibodies. A mono-

clonal antibody recognizing the carboxy terminal portion of the 16 amino acid peptide was developed. This is the first detailed description for production of a monoclonal antibody to the PSTAIR peptide and demonstrates the advantage of using an octameric MAP form as the immunogen.

Materials and methods

Synthesis of monomeric and octameric EGVPSTAIREISLLKE-amide (MAP) peptides Fmoc-PAL-polystyrene (0.08 mmol) and Pepsyn KMAPS (0.03 mmol) were placed in the reaction vessel of a M G / B model 9600 automatic peptide synthesizer. The synthesis was run using the BOP + HOBt coupling method, and 0.5 g scale synthesis program. The following amino acids were incorporated (coupling time in parentheses): Fmoc-Glu(OtBu)-OH (2 h), FmocLys(tBoc)-OH (2 h), Fmoc-Leu-OH (2 h), FmocLeu-OH (2 h), FMoc-Ser(tBu)-OH (2 h), FmocIle-OH (4 h), Fmoc-Glu(OtBu)-OH (4 h), FmocArg(Pmc)-OH (2 h), Fmoc-Ile-OH (4 h), FmocAla-OH (2 h), Fmoc-Thr(tBu)-OH (2 h), FmocSer(tBu)-OH (4 h), Fmoc-Pro-OH (2 h), FmocVal-OH (2 h), Fmoc-Gly-OH (2 h) and FmocGlu(OtBu)-OH (2 h). The terminal Fmoc group was removed, then the resin removed and placed in a beaker. Methylene chloride was added to the mixture, and after several minutes, decanted (taking with it the majority of the polystyrene). This process was repeated several times. The Pepsyn KMAPS, which remained at the bottom of the beaker during these treatments, was dried, then cleaved and deprotected using Reagent R (trifluoroacetic acid/thioanisole/ethane dithiol/anisole, 90 : 5 : 3 : 2 v/v) for 2 h. The octameric MAP was isolated by precipitation with cold dry ether, and filtration for purification by HPLC. For HPLC on a Varian 5000 HPLC, a 25 cm Waters Deltapak C-18 column was used with buffer A (0.1% trifluoroacetic acid in water) and buffer B (0.1% trifluoroacetic acid in acetonitrile). The gradient was 5% buffer B for 3 min followed by linear to 100% B over the next 20 min. Flow rate was 1.7 ml/min and detection at 230 nm.

165 The floated PAL-polystyrene was washed with methanol, dried and cleaved to yield monomeric peptide (Hudson et al., 1992).

Synthesis of PSTAIR monomer and three variations The 16 amino acid monomeric PSTAIR peptide and three variations of this peptide (gift of Steve Wolniak, University of Maryland) were synthesized using a Milligen/Biosearch 9600 peptide synthesizer according to the protocol provided by the manufacturer (MilliGen/Biosearch, Novato, CA). The peptides had the following amino acid sequences: Glu-Gly-Val-Pro-Ser-ThrAla-Ile-Arg-G lu-Ile-Ser-Leu-Leu-Lys-Glu (PSTAIR monomer, peptide 1), Glu-Gly-ThrPro-Ser-Thr-Ala-Ile-Arg-Glu-Ile-Ser-LeuMet-Lys-Glu (peptide 2), Glu-Gly-Thr-Pro-SerThr-Ala-Ile-Arg-Glu-Ile-Ser-Leu-Leu-Lys-Glu (peptide 3), Glu-Gly-Val-Pro-Ser-Thr-Ala-IleArg-Glu-Ile-Ser-Leu-Met-Lys-Glu (peptide 4). Synthesis of conjugated monomer Monomer peptide no. 1 was synthesized with an additional cysteine at the amino terminus and conjugated to either BSA or KLH by conventional methods with MBS as the linker (Kitagawa and Aikawa, 1976). Briefly described, the carrier protein and sulfo-MBS were suspended in PBS, pH 8.5 and mixed at 70 rpm for 20 min, 25°C. The peptide:carrier ratio was 50: 1, and the sulfo-MBS to peptide ratio was 2 : 1. Peptide dissolved in PBS, pH 5.0 was added dropwise to derivatized carrier, and the pH was maintained at 7.2 during the 2 h/25°C mixing. The conjugated peptide was then dialyzed for 16 h/4°C against PBS. Conjugation was performed by Berkeley Antibody (Richmond, CA). Expression of p34 cdc2 protein The protein used for Western blotting was p34 cdc2 expressed in E. coli. The vector pRK171 into which cdc2 was subcloned was a gift from K. Gould and P. Nurse (1989) (Microbiology Unit, University of Oxford, Oxford, UK). The plasmid was expressed as previously reported (Studier et al., 1990) in the bacteria strain BL21(DE3) which was a gift from F.W. Studier and A. Rosenberg (Brookhaven National Laboratory, Upton, NY).

Following induction of T7 RNA polymerase, the bacteria cells were centrifuged at 7700 × g for 5 min at 4°C, and cells were resuspended in Laemmli sample buffer, heated in boiling water 5 min. As a control, the vector pet 3-C (gift of F.W. Studier and A. Rosenberg) was also expressed and induced in BL21 (DE3), and the cells prepared in the same way as the cdc2-expressing cells for gel electrophoresis. Bacterial extracts in Laemmli buffer were used for coating ELISA plates (50 ixl bacterial extract/10 ml coating buffer, and 100 tzl per well).

Polyclonal antibody production Two New Zealand White rabbits were each initially injected via lymph node with 300 /xg of PSTAIR monomer conjugated to KLH in PBS emulsified with complete Freund's adjuvant. Subsequent intramuscular injections of 150 txg antigen per rabbit emulsified with incomplete Freund's adjuvant were administered every 2 weeks for 16 weeks. BALB/C mice were immunized with 30/xg of either PSTAIR monomer, octameric MAP, or monomer conjugated to KLH by intraperitoneal injection. The antigen, in PBS, was emulsified with an equal volume of incomplete Freund's adjuvant. Injections were given every 3 weeks for 21 weeks. At the last injection each mouse was also injected (intraperitoneal) with 300 txl P3/NS1/1-Ag4-1 (NS1) myeloma cell suspension (106 cells per mouse) and killed about 2 months afterwards by exsanguination during ether anesthesia. All animals were cared for according to federal, state and institutional guidelines. Monoclonal antibody production The mouse chosen for monoclonal antibody production was injected with the octameric MAP as above for seven injections followed by three weekly injections each consisting of 50 /xg octameric MAP in PBS emulsified in an equal volume of incomplete Freund's adjuvant, and then one final injection 1 week later (50 txg octameric MAP, no adjuvant) 4 days prior to splenectomy. The mouse was killed by cervical disarticulation following ether anesthesia and the spleen removed.

166 Unless stated otherwise, maintenance of myelomas, fusion of spleen and myeloma cells and selection of hybridomas were essentially similar to those previously described (Hsu and Lawson, 1985; Jordan, 1990). A modified complete medium containing Rosewell Park Memorial Institute (RPMI 1640) medium (Gibco Laboratories, Grand Island, NY), 1 mM L-glutamine, 1 mM pyruvate, 10% (v/v) fetal bovine serum (Hyclone Laboratory, Logan, UT), and 5% (v/v) each of Nu serum (Collaborative Research, Bedford, MA), CPSR-3 controlled process serum replacement (Sigma Chemical, St. Louis, MO), and 100 ppm gentamycin was used. About 1-2 weeks after fusion the culture supernatants were screened for the presence of antibodies to PSTAIR monomer, octameric MAP or p34 cdc2 by ELISA and to p34 cdc2 by Western blotting. Selected cultures were then cloned by limiting dilution. Class of the monoclonal antibody was determined by Ouchterlony immunodiffusion with rabbit antisera specific for mouse heavy and light chains (Litton Bionetics). Solid-phase ELISA For indirect ELISA (Hsu and Lawson, 1985) antigens were dissolved (2 ~ g / m l ) in coating buffer (0.05 M carbonate, pH 9.6) and 100 /xl antigen solution was incubated in each well (Maxisorb, Nunc) for 3-4 h. Wells were then washed four times, 5 min/wash, with 0.02 M Tris base, 0.15 M NaC1, pH 7.5, 0.05% Tween 20 (TBST). Wells were blocked for 30-60 min with blocking solution consisting of 1% (w/v) Carnation non-fat dry milk, 0.5% (w/v) BSA (fraction V, A-4503, Sigma) in TBS (TBS without Tween 20). After blocking, wells were washed once in TBST. Antisera diluted in blocking solution diluted 1/10 with TBS (100/zl/well) was added and incubated 16 h at 4°C. Following four washes, 5 rain/wash, with TBST, 100 /~1 of alkaline phosphataselabelled affinity purified goat antibody to either mouse IgG, IgA, IgM or rabbit IgG and IgM (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added to each well at a 1/2000 dilution and incubated 1-3 h followed by five washes with TBST. To each well 200 p.1 of 1 m g / m l p-nitrophenyl phosphate (N2765, tablets, Sigma) in substrate buffer (0.1 M di-

ethanolamine, 0.05 mM MgC126H20 , pH 9.8) was added. Colorimetric response was recorded by an ELISA plate reader at 405 nm after 30 min or 3 h at 25°C and 16 h at 4°C. Endpoint titer was defined as the reciprocal of the antiserum dilution which resulted in an absorbance greater than 0.1 and was two times higher than background. All incubations were at 25°C unless noted otherwise. Antigen competition assays The method used was as described above in solid-phase ELISA, except that competing antigen was incubated with antisera in solution for 1 h at 25°C prior to the addition of antisera to the antigen-coated ELISA plate. Western blotting Expressed proteins from E. coli (BL21-DE3) containing the pet 3-C vector with and without p34 cdc2 gene insert were electrophoresed on 12% acrylamide gels S D S / P A G E according to Laemmli (1970). After electrophoresis, protein was transferred to Immobilon P membrane (Milipore Corp., Bedford, MA) by electroblotting 16 h at 0.2 A, 30 V using transfer buffer (20 mM Tris base, 0.15 M glycine, 0.01% SDS). After blocking in the blocking solution for 90 min, blots were washed once in TBS and then incubated with antigen-specific primary antibody 16 h at 4°C. Blots were washed 3-4 times, 5-10 min each wash, in TBS. Alkaline phosphatase-labelled goat anti-mouse or rabbit IgG and IgM were added at a 1/2000 dilution for 90 min followed by 3-4 washes, 10 rain each, in TBS and one 10 min wash in substrate buffer (0.1 M Tris base, pH 9.5, 0.1 M NaC1, 5 mM MgCI2). Color was developed by adding 350 p.g/ml nitroblue tetrazolium (Sigma) and 175 /zg/ml 5-bromo-4-chloro-3-indoyl phosphate (Sigma). Protein determination Protein was precipitated following the addition of 10 vols. acetone, incubation at - 20°C for 16 h, centrifugation at 10,000 × g for 5 min. The protein pellet was resuspended in 1% SDS (w/v in water) and assayed using the BCA protein assay reagent according to the company's protocol (Pierce, Rockford, IL).

167

Results

Synthesis of monomeric and MAP peptide Using the MAP technique described in the materials and methods section both monomeric (200 mg after HPLC purification) and octameric MAP peptides (58 mg) were synthesized simultaneously and were readily separated post synthesis. The MAP synthesized had the same branching, octameric pattern as that described originally by Tam, but was produced differently. Tam synthesized the octameric MAP by using t-BOCmediated assembly of the peptide sequences on a polystyrene support with a core of branching lysine residues. Recently Hudson et al. (1992) developed a variation of this method for Fmocmediated synthesis, utilizing a similarly functionalized Pepsyn K support, which allowed the simultaneous synthesis of the monomeric peptide on admixed polystyrene. Amino acid analysis showed that the actual number of amino acids in the monomeric and octameric MAP peptides were

in close agreement with the theoretical number. The octameric MAP peptide was purified and shown by HPLC to be free of monomeric peptide (data not shown). The monomeric peptide was purified and shown by HPLC to have some octameric MAP content (data not shown).

Production of polyclonal antisera made to various forms of the peptide There was no appreciable antibody titer to either the monomer or octameric MAP peptides in solid-phase ELISA tests in antisera from two mice injected with the monomer peptide (Table I). In comparison antisera from the two mice injected with the octameric MAP form of the peptide had low endpoint titers of 320 to octameric peptide (Table I), and the antisera reacted with p34 cdc2 protein by Western blot (data not shown). Antisera from the two mice and two rabbits injected with PSTAIR monomer conjugated to KLH showed that only one mouse had a very low

TABLE I E N D P O I N T T I T E R S S H O W I N G I M M U N O G E N I C I T Y O F V A R I O U S F O R M S O F T H E P S T A I R PEPTIDE, T H E R E C O M B I N A N T cdc2 P R O T E I N A N D MBS L I N K E R U S E D IN C O N J U G A T I O N Endpoint titers are expressed as the reciprocal of the serum dilution that was two times higher than background with a m i n i m u m absorbance of 0.1 at 405 nm. A noncrossreacting m o u s e antiserum prepared against Spiroplasma, normal or p r e i m m u n e serum and all antisera samples titered against BSA or 600 n g / w e l l bacterial extract from BL21 (DE3) containing the pet 3-C vector without cdc2 gene insert were all used to assess background. Data is shown for each antigen-injected animal. Endpoint titers were determined 20 weeks after the initial injection for mice and after 12 weeks for the two rabbits. Octameric peptide (100 ng/well), monomeric P S T A I R peptide and BSA-conjugated peptide (800 ng/well), MBS-BSA (200 ng/well) and recombinant ode2 protein (800 ng/well) were used to coat the E L I S A plate. Absorbance readings were taken 60 min after adding substrate. Source of antiserum

Forms of peptide monomer

octamer

conjugate

Linker

Protein

MBS-BSA

cdc2

Normal mouse 1 P r e i m m u n e rabbit 1

0 0

0 0

0 0

0 0

0 0

Monomer-injected mouse 1 Monomer-injected m o u s e 2

0 0

0 0

0 0

0 0

0 0

Octamer-injected m o u s e 1 Octamer-injected m o u s e 2

0 0

320 320

0 0

0 0

320 320

Conjugate-injected mouse 1 Conjugate-injected m o u s e 2

20 0

20 0

2,560 10,240

5,120 10,240

0 0

Conjugate-injected rabbit 1 Conjugate-injected rabbit 2

0 270

0 0

14,580 21,870

21,870 65,610

0 0

0

0

0

0

0

Spiroplasma-injected mouse 1

168

endpoint titer of 20 to the monomeric peptide (Table I) and showed a faint alkaline phosphatase reaction to p34 cdc2 by Western blot (data not shown). Antisera from each of the four animals injected with the peptide conjugate showed a high titer endpoint ranging from 5,120 to 65,610 when MBS-BSA was the antigen in solid-phase ELISA. This indicated that some antibodies were most likely made to the sulfo-MBS bridge used to link the peptide with KLH or BSA (Table I). Competition assays (data not shown) showed that although the antibodies made to MBS did not account for all of the antibody titer reactive to monomer conjugated to BSA as the antigen in ELISA, these results indicated that MBS can ellicit an immunogenic response. Antisera from two mice injected with the monomer peptide, two mice injected with octameric MAP, or four animals (two rabbits and two mice) injected with K L H - c o n j u g a t e d monomer showed no increase in titer to PSTAIR monomer when the serum or ascites were assayed anytime during the 21-25 weeks following the initial injection for the mice and 16 weeks later for the two rabbits (data not shown).

Monoclonal antibody production to the octameric MAP One mouse injected with the octameric MAP was used to generate monoclonal hybridoma antibody-secreting cell lines. Initially 951 hybridoma cultures were screened by both ELISA and Western blot, and 67 tested positive to at least one antigen after the first screen. Five hybridoma cell lines producing monoclonal antibodies reactive to octameric MAP, PSTAIR lhonomer a n d / o r p34 cdc2 protein remained stable through subsequent cloning. One hyridoma cell line (12B4) grew well in culture and produced IgG1 antibodies which reacted strongly to p34 cdc2 by Western blot and ELISA assays, strongly to octameric MAP in ELISA and weakly to PSTAIR monomer in ELISA. Two hybridoma cell lines secreted IgM antibodies and their cell culture supernatants reacted weakly to both octameric MAP in ELISA and p34 cdc2 in Western blot. The IgG3 monoclonal antibody (6F3) produced in culture fluid reacted with a high anti-

o 0.5

0.0'

~'~ 0

1

'~ 2

3

4

5

6

7

8

9

RECIPROCAL DILUTION (LOG 2) Fig. 1. Reaction of hybridoma monoclonal antibody (6F3) to various antigens in solid-phase ELISA. The ELISA plates were coated with B, 800 n g / w e l l PSTAIR monomer; e, 100 n g / w e l l octameric MAP; A, 600 ng/well protein extract from bacteria expressing p34CdC2; zx, 600 ng/well protein extract from bacteria with pRK171 vector but without p34 cdc2 gene. Two-fold serial dilutions of culture supernatant .were added and absorbance readings taken 3 h after addition of substrate. ELISA antibody titer is the mean of duplicate samples run at the same time. The assay was done two times and overall results were similar for both experiments.

body titer (2048) to octameric MAP and low endpoint titer (32) to PSTAIR monomer in either solid-phase or antigen competition assay ELISA (Figs. 1 and 3). The antibody also reacted specifically to p34 cdc2 produced by expression in bacterial cells in both ELISA assay and Western blot assays (Figs. 1 and 2). Culture fluid containing a monoclonal antibody to a potyvirus reacted with the four antigens (monomeric and octameric MAP peptides, p34 cdc2, and BL21-DE3 bacterial extract) in ELISA assay at the same level as undiluted NS1 conditioned medium (maximum absorbance 0.01, data not shown). The monoclonal antibody (6F3) was used in a competition assay against three variations of the P S T A I R monomer (peptides 2-4), and the reactions indicated that the monoclonal antisera were reacting specifically to the carboxyl end of the peptide (Fig. 3).

169 100

Discussion

Our main difficulty was the poor immune response to the synthetic peptide which may have been because the peptide represents an evolutionarily conserved sequence present in all eukaryotic ceils. The immune response in rabbits and mice injected with the KLH-conjugated PSTAIR peptide as well as the unconjugated monomeric peptide was variable between animals and with very few antibodies being produced that reacted with the P S T A I R monomer. The octameric MAP form of the peptide gave the best

A 1

2

1

2

3

i

[

,

80

/ 1///

z

6o

~

40

g

,

2(3

I 1 10 50 100 COMPETING ANTIGEN CONCENTRATION (,uM)

B 3 4

,

4

t

Fig. 2. Immunoblot of monoclonal antibody to p34 cdc2 as expressed in BL21 (DE3). A: SDS-PAGE showing proteins from BL21 (DE3) expressing p34 cdc2 and stained with Coomassie Blue. Arrow indicates p34 cdc2. Molecular mass (M r) markers shown are 66, 45, 36, 29, 24, 20 kDa from top to bottom. B: immunoblot of proteins from BL21 (DE3) expressing p34 cdc2 (1), total proteins (16 /xg) from BL 21 (DE3) containing pRK171 vector but without p34 cJc2 gene insert (2), total proteins (12 ~g) from BL21 (DE3) without the pRK171 vector (3), total proteins (16/xg) from E. coli, JM 83 (4).

Fig. 3. Inhibition of hybridoma monoclonal antibody 6F3 diluted 1/32 to octamer by PSTAIR monomeric peptide and 3 variations (peptides 2-4). The ELISA plates were coated with 100 ng octamer MAP/well. Antibodies were allowed to react with zx, PSTAIR monomer; D, peptide 2; A, peptide 3; m, peptide 4; ©, BSA. Values are expressed as the mean from one representative experiment with duplicate assays. The experiment was repeated twice.

immune response. A total of three fusions each using the spleen from one mouse were performed for hybridoma cell production. Although only one of the three fusions was successful in hybridoma cell production, the polyclonal antisera from all three mice had reacted to p34 cdc2 by Western blot. Conjugation of a carrier protein through a linker to a hapten immunogen is commonly used to enhance immunogenicity of the synthetic peptides, but there has only been one report of the linker acting as a better immunogen than the haptenic peptide (Peeters et al., 1989). When the peptide used in conjugation is evolutionarily conserved as was the situation in this report, it is probably much more likely that the linker, rather than the conserved peptide, will be an immunogenic antigen. In such cases it is likely that the MAPs system which presents a high molecular weight form of the peptide, devoid of linkers or carrier, will be more successful. The octameric MAP form was critical for us in obtaining a monoclonal antibody to the PSTAIR peptide representing a conserved sequence of p34 cdc2.

170

Acknowledgements We thank Mary Ann Guaragna and Jeanne Williams for excellent technical assistance. Peptide syntheses were performed by Sara Bianc a l a n a in t h e M i l l i G e n / B i o s e a r c h research laboratories of Millipore Corporation.

References Del-Giudice, G., Tougne, C., Louis, J.A., Lambert, P.H., Bianchi, E., Conelli, R., Chiapinelli, L. and Pessi, A. (1990) A multiple antigen peptide from the repetitive sequence of the Plasmodium malariae circumsporozoite protein induces a specific antibody response in mice of various H-2 haplotypes. Eur. J. Immunol. 20, 1619. Gould, K.L. and Nurse, P. (1989) Tyrosine phosphorylation of the fission yeast cdc2 + protein kinase regulates entry into mitosis. Nature 342, 39. Harlow, E. and Lane, D. (1988) In: E. Harlow and D. Lane (Eds.), Antibodies, A Laboratory Manual. Cold Spring Harbor, Laboratories, Cold Spring Harbor, NY, pp. 82, 125, 153. Hsu, H.T. and Lawson, R.H. (1985) Comparison of mouse monoclonal antibodies and polyclonal antibodies of chicken egg yolk and rabbit for assay of Carnation etched ring virus. Phytopathology 75, 778. Hudson, D., Johnson, C.R., Barany, G., Biancalana, S., Calnan, B.J., Frankel, A.D., Cohn, W.B., Hayes, T., Dahl, C., Markus, M.A., Weiss, M.A., Hammer, R.P., Hsu, H.T., Jordan, R., Kamo, K.K., Lyttle, M.H., Toll, L., Tsou, D.S. and Wright, P.B. (1992) Tactics and strategies in solidphase peptide synthesis: New directions, methods and applications. In: Proceedings of the Innovations and Perspectives in Solid-Phase Peptide Synthesis Conference, Canterbury, England, in press. Jordan, R.L. (1990) Strategy and techniques for the production of monoclonal antibodies. In: R.O. Hampton, E. Call and S. DeBoer (Eds.), Serological Detection and Identification of Plant Viral and Bacterial Pathogens: A Laboratory Manual. American Pathological Society Press, Washington, DC, p. 55. Kitagawa, T. and Aikawa, T. (1976) Enzyme coupled immunoassay of insulin using a novel coupling reagent. J. Biochem. 79, 233. Kitajima, S., Seto-Ohshima, A., Sano, M. and Kato, K. (1983) Production of antibodies to calmodulin in rabbits and enzyme immunoassays for calmodulin and anti-calmodulin. J. Biol. Chem. 94, 559. Laemmli, U.K. (1970) Cleavage of structural proteins during

the assembly of the head of bacteriophage T4. Nature 227, 680. Lee, M.G. and Nurse, P. (1987) Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327, 31. Peeters, J.M., Hazendonk, T.G., Beuvery, E.C. and Tesser, G.I. (1989) Comparison of four bifunctional reagents for coupling peptides to proteins and the effect of the three moieties on the immunogenicity of the conjugates. J. Immunol. Methods 120, 133. Sacks, D.B., Porter, S.E., Ladenson, J.H. and McDonald, J.M. (1991) Monoclonal antibody to calmodulin: development, characterization and conparison with polyclonal anticalmodulin antibodies. Anal. Biochem. 194, 369. Studier, F.W., Rosenberg, A.H., Dunn, J.J. and Dubendorff, J.W. (1990) Use of T7 RNA polymerase to direct the expression of cloned genes. In: D.V. Goeddel (Ed.), Methods in Enzymology, Vol. 185, Academic Press, San Diego, CA, p. 60. Tam, J.P. (1988) Synthetic peptide vaccine design: synthesis and properties of a high density multiple antigenic peptide system. Proc. Natl. Acad. Sci. USA 85, 5409. Tam, J.P. (1989) Multiple antigen peptide system: a novel design for synthetic peptide vaccine and immunoassay. In: J.P. Tam and E.T. Kaiser (Eds.) Synthetic Peptides: Approaches to Biological Problems. Alan R. Liss, New York, p. 3. Troalen, F., Razafindratsita, A., Puisieux, A., Voeltzel, T., Bohuon, C., Belier, D. and Bidart, J.-M. (1990) Structural probing of human lutropin using antibodies raised against synthetic peptides constructed by classical and multiple antigen peptide system approaches. Mol. Immunol. 27, 363. Van Eldik, L.J. and Watterson, J.M. (1981) Reproducible production of antiserum against vertebrate calmodulin and determination of the immunoreactive site. J. Biol. Chem. 256, 4205. Wallace, R.W. and Cheung, W.Y. (1979) Calmodulin-production of an antibody in rabbit and development of a radioimmunoassay. J. Biol. Chem. 254, 6564. Winkler, M.A., Zysk, J.R. and Cheung, W.Y. (1987) Production and characterization of a monoclonal antibody crossreactive with calmodulin, calmodulin-dependent phosphodiesterase and protein phosphatase. In: A.R. Means and P.M. Conn (Eds.), Methods in Enzymology, Vol. 139. Academic Press, San Diego, CA, p. 505. Yamashita, M., Yoshikuni, M., Hirai, Y., Fukada, S. and Nagahama, Y. (1991) A monoclonal antibody against the PSTAIR sequence of p34 cdc2, catalytic subunit of maturaton-promoting factor and key regulator of the cell cycle. Dev. Growth Differ. 33, 617. Zavala, F. and Chai, S. (1990) Protective anti-sporozoite antibodies induced by a chemically defined synthetic vaccine. Immunol. Lett. 25, 271.