Detection of anti-p53 antibodies by ELISA using p53 synthetic or phage-displayed peptides

Journal of Immunological Methods 259 Ž2002. 65–75 www.elsevier.comrlocaterjim

Detection of anti-p53 antibodies by ELISA using p53 synthetic or phage-displayed peptides Jean-Michel Portefaix a , Cristina Fanutti b, Claude Granier a , Evelyne Crapez c , Richard Perham b, Jean Grenier c , Bernard Pau a , Maguy Del Rio a,) a

b

CNRS-UMR 5094, Centre de Recherche en Cancerologie, CRLC Val d’Aurelle, Montpellier, France ´ Department of Biochemistry, Cambridge Centre for Molecular Recognition, UniÕersity of Cambridge, 80, Tennis Court Road, Cambridge CB2 1GA, UK c Laboratoire de Radioanalyse, Centre de Recherche en Cancerologie, CRLC Val d’Aurelle, Montpellier, France ´ Received 15 February 2001; received in revised form 15 June 2001; accepted 30 July 2001

Abstract Anti-p53 antibodies have been detected in the sera of patients with various types of cancers. In this report, we describe the development of a new ELISA aimed at detecting anti-p53 antibodies using two peptides belonging to immunodominant epitopes of the p53 N-terminal region. We first tested the reactivity of the sera by an indirect ELISA using the peptides as a capture system. Then, the specificity of the reaction was confirmed by an inhibition assay. Two systems of peptide presentation, phage display and the streptavidinrbiotin system, were evaluated. Using a panel of sera from cancer patients, both systems were found to be equally reliable, demonstrating that both peptide-based ELISAs can be used for the specific detection of anti-p53 antibodies. The presence of anti-p53 antibodies was associated with p53 alteration whether it be mutation or accumulation. q 2002 Elsevier Science B.V. All rights reserved. Keywords: p53; Antibodies; ELISA; Peptide; Phage display

1. Introduction Mutations in the p53 gene are one of the most common genetic alterations found in human cancer ŽHollstein et al., 1991.. The tumor suppressor gene p53 codes for a 53 kDa nuclear phosphoprotein, which is implicated in regulation of normal cell growth, division and apoptosis ŽLevine et al., 1991.. Antibodies against the p53 protein have been detected in sera from patients with various cancers ) Corresponding author. Tel.: q33-4-6761-3745; fax: q33-46761-3787. E-mail address: [email protected] ŽM. Del Rio..

ŽAngelopoulou et al., 1994.. Their clinical interest as a serological marker of malignancy has been widely demonstrated. Indeed, it has been claimed that antip53 antibodies may predict a poor prognosis ŽHoubiers et al., 1995; Peyrat et al., 1995; Bourhis et al., 1996; Lai et al., 1998., may be of some value in patient monitoring ŽAngelopoulou et al., 1997; Hammel et al., 1997., may predate the clinical detection of neoplasia ŽSchlichtholz et al., 1994; Lubin et al., 1995b; Trivers et al., 1995, 1996. and may predict response to treatment ŽBergqvist et al., 1998; Zalcman et al., 1998.. Several studies have shown a good correlation between the presence of anti-p53 antibodies, accu-

0022-1759r02r$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 0 1 . 0 0 4 9 4 - X

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mulation of the mutant protein in tumor cells and the presence of a mutation in the gene ŽWinter et al., 1993; Wild et al., 1995; von Brevern et al., 1996.. However, the presence of a mutation or overexpression does not automatically lead to a p53 immune response. Only 30–50% of patients with tumors exhibiting mutations of their p53 gene exhibit an antibody response against the protein p53 ŽLubin et al., 1995a.. The reason why 100% of patients with mutated p53 protein have not been found to be positive for anti-p53 antibodies has not yet been elucidated. One explanation could be that the methodologies currently available are not sufficiently sensitive to detect serum antibodies in some patients. A second hypothesis is that the p53 antibody response is a function of the immune status of the patient and, in particular, is dependent on the specific combination of major histocompatibility complex ŽMHC. class I and II molecules expressed by each individual. Historically, the techniques for detecting anti-p53 antibodies in patient sera have been based on immunoprecipitation or immunoblotting ŽCrawford et al., 1982; Caron de Fromentel et al., 1987; Labrecque et al., 1993.. More recently, using p53 protein as antigen, several ELISA procedures have been developed, thereby permitting the evaluation of a large series of patients ŽAngelopoulou et al., 1994; Lubin et al., 1995a.. Here, we describe the generation of new ELISAs for the detection of anti-p53 antibodies in human sera. These ELISAs are based on the use of two peptides belonging to the immunodominant epitopes of the N-terminus of the p53 protein. Two different presentations of the peptides were tested, synthetic biotinylated peptides complexed with streptavidin or genetically engineered peptides displayed on the surface of filamentous bacteriophages ŽMalik and Perham, 1996.. Using both formats, we were able to specifically detect anti-p53 antibodies in the sera of patients with various types of cancer. 2. Materials and methods 2.1. Sera Sera from 26 patients with esophageal cancer, from 27 patients with rectal cancer and from 33

patients with lung cancer were collected at the time of diagnosis and prior to treatment. The study also included 40 sera from patients with diagnosed breast cancer, five sera from patients with carcinomas of unknown primary site and 50 sera from blood donors. All sera were stored at y80 8C until use. 2.2. Monoclonal antibodies We used purified mAb H447 ŽLegros et al., 1993. and mAb B17 ŽLegros et al., 1994., which recognize epitopes within the two immunodominant regions at the N-terminus of p53 protein, and mAb HR231 ŽLegros et al., 1994., directed against the C-terminus of p53. 2.3. Synthesis of soluble p53 peptides Peptides SQ, 15–29 ŽSQETFSDLWKLLPEN. and DD, 41–55 ŽDDLMLSPDDIEQWFT. were synthesized by the Fmoc strategy using an Abimed AMS 422 synthesizer. Peptides were deprotected and cleaved from the resin by trifluoroacetic acid treatment. A spacer sequence ŽGSGS. and a biotin residue were added to the N-terminus of each peptide. The same peptides without a biotin residue and acetylated at their N-terminus were also synthesized to serve as competitor peptides. A peptide from angiotensin II, DRVYIHPF, was also synthesized in both forms, i.e. acetylated and biotinylated, and used as control peptide. Peptides were lyophilized and purified to greater than 90% homogeneity by HPLC. 2.4. Construction of the phage-peptide An oligonucleotide coding for peptide SQ ŽSQET FSDLWKLLPEN. was cloned into the pfd8SHU plasmid ŽMalik and Perham, 1996.; an oligonucleotide coding for peptide DD ŽDDLMLSPDDIEQW FT. was cloned into pTfd8SHU plasmid. A DNA fragment coding for peptide SQ was also cloned into bacteriophage fdAMPLAY88. Hybrid bacteriophages were produced either by infecting cells carrying one of these plasmids with bacteriophage fdISPLAY or by infecting TG1 cells with the modified bacteriophages fdAMPLAY88-SQ bearing the SQ peptides. Double hybrid bacteriophages were also produced by infecting cells carry-

J.-M. Portefaix et al.r Journal of Immunological Methods 259 (2002) 65–75

ing the pTfd8SHU-DD plasmid with fdAMPLAY88SQ bacteriophages. Expression of the insert peptides was controlled by SDS-PAGE in 16% Žwtrvol. acrylamide gels ŽSchagger and von Jagow, 1987. and silver staining ŽMorrissey, 1981.. The virions were prepared and purified as described by Malik et al. Ž1996.. 2.5. Enzyme immunoassays for serum anti-p53 antibodies p53-ELISA: Anti-p53 antibodies were detected with a commercially available ELISA ŽPharmacell, Immunotech, Marseille, France. using microtiter plates coated with recombinant wild-type human p53 protein or control protein Žto detect non-specific interactions.. In brief, samples were diluted 1r100 and incubated for 1 h with the bound protein. A peroxidase-conjugated goat anti-human IgG was added and incubated for another hour. Bound antibody was detected by addition of a chromogenic substrate. To determine the presence of anti-p53 antibodies in samples, an index value was used according to the manufacturer’s instructions. A sample was considered positive for anti-p53 antibodies if it’s index value was greater than 1.1. Peptide-ELISA: Standard 96-well microtiter plates ŽMaxisorp, Nunc, Roskilde, Denmark. were coated overnight at 4 8C with 100 ml of a mixture of 3 mg streptavidinrml ŽBoerhinger-Mannheim, Germany. and 0.5 mgrml biotinylated peptide SQ or DD diluted in phosphate-buffered saline ŽPBS.. Plates were subsequently washed three times with washing buffer wPBS containing 0.1% Žvolrvol. Tween 20x. Excess binding sites were blocked for 90 min at 37 8C using 100 ml of a solution of 20 mM Tris–HCl, pH 7.5, 150 mM NaCl ŽTBS. containing 1% BSA. Wells were washed and 100 ml of serum diluted 1r100 in 50 mM borate buffer, pH 6.8, containing 0.1% Tween 20, 1% BSA, 5% saccharose and 10 mg streptavidinrml was added in duplicate and incubated for 90 min at 37 8C. Plates were washed four times with washing buffer, incubated for another 60 min at 37 8C with 100 ml of peroxidase-conjugated goat antihuman IgG antibodies ŽSigma, St Louis, MO, USA., diluted 1r4000 and again washed four times. The peroxidase activity retained in the wells was assayed by the addition of 100 ml of a 4 mgrml ortho-phen-

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ylene diamine solution for 20 min in the dark at room temperature. The reaction was stopped by adding 50 ml 4 N H 2 SO4 per well and the absorbance ŽA. in each well was measured at 490 nm in a microtiter plate reader ŽMR 5000, Dynatech, Denkendorf, Germany.. Each plate included a positive and a negative control. For competition experiments, each serum diluted at 1r100 Žfinal dilution. was incubated prior to the ELISA with acetylated peptide SQ or DD at 50 mgrml Žfinal concentration. for 2 h at room temperature. Then, the ELISA procedure was carried out as described above. Phage-ELISA: Microtiter plates were coated overnight at 4 8C with 100 ml of the phage solution diluted 1r10 in PBS. Non-specific binding sites were blocked by the addition of 200 ml of PBS–0.1% Tween 20 containing 5% nonfat dry milk. Then, 100 ml of serum diluted 1r100 in 50 mM borate buffer, pH 6.8 containing 0.1% Tween, 1% BSA, 5% saccharose and a 1r10 bacterial lysate containing wildtype phages was added to the plates, which were then incubated for 90 min at 37 8C. Immunoreactivity was assayed as described above for the peptideELISA. 2.6. Immunoblotting For immunoblotting experiments, wild-type human p53 protein produced in insect cells infected with a recombinant baculovirus was used. Recombinant p53 from the cell extract was denatured by boiling in SDS-PAGE buffer, then electrophoretically separated on 10% SDS-PAGE and subsequently transferred to a nitrocellulose filter ŽSchleicher and Schuell, Keene, NH. in Trisrglycine buffer w2.5 mM Tris, 192 mM glycine and 20% Žvolrvol. methanolx using the Bio-Rad electrotransfer system. The filters were cut into strips which were then incubated for 1 h at room temperature in a solution of 10 mM Tris–HCl, pH 7.5, 150 mM NaCl ŽTBS., 0.05% Žvolrvol. Tween 20 ŽTBS-T. containing 5% Žwtrvol. nonfat dry milk to block nonspecific binding sites. After washing with TBS-T, each strip was incubated for 2 h at room temperature with individual patient or control serum diluted 1r50 or 1r100 in TBS-T–0.5% nonfat dry milk, followed after five washings by the addition of the pero-

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labelled primer cycle sequencing kit with 7-deazadGTP ŽAmersham Pharmacia Biotech.. The reaction mix was prepared according to the instructions provided by the manufacturer.

3. Results

Fig. 1. Reactivity of peroxidase-labeled mAb B17 with peptide SQ presented either directly coated onto the plate Žl., biotinylated and complexed with streptavidin ŽB. or displayed on the surface of bacteriophages Ž'..

xidase-conjugated goat anti-human IgG ŽSigma. and incubation for 1 h at room temperature. A tablet of 4-chloro-1-naphthol ŽSigma. in 50 ml TBS, 10 ml methanol and 0.1% Žvolrvol. H 2 O 2 was used as substrate for enzyme detection. 2.7. Immunohistochemistry For each patient, two thick cryostat sections were used to detect nuclear expression of the p53 protein with two anti-p53 mAbs, Pab1801 ŽOncogen Research Products, Germany. and DO7 ŽDAKO, Denmark.. The alkaline phosphatase anti-alkaline phosphatase ŽAPAAP. staining procedure was used ŽCordell et al., 1984. and a tumor sample which showed strong nuclear staining with the two anti-p53 mAbs was used as a positive control. 2.8. Detection of p53 mutation Direct sequencing of complete p53 cDNA was carried out using the Thermo Sequenase fluorescent

Previous studies have indicated that two immunodominant regions localized in the amino terminus of the p53 protein, 11–35 ŽEPPLSQETFSDLWKL LPENNVLSPL. and 41–60 ŽDDLMLSPDDIEQ WFTEDPGP. are recognized by serum antibodies ŽLubin et al., 1993.. In an attempt to more precisely define the epitopes recognized by anti-p53 antibodies, decapeptides, overlapping by nine amino acids and corresponding to the N-terminal part of the p53 protein, were synthesized using Spot technology ŽFrank, 1992; Molina et al., 1996.. Two sera and purified IgG from p53 antibody-positive patients were found to recognize peptides containing residues ŽFSDLW. and residues ŽDIEQWF. Žunpublished results.. On the basis of these results, we synthesized two peptides containing the following interacting regions: peptide SQ ŽSQETFSDLWKLLPEN. and peptide DD ŽDDLMLSPDDIEQWFT.. In a second step, we developed immunoassays based on the reactivity of these two peptides. We tested three different presentations of the peptides: peptides coated alone, biotinylated peptides complexed with streptavidin and peptides displayed on the surface of filamentous bacteriophages. 3.1. Analysis of phages displaying p53 peptides Four types of phage peptides were constructed. All peptides were cloned in position q3 of the pVIII protein. In constructions 1 and 2, peptides SQ and DD were expressed by plasmids pfd8SHU and PTfd8SHU, respectively. In construction 3, peptide SQ was incorporated directly into the genome of

Fig. 2. Detection of anti-p53 antibodies in human serum samples. ŽA. Immunoblotting using wild-type human p53 protein. The sera were diluted 1r50; mAb HR231 was diluted 1r5000. C y represents a conjugate control Žwithout serum. and C q is a p53 antibody positive serum. ŽB. Peptide-ELISA: reactivity of human sera diluted 1r100 and tested on two p53 peptides and on an irrelevant peptide. ŽC. Inhibition of the peptide-ELISA assay. Human sera diluted 1r100 Žfinal dilution. were preincubated with acetylated peptide SQ ŽB. or acetylated peptide DD ŽI. at 50 mgrml Žfinal concentration. and tested for reactivity with the respective peptide.

J.-M. Portefaix et al.r Journal of Immunological Methods 259 (2002) 65–75

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fdAMPLAY88. This latter construction gave a better expression of the insert SQ than construction 1 and was therefore considered for the production of SQ hybrid bacteriophages. Consequently, construction 4, corresponding to the double hybrid display of both peptides, was produced by infecting cells carrying the pTfd8SHU-DD plasmid with fdAMPLAY-SQ phage. Control of the expression was checked by electrophoresis. The amount of recombinant expressed peptide was low compared to wild-type pVIII Ždata not shown.. The copy number of the displayed peptides in each construction was estimated by N-terminal sequencing and was found to be 5% for both peptides.

and on an irrelevant peptide. As shown in Fig. 2ŽB., the same two sera, 729 and 741, contained anti-p53 antibodies and specifically recognized peptide SQ and DD, respectively. All the sera from healthy donors and three of the sera from lung cancer patients gave a similar signal with the three peptides, indicating that their reactivity was not specific for p53. The specificity of recognition by sera 729 and 741 was confirmed by an inhibition assay involving the preincubation of the serum with the homologous

3.2. Influence of peptide presentation on mAb recognition First, we evaluated the reactivity of two p53 peroxidase-labelled mAbs, B17 and H447, with peptides SQ and DD in three different presentations. These mAbs recognize epitopes FSDLW and DIEQW, respectively ŽPortefaix et al., 2000.. The results show ŽFig. 1. that mAb B17 reacted strongly with peptide SQ when presented by the bacteriophage or when complexed with streptavidin. A weak reactivity was observed when the peptide was passively adsorbed onto the plate. The same kinds of results were obtained with peptide DD and mAb H447 Ždata not shown.. These data suggest that a particular peptide presentation is necessary for recognition. 3.3. Peptide-ELISA In a preliminary study, using streptavidinrbiotinylated peptide coated plates Žcalled peptideELISA., we tested the binding on peptides of 10 serum samples Žfive from diagnosed lung cancer patients and five from healthy donors.. The presence of anti-p53 antibodies in these sera was first determined by performing an immunoblot assay on human recombinant p53 protein. The sera were tested at a dilution of 1r50. Only sera 729 and 741 reacted with p53 protein ŽFig. 2ŽA... Then, the reactivity of the sera was tested in parallel on peptides SQ, DD

Fig. 3. Phage-ELISA: ŽA. reactivity of human sera from cancer patients and healthy donors diluted 1r100 and tested on phage fdAMPLAY-SQ without preincubation ŽI. or after preincubation of sera with peptide SQ ŽB. or peptide DD Ž .. ŽB. Comparison of reactivity of human sera diluted 1r100 and tested with peptide SQ either biotinylated ŽB. or displayed on the surface of bacteriophages ŽI.. All serum samples were tested in duplicate, in two experiments, and inhibition studies were always performed in parallel, in the same plate.

Tumor type

Ob O Rc R O O R

Serum number

9501 9502 9614 9617 9621 9625 9716

p53-ELISA index

3.29 1.94 15.10 0.59 1.74 4.73 ) 20

Peptide-ELISA Peptide SQ

Phage-ELISA Peptide DD

Immunoblot

IHC a

Mutation

NEG NEG POS POS NEG POS POS

q q qqq qqq y qqq qqq

y q q q y q y

Peptide SQ

A Ž490 nm.

Inhibition Ž%.

A Ž490 nm.

Inhibition Ž%.

A Ž490 nm.

Inhibition Ž%.

0.50 0.40 1.90 1.00 0.37 0.57 3.40

60 0 95 87 0 81 96

0.20 0.30 0.98 0.09 0.40 0.29 0.19

0 0 94 0 0 90 88

0.52 0.17 1.30 0.73 0.29 0.23 3.5

82 0 86 64 0 76 85

Twenty-six patients with esophageal cancer and 27 patients with rectal cancer were tested. Only the results from positive sera are shown. a Immunohistochemistry: q -10%, qq - 20% and qqq ) 20%. b O, oesophagus. c R, rectum.

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Table 1 Evaluation of anti-p53 antibodies in human sera by three ELISA procedures and by immunoblotting and correlation with p53 status

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acetylated peptide prior to ELISA. The results in Fig. 2ŽC. show that after preincubation with the peptide, the ELISA reactivity of these sera was significantly reduced Ž59% and 93%, respectively., demonstrating that their reactivity was specific for p53 peptides. 3.4. Phage-ELISA The ELISA procedure using the peptides displayed on the surface of the filamentous bacteriophage Žcalled phage-ELISA. was also evaluated by checking the reactivity of five anti-p53 positive sera from cancer patients and four sera from healthy donors. The results in Fig. 3ŽA. show that the antibody-positive sera reacted strongly with phagepeptide SQ. Moreover, only the homologous acetylated peptide was able to inhibit this reactivity, demonstrating that the serum antibodies were specifically directed against p53 peptide. Results obtained with other constructions Žphages expressing peptide DD or phages with double display. gave very similar results Ždata not shown.. Next, the sensitivity of the two ELISA formats, i.e. peptide-ELISA and phageELISA, were compared using the same sera. As shown in Fig. 3ŽB., the phage ELISA was slightly more sensitive than the peptide-ELISA. Taken together, these preliminary results demonstrate that peptide-ELISA, as well as the phageELISA, can detect anti-p53 antibodies in a specific manner. 3.5. EÕaluation using human sera We then assayed for the presence of anti-p53 antibodies in sera from a larger series of well-characterized patients Ž26 patients with esophageal cancer and 27 patients with rectal cancer. whose p53 status was known, i.e. p53 mutation, p53 accumulation and anti-p53 antibodies Ždetected with a com-

mercially available ELISA. ŽCrapez et al., manuscript in preparation. and in 50 sera from healthy blood donors. We considered as p53 antibody-positive, a serum whose reactivity with at least one p53 peptide was inhibited by 50% or more by the homologous acetylated peptide. As shown in Table 1, the sera from seven cancer patients were positive in at least one of the ELISA formats. Four sera Ž9501, 9614, 9625 and 9716. were positive by the three ELISA procedures, sera 9502 and 9621 were positive only in the p53-ELISA and 9617 only in the peptide-ELISA and the phage-ELISA. In addition, questionable positive, i.e. different results in the three ELISAs, and some of the negative ELISA results were verified by immunoblotting using wild-type p53 as antigen: the presence of anti-p53 antibodies was confirmed in sera 9614, 9617, 9625 and, 9716 but not in sera 9501, 9502 and 9621 ŽTable 1.. The sera from healthy donors did not give any signal either by ELISA or by immunoblotting. Both ELISA procedures using peptides gave exactly the same results but some discrepancies were observed between the p53-ELISA and the peptide-ELISA. As shown in Table 2, the concordance of the results between detection of anti-p53 antibodies by the peptideELISA and accumulation of altered p53 andror mutated p53 gene was 41.5% wboth positive Ž9.5%. and both negative Ž32%.x. An additional 58.5% of patients presented with a p53 alteration and negative serology. Interestingly, we never found anti-p53 antibodies in sera from patients devoid of p53 alterations, demonstrating the high specificity of our assay. The peptide-ELISA was also used to assess the presence of anti-p53 antibodies in sera from lung cancer patients and from breast cancer patients. We found 5r33 and 6r40 anti-p53 positive sera in lung cancer patients Ž15.1%. and breast cancer patients Ž15%., respectively Ždata not shown..

Table 2 Relationship between serology and p53 alteration Detection of p53 antibodies by phage- or peptide-ELISA

Positive

Accumulation of p53 protein andror mutation of the p53 gene Negative

Total

Positive Negative Total

5 Ž9.5%. 31 Ž58.5%. 36 Ž68%.

0 Ž0%. 17 Ž32%. 17 Ž32%.

5 Ž9.5%. 48 Ž90.5%. 53 Ž100%.

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4. Discussion p53 antigenic sites recognized by serum antibodies were studied by Lubin et al. Ž1993. and Schlichtholz et al. Ž1994. using peptide scanning analysis ŽPEPSCAN.. Briefly, 77 peptides Ž15-mer overlapping by 10 amino acids. spanning the entire human p53 sequence were used for serum screening. These authors demonstrated that anti-p53 antibodies are predominantly directed against epitopes in the highly antigenic amino-terminal region of the p53 protein ŽSchlichtholz et al., 1994. and that the majority of sera Ž98%. react with one of the amino-terminal peptides ŽLubin et al., 1993.. By using Spot technology, we confirmed that most peptides recognized by p53 antibody-positive sera contain the sequences FSDLW and DIEQWF, which are also the targets of various recently characterized p53-specific mAbs ŽPortefaix et al., 2000.. In the present report, we describe the development of new ELISA procedures using two peptides belonging to the immunodominant regions of the amino-terminal part of the p53 protein. The peptides were prepared either as biotinylated synthetic peptides or displayed on the surface of filamentous bacteriophages as described by Malik and Perham Ž1997.. Indeed, it has been shown that a high density of peptides can be displayed when the peptides are incorporated into the major coat pVIII protein of filamentous bacteriophages ŽPerham et al., 1995.. Furthermore, peptides displayed in this way are remarkably effective structural mimics of the natural epitope Ždi Marzo Veronese et al., 1994.. Our results indicate that peptide recognition is dependent on the manner of presentation. Peptides were correctly recognized when expressed on the phage surface or when presented by the streptavidinrbiotin system, but not when they were presented coated directly, probably due to an incorrect conformation of the peptides andror inefficient coating. We investigated the sensitivity and specificity of the peptide-ELISA and phage-ELISA using samples from a series of healthy donors and patients with various types of cancer. The series of esophageal and rectal carcinoma patients had a well-characterized p53 status Žp53 mutation, p53 accumulation and anti-p53 antibodies.. The results obtained by both tests were similar, indicating that reactivity was

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specifically directed towards p53 and was independent of peptide presentation Žphage or biotinrstreptavidin.. Furthermore, binding of serum anti-p53 antibodies was blocked by the homologous peptide, whereas non-homologous peptides did not abolish the reactivity. In addition, in the series of esophageal and rectal carcinoma patients, the presence of antip53 antibodies was associated with p53 alterations. As previously demonstrated ŽLubin et al., 1995a., none of the sera from healthy donors were positive for anti-p53 antibodies. Similar frequencies of anti-p53 antibodies were detected by the peptide-ELISA Ž9.5%. and by the p53-ELISA Ž11.3%.. However, one serum Ž9617. was found to be positive only in the peptide-ELISA and two sera Ž9502 and 9621. were found to be positive only in the p53-ELISA. For serum 9617, anti-p53 positivity was confirmed by immunoblotting and was associated with both a p53 gene mutation and a strong p53 accumulation, demonstrating that the peptide-ELISA was more sensitive than the p53-ELISA. Sera 9502 and 9621 gave borderline results in the p53-ELISA, with index values of 1.94 and 1.74, respectively. Furthermore, for serum 9621, additional results such as a negative immunoblotting, an absence of a p53 gene mutation and p53 accumulation could suggest false positivity. It is difficult to reach a conclusion for patient 9502 because a p53 gene mutation and a weak p53 accumulation were found but immunoblotting was negative. We cannot rule out the presence of anti-p53 antibodies directed specifically against the core domain or the C-terminus of the protein even if some studies have indicated that no serum is exclusively specific for the Cterminus ŽSchlichtholz et al., 1992; Zalcman et al., 1998.. Overall, the frequency of anti-p53 antibodies in the esophageal cancer patients included in our study was 15.4%, which is lower than in previously published studies. von Brevern et al. Ž1996. in a series of 65 patients reported p53 antibody positivity in 25% of the population. Three more recent studies reported positivities of 30% ŽSobti and Parashar, 1998., 33% ŽCawley et al., 1998. and up to 58% ŽShimada et al., 1998.. All of these studies used a p53-ELISA for the detection of anti-p53 antibodies, and two of them ŽShimada et al., 1998; Sobti and Parashar, 1998. used the same p53-ELISA commer-

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cial test that was used in our study. These discordances may be dependent on the differences in population investigated andror a lack in standardization procedures. We also detected anti-p53 antibodies in 3 of 27 Ž11.1%. sera from rectal cancer patients, in 5 of 33 Ž15.1%. sera from lung cancer patients and in 6 of 40 Ž15%. sera from breast cancer patients. These frequencies are in accordance with published data. In conclusion, we have developed two inhibition ELISA tests to assay anti-p53 antibodies in serum, and we have demonstrated that both are specific for the detection of such antibodies. The frequencies of anti-p53 antibodies found were very similar in both assays, suggesting that the reactivities were very specific for the displayed peptides. The inhibition assay allowed us to eliminate false positives. Furthermore, these tests were easy to perform, although dependent on the competence of the laboratory in phage display or peptide engineering. We suggest that the development of inhibition assays will greatly help to reduce discrepancies in the results obtained by different teams of investigators.

Acknowledgements We thank Dr. L.C. Packman, Dr. C. Hill and Mr. Walden for protein sequence analysis and oligonucleotide synthesis. Mr. J. Lester for automated DNA sequencing and Mr. C. Fuller for skilled technical assistance. We are grateful to Dr. S.L. Salhi for critical reading of the manuscript. This work was supported by a grant from the Association pour la Recherche sur le Cancer. JMP was supported by a doctoral fellowship from this same Association.

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