WO1994010306A1 - P53 protein fragments and use thereof for detecting and monitoring diseased conditions - Google Patents

P53 protein fragments and use thereof for detecting and monitoring diseased conditions Download PDF

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Publication number
WO1994010306A1
WO1994010306A1 PCT/FR1993/001082 FR9301082W WO9410306A1 WO 1994010306 A1 WO1994010306 A1 WO 1994010306A1 FR 9301082 W FR9301082 W FR 9301082W WO 9410306 A1 WO9410306 A1 WO 9410306A1
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ser
leu
lys
seq
pro
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PCT/FR1993/001082
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French (fr)
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Thierry Soussi
Richard Lubin
Yann Legros
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Laboratoires Eurobio
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Priority to AU54246/94A priority Critical patent/AU5424694A/en
Priority to JP6510784A priority patent/JPH08502414A/en
Priority to EP93924668A priority patent/EP0666913A1/en
Publication of WO1994010306A1 publication Critical patent/WO1994010306A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes

Definitions

  • the present application relates to fragments of the p53 protein corresponding to immunodominant epitopes of the human p53 protein.
  • Mutations in the p53 gene are the most commonly encountered gene alterations in human cancers.
  • the frequency of mutations in the p53 gene is high for cancer of the colon (1), breast (2) and lung (3) as well as in leukemias (4), osteosarcomas (5), cancers of the ovary (6), stomach (7), and brain (8).
  • Hereditary mutations have recently been demonstrated to support Li-Fraumeni family cancer syndrome
  • the applicant has surprisingly shown that the regions recognized by the antibodies of the patients do not correspond to the regions of p53 modified in cancer but to other regions of the human p53 protein which are located in the amino and carboxy-terminal ends of p53. This original result could not be predicted by any previous study.
  • WADE-EVANS and JENKINS (30) have mapped the epitopes of 4 monoclonal antibodies directed against mouse p53.
  • the results are as follows: the antibody PAb242 recognizes an epitope located between amino acids 9 and 25, the antibody PAb246 recognizes an epitope located between amino acids 88 and 109, the antibody PAb248 recognizes a epitope located between amino acids 157 and 192 and the antibody PAb421 recognizes an epitope located between amino acids 370 and 378.
  • the subject of the present invention is peptides or protein fragments, excluding the protein p53, having a specific reaction towards anti-p53 antibodies present in the biological fluids of precancerous patients or suffering from cancer whatever its origin.
  • such a peptide is included in the amino acid sequence of the terminal amino region (residues 1 to 112) or in the terminal carboxy region (residues 350 to 393) of the p53 protein.
  • the sequence of the wild-type human p53 protein is described by SOUSSI et al (26).
  • such a peptide comprises in part or in whole one of the following sequences:
  • the determination of the fragments of the p53 protein exhibiting a specific reaction vis-à-vis the antibodies of cancer patients can be done by the means made available to those skilled in the art and in particular by immunoenzymatic test, radio -immunological, by immunoprecipitation or by immunoblot.
  • the present invention also relates to a nucleotide, ribonucleotide (RNA) or deoxyribonucleotide (DNA) sequence, coding for the synthesis of one of the peptides object of the present application.
  • RNA ribonucleotide
  • DNA deoxyribonucleotide
  • Such peptides can be used for the detection or the follow-up of pathological states, and in particular for the detection or the follow-up of cancerous and precancerous states.
  • tissue or the fluid and one of the peptides which are the subject of the invention described above are brought into contact, and
  • the presence of either the antibody, the peptide or the antibody-peptide pair is determined and the corresponding assay is carried out.
  • the assay of the antibody-peptide complexes can be carried out either by assaying the antibody peptide bond, or by assaying the peptide or the antibody captured and entering the antibody-peptide complex.
  • This assay can be carried out by methods known to those skilled in the art and in particular by physical or immunological methods such as immunoenzymological, radioimmunological or chemoimmunological methods or by more recent approaches which are based on new molecular biology techniques such as than immuno-PCR.
  • the antigen-antibody complex is detected by a PCR reaction which involves an oligonucleotide linked to the revealing antibody. The complex is then visualized by electrophoresis.
  • the antibodies whose presence is determined or which are assayed are antibodies representative of pathological states and in particular of cancerous or pre-cancerous states.
  • the peptide can be fixed on a solid phase.
  • the complexes formed can be revealed using an antibody reacting specifically with the peptide or antibodies to be assayed or detected.
  • This antibody allowing the revealing of the complexes is advantageously conjugated to a marker in order to highlight the antibody-peptide complex.
  • Such a marker can be an enzyme, a chemiluminescent, bioluminescent, fluorescent or radioactive molecule.
  • the antibody peptide complex formed during the reaction is advantageously revealed using an antibody conjugated to a marker as defined above or using any molecules capable of detecting the presence of such a complex.
  • the peptides which are the subject of the present invention are preferably obtained by synthesis from amino acids by any means known to those skilled in the art.
  • They can also be obtained by genetic engineering or by enzymatic or chemical cutting of the p53 protein.
  • the antibodies allowing the revelation of the complex formed between the peptide and the antibodies are advantageously directed against antigenic determinants specific for the species from which the fluid or tissue originates.
  • the antibodies allowing the revelation of the complex will be anti-human antibodies.
  • FIG. 1 represents the interaction profiles between the fragments of the p53 protein and antibodies present in the serum of different patients with cancer.
  • the antibodies used for the hybridizations of FIGS. A to IF are respectively a CM1 rabbit serum immunized with the p53 protein, a mouse serum immunized with the p53 protein, three sera from patients who were determined to have anti-p53 antibody and a control consisting of an anti-alkaline phosphatase antibody.
  • FIGS. 2 to 7 represent the reactivity of 77 peptides corresponding to the protein p53, vis-à-vis sera of three patients with lung cancers numbered 84, 37, 109, and three patients with breast cancer numbered 57 , 27 and 29.
  • the antibody / peptide complex is revealed by colorimetry and the optical density is measured by a microplate reader.
  • FIG. 8 illustrates the frequency of recognition (on the ordinate) of 77 peptides (on the abscissa) by different sera of patients suffering from cancer.
  • HCD highly conserved domain of the p53 protein
  • the sera of 80 patients with invasive carcinoma of the breast and 20 patients with metastases were pooled after histopathological diagnosis and stored at -20 ° C.
  • mice of haplotype H-2d are immunized intraperitoneally with 100 ⁇ g of normal human p53.
  • a booster injection is given intravenously with 80 ⁇ g of protein of the same origin.
  • the splenocytes are removed and fused with NS-1 myeloma cells in the presence of polyethylene glycol (PEG 1500 Boehringer).
  • PEG 1500 Boehringer polyethylene glycol
  • HAT Hypoxanthine Aminopterin Thymine
  • the secretion of antibodies by the hybridomas was sought by an ELISA test using human p53 as antigen.
  • Hybridomas secreting antibodies capable of recognizing p53 were cloned by the limit dilution technique.
  • the H111 antibody recognizes an epitope between amino acids 291 and 300
  • the HR231 antibody recognizes an epitope between amino acids 371 and 380
  • HT 216 recognizes an epitope between amino acids 1 to 64.
  • the vector pLip4 was used for the production of the hybrid proteins.
  • This plasmid is derived from pLipl (21) into which two Sac I and Sal I restriction sites are introduced between the codons +6 and +7 of the E.coli PhOA gene. After cloning, the fusion protein PhoA is exported to the bacterial periplasm and can be extracted therefrom by cold osmotic shock (21).
  • Fragments 2 (residues 108 to 162), 3 (residues 158 to 219), 4 (residues 215 to 267) and 5 (residues 263 to 310) respectively contain the regions HCD II to V and correspond to the preferred sites for mutations ( HOT-SPOT regions) in human cancers (11,12).
  • Fragments 1 correspond to the amino- and carboxy-terminal regions of the protein and are generally away from mutations.
  • the six fragments fused to the PhoA gene can be expressed in E. coli as soluble proteins.
  • the expression yield of fusion protein with, fragment 6 is lower (2 to 5 times) than for the other fragments.
  • the antigenicity of the expressed protein is established by its reactivity with different monoclonal antibodies.
  • PAb1801 (22) and HT 216 are specific for fragment 1; the PAb240 epitope is located on fragment 3 (23). H111 is on fragment 5; HR 231 and PAb122 (24) are specific for the carboxy-terminal region of p53 (fragment 6). All these monoclonal antibodies react in immunoblot and immunoprecipitation with the fusion protein pLIP4 in accordance with the localization of their epitope (see table 2).
  • the Immunoblot was performed as described by
  • the proteins are separated by electrophoresis in polyacrylamide gel (10% acrylamide) in Tris-Glycine buffer containing 0.1% SDS, pH 8.3. The proteins are transferred to a nitrocellulose membrane (2 hours, 40 volts); the membrane is immersed in PBSTM buffer to saturate non-specific binding sites and then washed 5 times with
  • the sera are first incubated with the extract bacterial expressing alkaline phosphatase E.coli for one hour at + 4 ° C and then on a nitrocellulose membrane containing a bacterial extract (without fusion protein) 12 hours at + 4 ° C.
  • the nitrocellulose membrane with the hybrid proteins is incubated with the sera prepared and diluted from 1/100 to 1/500 for 1 hour in PBSTM buffer at room temperature. After 5 washes in PBSTM, the membranes are incubated for 1 hour with a peroxidase conjugated anti-mouse rabbit antibody (diluted 1/4000 in PBSTM buffer), washed 5 times in PBS buffer and revealed by Luminol (Amersham R ).
  • the filters are checked by immunological reaction (Immunoblot) for the quantity of fusion protein actually deposited by incubating the membrane with an anti-alkaline phosphatase antibody.
  • CM1 is a rabbit polyclonal antibody obtained by immunization with highly purified human p53 and frequently used in immunocytochemistry (24). Western blot techniques have shown that the CM1 serum contains antibodies which mainly recognize fragments 1 and 6 and to a lesser degree fragment 5. Similar results are obtained by immunoprecipitation.
  • a control experiment with an anti-alkaline phosphatase antibody makes it possible to establish that identical quantities of the various fusion proteins are indeed present on the gel (FIG. 1).
  • amino terminal part of p53 contains one or more dominant epitopes involved in the response.
  • the terminal carboxy region also contains one or more immunodominant epitopes, but their importance is less compared to the amino terminal region.
  • microtiter plates used are 96-well flat bottom polyvinyl chloride plates. Ref: M129B, Dynatech company.
  • a series of overlapping peptides (length of 15 amino acids (aa), overlap of 10 aa) covering the entire p53 protein.
  • the first 76 peptides from the N-terminus are 15 amino acids in length each and the 77th peptide only comprises 13 amino acids (SEQ ID NO: 15).
  • control peptides not corresponding to the p53 protein are used as a negative control.
  • streptavidin concentration 5 ⁇ g / ml in distilled water
  • the plates are then incubated at 37oC in a dry oven for 48 hours. After this dehydration stage, they can be individually wrapped and stored for several months at 4 ° C.
  • the dehydrated plates are washed 5 times with PBS (phosphate buffered saline) containing 0.05% Tween 20.
  • PBS phosphate buffered saline
  • This PBS buffer with Tween (PBS-T) will be used throughout the experiment as a washing solution.
  • the 5 washes are carried out in the usual way using 200 ⁇ l of PBS-T buffer per well.
  • the plate After the last wash, the plate is dried on absorbent paper. All washes are done this way.
  • the plates are then incubated for 1 hour at 37 ° C with gentle shaking and are then washed as indicated above.
  • peptide solution 125 nanograms of peptide diluted in PBS buffer containing 0.1% bovine serum albumin and 0.02% sodium azide are added to each well.
  • Each well receives a different peptide corresponding to a specific region of p53.
  • the plates are incubated for 1 hour at 20oC with gentle shaking and are then washed as indicated above.
  • test sera are diluted 1/50 in a PBS solution containing 5% milk.
  • the plates are incubated for 1 hour at 20 ° C. with gentle shaking and are then washed as indicated above.
  • the plates are incubated for 30 minutes at 37 ° C with gentle shaking and are then washed as indicated above.
  • the plates are incubated at 20 ° C with gentle shaking.
  • the result is read in a reading device
  • Example 1 Two phenomena were highlighted.
  • the anti-p53 antibodies mainly recognize a region of 112 amino acids located in the amino terminal part of p53 as well as a carboxy terminal region located between residues 306-393. The study could not provide more detailed information on the region recognized by anti-p53 antibodies.
  • PA pancreatic cancer
  • OV ovarian cancer
  • L leukemia
  • PR prostate cancer.
  • FIG. 8 illustrates the frequencies obtained for each of these peptides.
  • This histogram shows the frequency of recognition of each peptide by the 47 sera studied (see Table 5). It shows, for example, that 40% of the sera recognize at least peptide 3 and that 63% recognize at least peptide 4. 98% of the sera recognize at least one of the 5 peptides of the amino terminal region (N-ter) while only 47% recognize one of the peptides of the carboxy terminal region (C-ter). Of course, 100% of the sera recognize one of the 13 peptides described in this table.

Abstract

A p53 protein fragment peptide specifically reacting with antibodies to p53 in the body fluids of cancer patients or precarcinomatous individuals. The peptide is preferably included in amino acid sequence 1-112 or 350-393 of protein p53, and may be used for detecting or monitoring diseased conditions, particularly carcinous conditions.

Description

FRAGMENTS DE LA PROTEINE p53 ET LEURS UTILISATIONS DANS LA DETECTION ET LE SUIVI D'ETATS PATHOLOGIQUES  FRAGMENTS OF PROTEIN p53 AND THEIR USES IN THE DETECTION AND MONITORING OF CONDITIONS
La présente demande a pour objet des fragments de la protéine p53 correspondants à des épitopes immunodominants de la protéine p53 humaine.  The present application relates to fragments of the p53 protein corresponding to immunodominant epitopes of the human p53 protein.
Elle est d'autre part relative à leurs utilisations dans la détection et le suivi d'états pathologiques et en particulier d'états cancéreux et pré-cancéreux.  It also relates to their uses in the detection and monitoring of pathological conditions and in particular of cancerous and pre-cancerous states.
Les mutations du gène p53 sont les altérations géniques les plus souvent rencontrées dans les cancers humains. La fréquence des mutations du gène p53 est élevée pour le cancer du colon (1), du sein (2) et du poumon (3) de même que dans les leucémies (4), les ostéosarcomes (5), les cancers de l'ovaire (6), de l'estomac (7), et du cerveau (8). Des mutations héréditaires ont été récemment mises en évidence comme support du syndrome du cancer familial de Li-Fraumeni Mutations in the p53 gene are the most commonly encountered gene alterations in human cancers. The frequency of mutations in the p53 gene is high for cancer of the colon (1), breast (2) and lung (3) as well as in leukemias (4), osteosarcomas (5), cancers of the ovary (6), stomach (7), and brain (8). Hereditary mutations have recently been demonstrated to support Li-Fraumeni family cancer syndrome
(9,10). Rapportées aux 10 cancers les plus fréquents dans le monde, les altérations du gène p53 sont retrouvées chez 40 à 45% des cancéreux. Les altérations les plus fréquentes sont des mutations ponctuelles situées dans 4 des 5 régions phylogénétiquement conservées (HCD) de la protéine p53 (11,12). (9.10). Reported to the 10 most common cancers in the world, alterations in the p53 gene are found in 40 to 45% of cancer patients. The most frequent alterations are point mutations located in 4 of the 5 phylogenetically conserved regions (HCD) of the p53 protein (11,12).
En 1982, Benchimol et al. ont montré par une technique radio-immunologique que la protéine p53 est spécifiquement surexprimée dans les cellules transformées et indécelable dans les cellules normales (13). De nombreuses études ont confirmé depuis ces résultats et montré que l'accumulation de la protéine p53 est due à sa stabilisation . On sait maintenant que cette stabilisation est due dans presque tous les cas à une mutation ponctuelle qui modifie la conformation et la stabilité de la protéine. Ces observations encouragent l'étude systématique par immunohistologie de l'expression de la protéine p53 sur une large gamme de tumeurs car il semble y avoir une bonne corrélation entre la mutation du gène p53 et l'accumulation de la protéine (14, 15). In 1982, Benchimol et al. have shown by radioimmunoassay that the p53 protein is specifically overexpressed in transformed cells and undetectable in normal cells (13). Many studies have since confirmed these results and shown that the accumulation of the p53 protein is due to its stabilization. We now know that this stabilization is due in almost all cases to a point mutation which modifies the conformation and the stability of the protein. These observations encourage systematic study by immunohistology expression of the p53 protein over a wide range of tumors as there appears to be a good correlation between the mutation in the p53 gene and the accumulation of the protein (14, 15).
En 1982, Crawford et al. ont détecté des anticorps anti-p53 chez des patients présentant un cancer du sein (16). Caron de Fromentel et al. ont ensuite montré en 1987 que de tels anticorps étaient présents dans le sérum d'enfants présentant des cancers très divers (17). La fréquence moyenne est de 12% avec une fréquence particulière de 20% dans le lymphome de Burkitt (17). Plus récemment, Davidoff et al. (18) ont montré que la présence d'anticorps anti-p53 est associée avec des mutations spécifiques des exons 5 et 6 du gène. Ces protéines mutées sont connues pour s'associer à la protéine hsp70 et sont encore plus oncogéniques in vitro et in vivo.  In 1982, Crawford et al. have detected anti-p53 antibodies in patients with breast cancer (16). Caron de Fromentel et al. then showed in 1987 that such antibodies were present in the serum of children with very diverse cancers (17). The average frequency is 12% with a specific frequency of 20% in Burkitt's lymphoma (17). More recently, Davidoff et al. (18) have shown that the presence of anti-p53 antibodies is associated with specific mutations of exons 5 and 6 of the gene. These mutated proteins are known to associate with the hsp70 protein and are even more oncogenic in vitro and in vivo.
L'importance de ces anticorps sériques a été récemment soulevée par la découverte que leur présence est généralement corrélée avec un mauvais pronostic pour le patient.  The importance of these serum antibodies has recently been raised by the discovery that their presence is generally correlated with a poor prognosis for the patient.
La partie de la protéine p53 humaine présentant le plus de mutations étant située environ au milieu de la séquence de la protéine p53, on aurait pu supposer que les anticorps de patients atteints de cancer et produisant une telle protéine mutée reconnaissent spécifiquement cette partie centrale de la protéine présentant des mutations. En effet, cette région de la protéine p53 humaine étant différente chez des patients atteints de cancers, il semblait logique de concevoir que la réponse immunitaire de l ' organisme serait préférentiellement dirigée contre la région modifiée.  The part of the human p53 protein with the most mutations being located approximately in the middle of the p53 protein sequence, it could have been supposed that the antibodies of cancer patients and producing such a mutated protein specifically recognize this central part of the protein with mutations. Indeed, since this region of the human p53 protein is different in cancer patients, it seemed logical to imagine that the body's immune response would preferentially be directed against the modified region.
Le demandeur a montré de manière surprenante que les régions reconnues par les anticorps des patients ne correspondent pas aux régions de la p53 modifiées dans les cancers mais à d'autres régions de la protéine p53 humaine qui sont situées dans les extrémités amino et carboxy-terminales de la p53. Ce résultat original ne pouvait être prédit par aucune étude antérieure. The applicant has surprisingly shown that the regions recognized by the antibodies of the patients do not correspond to the regions of p53 modified in cancer but to other regions of the human p53 protein which are located in the amino and carboxy-terminal ends of p53. This original result could not be predicted by any previous study.
On notera que dans les années 1980-1985, de nombreux anticorps monoclonaux murins ont été produits soit contre la p53 murine, soit contre la p53 humaine. Deux études ont porté sur la caractérisation de ces anticorps monoclonaux. Banks et al. (22) ont décrit la production d'anticorps monoclonaux murins dirigés contre la p53 humaine. L'un de ces anticorps (PAb1801) est très utilisé dans divers laboratoires à l'heure actuelle. En utilisant des protéines p53 tronquées, ces auteurs ont montré que l'épitope reconnu par pAb1801 ou Pab1803 est compris entre les acides aminés 32 et 79.  It will be noted that in the years 1980-1985, numerous murine monoclonal antibodies were produced either against murine p53 or against human p53. Two studies focused on the characterization of these monoclonal antibodies. Banks et al. (22) described the production of murine monoclonal antibodies against human p53. One of these antibodies (PAb1801) is widely used in various laboratories today. By using truncated p53 proteins, these authors have shown that the epitope recognized by pAb1801 or Pab1803 is between amino acids 32 and 79.
Dans un autre article, WADE-EVANS et JENKINS (30) ont cartographié les épitopes de 4 anticorps monoclonaux dirigés contre la p53 de souris. Pour cette étude, ils ont utilisé des fragments de protéine p53 murine. Les résultats sont les suivants: l'anticorps PAb242 reconnaît un épitope situé entre les acides aminés 9 et 25, l'anticorps PAb246 reconnaît un épitope situé entre les acides aminés 88 et 109, l'anticorps PAb248 reconnaît un épitope situé entre les acides aminés 157 et 192 et l'anticorps PAb421 reconnaît un épitope situé entre les acides aminés 370 et 378.  In another article, WADE-EVANS and JENKINS (30) have mapped the epitopes of 4 monoclonal antibodies directed against mouse p53. For this study, they used murine p53 protein fragments. The results are as follows: the antibody PAb242 recognizes an epitope located between amino acids 9 and 25, the antibody PAb246 recognizes an epitope located between amino acids 88 and 109, the antibody PAb248 recognizes a epitope located between amino acids 157 and 192 and the antibody PAb421 recognizes an epitope located between amino acids 370 and 378.
L'ensemble de ces études montre que les épitopes reconnus par ces anticorps monoclonaux sont répartis sur tout le long de la protéine.  All of these studies show that the epitopes recognized by these monoclonal antibodies are distributed along the entire length of the protein.
La présente invention a pour objet des peptides ou des fragments protéiques, à l'exclusion de la protéine p53, présentant une réaction spécifique vis- à-vis des anticorps anti-p53 présents dans les fluides biologiques de patients précancéreux ou atteints d'un cancer quel que soit son origine . The subject of the present invention is peptides or protein fragments, excluding the protein p53, having a specific reaction towards anti-p53 antibodies present in the biological fluids of precancerous patients or suffering from cancer whatever its origin.
Avantageusement , un tel peptide est compris dans la séquence des acides aminés de la région amino terminale (résidus 1 à 112) ou dans la région carboxy terminale (résidus 350 à 393) de la protéine p53. La séquence de la protéine p53 humaine sauvage est décrite par SOUSSI et al (26).  Advantageously, such a peptide is included in the amino acid sequence of the terminal amino region (residues 1 to 112) or in the terminal carboxy region (residues 350 to 393) of the p53 protein. The sequence of the wild-type human p53 protein is described by SOUSSI et al (26).
Il comprend avantageusement en partie ou en totalité l'une des séquences suivantes :  It advantageously comprises in part or in whole one of the following sequences:
Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu ( SEQ ID NO:1)  Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu (SEQ ID NO: 1)
Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro (SEQ ID NO : 2).  Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro (SEQ ID NO: 2).
Préférentiellement , un tel peptide comprend en partie ou en totalité l'une des séquences suivantes :  Preferably, such a peptide comprises in part or in whole one of the following sequences:
Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu (SEQ ID NO: 3)  Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu (SEQ ID NO: 3)
Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn (SEQ ID NO: 4)  Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn (SEQ ID NO: 4)
Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
Val Leu Ser Pro Leu (SEQ ID NO: 5) Val Leu Ser Pro Leu (SEQ ID NO: 5)
Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr (SEQ ID NO 6)  Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr (SEQ ID NO 6)
Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro (SEQ ID NO:7).  Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro (SEQ ID NO: 7).
Si un tel peptide est compris dans la séquence des acides aminés 350 à 393 de la protéine p53, il comprend alors préférentiellement en partie ou en totalité l'une des séquences suivantes :  If such a peptide is included in the sequence of amino acids 350 to 393 of the protein p53, it then preferentially comprises in part or in whole one of the following sequences:
Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly (SEQ ID NO : 8) Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly (SEQ ID NO: 8)
Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His (SEQ ID NO: 9)  Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His (SEQ ID NO: 9)
Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys (SEQ ID NO: 10)  Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys (SEQ ID NO: 10)
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln (SEQ ID NO: 11)  Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln (SEQ ID NO: 11)
Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His (SEQ ID NO: 12)  Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His (SEQ ID NO: 12)
Ser Lys Lys Gly Gln Ser Thr Ser Arg His Ser Lys Lys Gly Gln Ser Thr Ser Arg His
Lys Lys Leu Met Phe (SEQ ID NO: 13 ) Lys Lys Leu Met Phe (SEQ ID NO: 13)
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro (SEQ ID NO: 14)  Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro (SEQ ID NO: 14)
Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp (SEQ ID NO: 15).  Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp (SEQ ID NO: 15).
La détermination des fragments de la protéine p53 présentant une réaction spécifique vis-à-vis des anticorps de patients atteints d'un cancer peut se faire par les moyens mis à la disposition de l'homme du métier et en particulier par test immunoenzymatique, radio-immunologique, par immuno-précipitation ou par immunoblot.  The determination of the fragments of the p53 protein exhibiting a specific reaction vis-à-vis the antibodies of cancer patients can be done by the means made available to those skilled in the art and in particular by immunoenzymatic test, radio -immunological, by immunoprecipitation or by immunoblot.
De tels tests sont bien connus de l'homme du métier et sont décrits dans des manuels généraux (28,29).  Such tests are well known to those skilled in the art and are described in general manuals (28,29).
La présente invention est en outre relative à une séquence nucléotidique , ribonucléotidique (ARN) ou désoxyribonucléotidique (ADN), codant pour la synthèse d'un des peptides objet de la présente demande .  The present invention also relates to a nucleotide, ribonucleotide (RNA) or deoxyribonucleotide (DNA) sequence, coding for the synthesis of one of the peptides object of the present application.
De tels peptides peuvent être utilisés pour la détection ou le suivi d'états pathologiques, et en particulier pour la détection ou le suivi d'états cancéreux et précancéreux.  Such peptides can be used for the detection or the follow-up of pathological states, and in particular for the detection or the follow-up of cancerous and precancerous states.
Un autre objet de la présente invention est un procédé pour la détermination de la présence d'anticorps ou pour leur dosage dans un tissu ou un fluide caractérisé en ce que : Another object of the present invention is a process for the determination of the presence of antibodies or for their determination in a tissue or a fluid characterized in that:
- on met en présence le tissu ou le fluide et l'un des peptides objet de l'invention précédemment décrits , et  the tissue or the fluid and one of the peptides which are the subject of the invention described above are brought into contact, and
- on détermine la présence soit de l'anticorps, soit du peptide soit du couple anticorps-peptide et on effectue le dosage correspondant .  - the presence of either the antibody, the peptide or the antibody-peptide pair is determined and the corresponding assay is carried out.
Le dosage des complexes anticorps-peptides peut être effectué soit par dosage de la liaison anticorps peptide, soit par dosage du peptide ou de l'anticorps capté et rentrant dans le complexe anticorps-peptide.  The assay of the antibody-peptide complexes can be carried out either by assaying the antibody peptide bond, or by assaying the peptide or the antibody captured and entering the antibody-peptide complex.
Ce dosage peut être effectué par les méthodes connues de l'homme du métier et en particulier par des méthodes physiques ou immunologiques telles que les méthodes immunoenzymologiques, radioimmunologiques ou chimioimmunologiques ou par des approches plus récentes qui sont basées sur les nouvelles techniques de biologie moléculaire telles que l'immuno-PCR. Selon cette technique, le complexe antigène-anticorps est détecté par une réaction de PCR qui met en jeu un oligonucléotide lié à l'anticorps de révélation. Le complexe est ensuite visualisé par électrophorèse.  This assay can be carried out by methods known to those skilled in the art and in particular by physical or immunological methods such as immunoenzymological, radioimmunological or chemoimmunological methods or by more recent approaches which are based on new molecular biology techniques such as than immuno-PCR. According to this technique, the antigen-antibody complex is detected by a PCR reaction which involves an oligonucleotide linked to the revealing antibody. The complex is then visualized by electrophoresis.
Avantageusement, les anticorps dont on détermine la présence ou que l'on dose sont des anticorps représentatifs d'états pathologiques et en particulier d'états cancéreux ou pré-cancéreux.  Advantageously, the antibodies whose presence is determined or which are assayed are antibodies representative of pathological states and in particular of cancerous or pre-cancerous states.
Afin de mettre en oeuvre le procédé, le peptide peut être fixé sur une phase solide.  In order to carry out the method, the peptide can be fixed on a solid phase.
Les complexes formés peuvent être révélés à l'aide d'un anticorps réagissant spécifiquement avec le peptide ou les anticorps à doser ou à détecter . Cet anticorps permettant la révélation des complexes est avantageusement conjugué à un marqueur afin de mettre en évidence le complexe anticorps-peptide .The complexes formed can be revealed using an antibody reacting specifically with the peptide or antibodies to be assayed or detected. This antibody allowing the revealing of the complexes is advantageously conjugated to a marker in order to highlight the antibody-peptide complex.
Un tel marqueur peut être un enzyme, une molécule chimioluminescente, bioluminescente, fluorescente ou radioactive. Such a marker can be an enzyme, a chemiluminescent, bioluminescent, fluorescent or radioactive molecule.
Un tel procédé peut être avantageusement mis en oeuvre à l'aide d'un coffret pour le dosage et à la détermination d'anticorps comprenant au moins:  Such a method can advantageously be implemented using a kit for the assay and determination of antibodies comprising at least:
une préparation d'un ou plusieurs des peptides tels que précédemment décrits et  a preparation of one or more of the peptides as previously described and
- un moyen permettant la révélation de la réaction ou de l'absence de réaction entre l'un de ces peptides et les anticorps à doser .  a means allowing the revelation of the reaction or the absence of reaction between one of these peptides and the antibodies to be assayed.
Le complexe peptide anticorps formé lors de la réaction est avantageusement révélé à l'aide d'un anticorps conjugué à un marqueur tel que défini cidessus ou à l'aide de toutes molécules capables de détecter la présence d'un tel complexe.  The antibody peptide complex formed during the reaction is advantageously revealed using an antibody conjugated to a marker as defined above or using any molecules capable of detecting the presence of such a complex.
Les peptides objets de la présente invention sont préférentiellement obtenus par synthèse à partir d'acides aminés par tous moyens connus de l'homme du métier .  The peptides which are the subject of the present invention are preferably obtained by synthesis from amino acids by any means known to those skilled in the art.
Ils peuvent aussi être obtenus par génie génétique ou par découpage enzymatique ou chimique de la protéine p53.  They can also be obtained by genetic engineering or by enzymatic or chemical cutting of the p53 protein.
Les anticorps permettant la révélation du complexe formé entre le peptide et les anticorps sont avantageusement dirigés contre des déterminants antigéniques spécifiques de l'espèce dont est originaire le fluide ou le tissu . Ainsi, dans le cadre de dosage d'anticorps d'origine humaine, les anticorps permettant la révélation du complexe seront des anticorps anti-humain.  The antibodies allowing the revelation of the complex formed between the peptide and the antibodies are advantageously directed against antigenic determinants specific for the species from which the fluid or tissue originates. Thus, in the framework of assaying of antibodies of human origin, the antibodies allowing the revelation of the complex will be anti-human antibodies.
La présente invention est illustrée sans pour autant être limitée par les exemples qui suivent dans lesquels : La figure 1 représente les profils d'interaction entre les fragments de la protéine p53 et des anticorps présents dans le sérum de différents patients ayant un cancer. Les anticorps utilisés pour les hybridations des figures l'A à IF sont respectivement un sérum de lapin CM1 immunisé avec la protéine p53, un sérum de souris immunisé avec la protéine p53, trois sérums de patients dont il avait été déterminé qu'ils possédaient des anticorps anti- p53 et un témoin constitué par un anticorps anti- phosphatase alcaline. The present invention is illustrated without however being limited by the following examples in which: FIG. 1 represents the interaction profiles between the fragments of the p53 protein and antibodies present in the serum of different patients with cancer. The antibodies used for the hybridizations of FIGS. A to IF are respectively a CM1 rabbit serum immunized with the p53 protein, a mouse serum immunized with the p53 protein, three sera from patients who were determined to have anti-p53 antibody and a control consisting of an anti-alkaline phosphatase antibody.
Les figures 2 à 7 représentent la réactivité de 77 peptides correspondant à la protéine p53, vis-à-vis de sérums de trois patients atteints de cancers du poumon numérotés 84, 37, 109, et de trois patients atteints de cancer du sein numérotés 57, 27 et 29.  FIGS. 2 to 7 represent the reactivity of 77 peptides corresponding to the protein p53, vis-à-vis sera of three patients with lung cancers numbered 84, 37, 109, and three patients with breast cancer numbered 57 , 27 and 29.
Le complexe anticorps/peptides est révélée par colorimétrie et la densité optique est mesurée par un lecteur de microplaque.  The antibody / peptide complex is revealed by colorimetry and the optical density is measured by a microplate reader.
La figure 8 illustre la fréquence de reconnaissance (en ordonnée) de 77 peptides ( en abscisse) par différents sérums de patients atteints de cancers.  FIG. 8 illustrates the frequency of recognition (on the ordinate) of 77 peptides (on the abscissa) by different sera of patients suffering from cancer.
Les abréviations suivantes sont utilisées dans les exemples :  The following abbreviations are used in the examples:
ER, récepteur oestrogène  ER, estrogen receptor
HCD, Domaine Hautement Conservé (highly conserved domain) de la protéine p53  HCD, highly conserved domain of the p53 protein
hsp70, protéine du choc thermique de 70 kilodaltons MAB, anticorps monoclonal hsp70, 70 kilodalton heat shock protein MAB, monoclonal antibody
PBS, tampon phosphate PBS, phosphate buffer
PBST, PBS avec 0,1 % Tween 20 PBST, PBS with 0.1% Tween 20
PBSTM, PBS avec 0,1% Tween 20 et 3% poudre de lait écrémé  PBSTM, PBS with 0.1% Tween 20 and 3% skim milk powder
PCR, réaction de polymérisation en chaîne (polymerase chain reaction). PCR, polymerase chain reaction (polymerase chain reaction).
PhoA, Phosphatase alcaline d'E.coli  PhoA, E.coli alkaline phosphatase
PR, récepteur progestérone  PR, progesterone receptor
SDS, sodium dodécyl sulfate  SDS, sodium dodecyl sulfate
EXEMPLE 1 - EXAMPLE 1 -
Mise en évidence et signification des anticorps anti-p53 présents dans le sérum de patients atteints de cancer et localisation des régions de la protéine p53 reconnues par ces anticorps. Demonstration and significance of anti-p53 antibodies present in the serum of cancer patients and localization of the regions of the p53 protein recognized by these antibodies.
1) MATERIELS ET METHODES  1) MATERIALS AND METHODS
1.1 SERUMS.  1.1 SERUMS.
Les sérums de 80 malades présentant un carcinome invasif du sein et de 20 malades avec des métastases ont été réunis après diagnostic histopathologique et conservés à -20°C.  The sera of 80 patients with invasive carcinoma of the breast and 20 patients with metastases were pooled after histopathological diagnosis and stored at -20 ° C.
1.2 Anticorps monoclonaux  1.2 Monoclonal antibodies
Des souris BALB/c d'haplotype H-2d sont immunisées par voies intrapéritonéale avec 100 μg de p53 humaine normale. Une injection de rappel est effectuée par voie intraveineuse avec 80μg de protéine de même origine. Quatre jours après le rappel, les splénocytes sont prélevés et fusionnés avec des cellules de myélome NS-1 en présence de polyéthylène glycol (PEG 1500 Boehringer). Après sélection en milieu additionné de 1 % d'Hypoxanthine Aminopterine Thymine (HAT) (Gibco), la sécrétion d'anticorps par les hybridomes a été recherchée par un test ELISA en utilisant la p53 humaine comme antigène. Les hybridomes sécrétant des anticorps capables de reconnaître la p53 ont été clones par la technique de dilution limite.  BALB / c mice of haplotype H-2d are immunized intraperitoneally with 100 μg of normal human p53. A booster injection is given intravenously with 80 μg of protein of the same origin. Four days after the recall, the splenocytes are removed and fused with NS-1 myeloma cells in the presence of polyethylene glycol (PEG 1500 Boehringer). After selection in medium supplemented with 1% of Hypoxanthine Aminopterin Thymine (HAT) (Gibco), the secretion of antibodies by the hybridomas was sought by an ELISA test using human p53 as antigen. Hybridomas secreting antibodies capable of recognizing p53 were cloned by the limit dilution technique.
Pour la localisation des épitopes sur la p53 humaine, nous avons utilisé une série de peptides chevauchants (longueur de 15 acides aminés (aa), chevauchement de 10 aa) couvrant l'intégralité de la protéine p53. On dispose d'une série de 77 peptides biotinylés correspondant à la totalité de la p53. Les 76 premiers peptides à partir de l'extrémité N- terminale ont une longueur de 15 acides aminés chacun et le 77ème peptide ne comprend que 13 acides aminés. For the localization of epitopes on human p53, we used a series of overlapping peptides (length of 15 amino acids (aa), overlap of 10 aa) covering the entire protein p53. We have a series of 77 biotinylated peptides corresponding to all of p53. The first 76 peptides from the N-terminus have a length of 15 amino acids each and the 77th peptide only comprises 13 amino acids.
L'anticorps H111 reconnaît un épitope compris entre les acides aminés 291 et 300, l'anticorps HR231 reconnaît un épitope compris entre les acides aminés 371 et 380 et HT 216 reconnaît un épitope compris entre les acides aminés 1 à 64.  The H111 antibody recognizes an epitope between amino acids 291 and 300, the HR231 antibody recognizes an epitope between amino acids 371 and 380 and HT 216 recognizes an epitope between amino acids 1 to 64.
1.3 EXPRESSION DE FRAGMENT DE P53  1.3 P53 FRAGMENT EXPRESSION
Le vecteur pLip4 a été utilisé pour la production des protéines hybrides. Ce plasmide est dérivé de pLipl (21) dans lequel deux sites de restriction Sac I et Sal I sont introduits entre les codons +6 et +7 du gène E.coli PhOA. Après clonage, la protéine de fusion PhoA est exportée dans le périplasme bactérien et peut en être extraite par choc osmotique à froid (21).  The vector pLip4 was used for the production of the hybrid proteins. This plasmid is derived from pLipl (21) into which two Sac I and Sal I restriction sites are introduced between the codons +6 and +7 of the E.coli PhOA gene. After cloning, the fusion protein PhoA is exported to the bacterial periplasm and can be extracted therefrom by cold osmotic shock (21).
La protéine p53 a été découpée en 6 fragments bien définis. Les fragments 2 (résidus 108 à 162), 3 (résidus 158 à 219), 4 (résidus 215 à 267) et 5 (résidus 263 à 310) contiennent respectivement les régions HCD II à V et correspondent aux sites privilégiés pour les mutations (régions HOT-SPOT) dans les cancers humains (11,12).  The p53 protein was cut into 6 well-defined fragments. Fragments 2 (residues 108 to 162), 3 (residues 158 to 219), 4 (residues 215 to 267) and 5 (residues 263 to 310) respectively contain the regions HCD II to V and correspond to the preferred sites for mutations ( HOT-SPOT regions) in human cancers (11,12).
Les fragments 1 (résidus 1 à 112) et 6 (résidus 306 à 393) correspondent aux régions amino- et carboxy-terminales de la protéine et sont généralement à l'écart des mutations.  Fragments 1 (residues 1 to 112) and 6 (residues 306 to 393) correspond to the amino- and carboxy-terminal regions of the protein and are generally away from mutations.
Les fragments précis d'ADNc de la p53 humaine (clone H8, Varda Rotter, Institut Weissman, Israël) ont été amplifiés par PCR utilisant la Vent-DNA polymerase pour réduire les erreurs d'incorporation puis sous clones dans un vecteur pLIP4. Les clones positifs sont contrôlés pour l'activité phosphatase alcaline et séquences directement. The precise fragments of human p53 cDNA (clone H8, Varda Rotter, Weissman Institute, Israel) were amplified by PCR using Vent-DNA polymerase to reduce incorporation errors and then under clones in a vector pLIP4. The clones positives are checked for alkaline phosphatase activity and sequenced directly.
Les six fragments fusionnés au gène PhoA peuvent être exprimés dans E.coli comme des protéines solubles. Le rendement d'expression' de protéine de fusion avec, le fragment 6 est plus bas (2 à 5 fois) que pour les autres fragments .  The six fragments fused to the PhoA gene can be expressed in E. coli as soluble proteins. The expression yield of fusion protein with, fragment 6 is lower (2 to 5 times) than for the other fragments.
L'antigénicité de la protéine exprimée est établie par sa réactivité avec différents anticorps monoclonaux . (voir tableau 2). PAb1801 (22) et HT 216 sont spécifiques du fragment 1; l' épitope PAb240 est localisé sur le fragment 3 (23). H111 est sur le fragment 5; HR 231 et PAb122 (24) sont spécifiques de la région carboxy-terminale de la p53 (fragment 6). Tous ces anticorps monoclonaux réagissent en immunoblot et immunoprécipitation avec la protéine de fusion pLIP4 en accord avec la localisation de leur épitope (voir tableau 2).  The antigenicity of the expressed protein is established by its reactivity with different monoclonal antibodies. (see table 2). PAb1801 (22) and HT 216 are specific for fragment 1; the PAb240 epitope is located on fragment 3 (23). H111 is on fragment 5; HR 231 and PAb122 (24) are specific for the carboxy-terminal region of p53 (fragment 6). All these monoclonal antibodies react in immunoblot and immunoprecipitation with the fusion protein pLIP4 in accordance with the localization of their epitope (see table 2).
1.4 METHODE D'ANALYSE DES FRAGMENTS P53 PAR IMMUNOBLOT.  1.4 METHOD OF ANALYSIS OF P53 FRAGMENTS BY IMMUNOBLOT.
L'Immunoblot a été réalisé tel que décrit par The Immunoblot was performed as described by
Towbin et al. (20). Les protéines sont séparées par électrophorèse en gel de polyacrylamide (10% acrylamide) en tampon Tris-Glycine contenant 0,1 % de SDS, pH 8,3. Les protéines sont transférées sur une membrane de nitrocellulose (2 heures, 40 volts); la membrane est immergée en tampon PBSTM pour saturer les sites de fixation non spécifiques puis lavée 5 fois auTowbin et al. (20). The proteins are separated by electrophoresis in polyacrylamide gel (10% acrylamide) in Tris-Glycine buffer containing 0.1% SDS, pH 8.3. The proteins are transferred to a nitrocellulose membrane (2 hours, 40 volts); the membrane is immersed in PBSTM buffer to saturate non-specific binding sites and then washed 5 times with
PBST. PBST.
Pour le Western blot du sérum du patient et de l'extrait d'E.coli, plusieurs pré-incubations sont nécessaires pour diminuer les liaisons non spécifiques à la membrane de nitrocellulose et éliminer les anticorps anti-phosphatase présents dans certains sérums. Les sérums sont d'abord incubés avec l'extrait bactérien exprimant la phosphatase alcaline E.coli pendant une heure à +4°C puis sur une membrane de nitrocellulose contenant un extrait bactérien (sans protéine de fusion) 12 heures à +4°C. For the Western blot of the patient's serum and the E. coli extract, several pre-incubations are necessary to reduce the non-specific bonds to the nitrocellulose membrane and to eliminate the anti-phosphatase antibodies present in certain sera. The sera are first incubated with the extract bacterial expressing alkaline phosphatase E.coli for one hour at + 4 ° C and then on a nitrocellulose membrane containing a bacterial extract (without fusion protein) 12 hours at + 4 ° C.
La membrane de nitrocellulose avec les protéines hybrides est incubée avec les sérums préparés et dilués du 1/100 au 1/500 pendant 1 heure en tampon PBSTM à température de la pièce . Après 5 lavages en PBSTM, les membranes sont incubées 1 heure avec un anticorps de lapin anti-souris conjugué peroxydase (dilué au 1/4000 en tampon PBSTM), lavées 5 fois en tampon PBS et révélées par le Luminol (AmershamR). The nitrocellulose membrane with the hybrid proteins is incubated with the sera prepared and diluted from 1/100 to 1/500 for 1 hour in PBSTM buffer at room temperature. After 5 washes in PBSTM, the membranes are incubated for 1 hour with a peroxidase conjugated anti-mouse rabbit antibody (diluted 1/4000 in PBSTM buffer), washed 5 times in PBS buffer and revealed by Luminol (Amersham R ).
Après lecture et interprétation , les filtres sont contrôlés par réaction immunologique (Immunoblot) pour la quantité de protéine de fusion effectivement déposée en incubant la membrane avec un anticorps anti-phosphatase alcaline.  After reading and interpretation, the filters are checked by immunological reaction (Immunoblot) for the quantity of fusion protein actually deposited by incubating the membrane with an anti-alkaline phosphatase antibody.
L'immunoprécipitation et l'électrophorèse en gel de polyacrylamide-SDS ( SDS-PAGE) ont été réalisés selon la technique décrite par Soussi et al. (19).  Immunoprecipitation and electrophoresis in polyacrylamide-SDS gel (SDS-PAGE) were carried out according to the technique described by Soussi et al. (19).
2) RESULTATS  2) RESULTS
2.1 Anticorps anti-p53 et résultats cliniques. 2.1 Anti-p53 antibodies and clinical results.
Les sérums de 100 patients avec un cancer du sein sont testés pour la présence d'anticorps par immunoprécipitation et Western blot (Tableau 1) . Les anticorps circulant sont détectés chez 14% des patients tous stades cliniques confondus. The sera of 100 patients with breast cancer are tested for the presence of antibodies by immunoprecipitation and Western blot (Table 1). Circulating antibodies are detected in 14% of patients in all clinical stages.
La fréquence est légèrement plus élevée que celle rapportée par Crawford et al. (10%) (16), en raison sûrement de l'utilisation de protéine purifiée recombinante p53 à la place de lysat cellulaire.  The frequency is slightly higher than that reported by Crawford et al. (10%) (16), probably due to the use of purified recombinant protein p53 in place of cell lysate.
Il n'y a pas de corrélation entre la présence d'anticorps et le stade clinique de la maladie. Aucune différence de fréquence en fonction de la taille de la tumeur ou de la dissémination ganglionnaire n'a été montrée. Il n'y a pas de corrélation avec d'autres paramètres cliniques tels que l'âge , le statut hormonal. Par contre une corrélation a été établie avec le stade histologique de la tumeur (SBR stade 3), un marqueur pronostic indépendant dans les cancers primitifs du sein . Ceci est confirmé par l'association entre la présence d'anticorps et l'absence de récepteurs hormonaux (ER-PR). There is no correlation between the presence of antibodies and the clinical stage of the disease. No difference in frequency depending on the size of the lymph node spread has not been shown. There is no correlation with other clinical parameters such as age, hormonal status. On the other hand, a correlation has been established with the histological stage of the tumor (SBR stage 3), an independent prognostic marker in primary breast cancers. This is confirmed by the association between the presence of antibodies and the absence of hormone receptors (ER-PR).
2.2 Analyse des régions de la protéine p53 impliquées dans la réponse immune.  2.2 Analysis of the regions of the p53 protein involved in the immune response.
Pour déterminer si la réponse immunitaire est dirigée contre la p53 mutante ou sauvage, tous les sérums positifs et plusieurs sérums négatifs sont contrôlés par immunoprécipitation contre différents mutants p53 (His175, Val 143 et Pro156). La reconnaissance des types sauvages et mutants par les sérums est identique.  To determine whether the immune response is directed against the mutant or wild-type p53, all the positive sera and several negative sera are checked by immunoprecipitation against different p53 mutants (His175, Val 143 and Pro156). The recognition of wild and mutant types by sera is identical.
Dans une seconde série d'expériences , le sérum CM1 (25) a été contrôlé . CM1 est un anticorps polyclonal de lapin obtenu par immunisation avec la p53 humaine hautement purifiée et fréquemment utilisée en immunocytochimie (24). Les techniques de Western blot ont montré que le sérum CM1 contient des anticorps qui reconnaissent principalement les fragments 1 et 6 et à un moindre degré le fragment 5. Des résultats similaires sont obtenus par immunoprécipitation . Une expérience de contrôle avec un anticorps anti-phosphatase alcaline permet d'établir que des quantités identiques des différentes protéines de fusion sont bien présentes sur le gel (figure 1). Pour élargir des observations nous avons contrôlé le sérum d'une souris immunisée avec de la p53 immunopurifiée. Le profil de reconnaissance est quasiment identique à celui du sérum CM1. Des résultats identiques ont été obtenus avec trois autres souris. In a second series of experiments, the CM1 serum (25) was checked. CM1 is a rabbit polyclonal antibody obtained by immunization with highly purified human p53 and frequently used in immunocytochemistry (24). Western blot techniques have shown that the CM1 serum contains antibodies which mainly recognize fragments 1 and 6 and to a lesser degree fragment 5. Similar results are obtained by immunoprecipitation. A control experiment with an anti-alkaline phosphatase antibody makes it possible to establish that identical quantities of the various fusion proteins are indeed present on the gel (FIG. 1). To broaden our observations, we checked the serum of a mouse immunized with immunopurified p53. The recognition profile is almost identical to that of CM1 serum. of the identical results were obtained with three other mice.
L'ensemble de ces résultats permet d'établir que les parties amino et carboxy terminales de la protéine p53 sont très immunogènes chez l'animal immunisé et correspondent à des réponses préférentielles alors que le fragment 5 a une réponse plus faible . Des temps d'exposition radiographique allongés n'ont pas permis de mettre en évidence une reconnaissance des fragments 2 à 4 tant par immunoprécipitation que par immunohybridation (immunoblot).  All of these results make it possible to establish that the amino and carboxy terminal parts of the p53 protein are very immunogenic in the immunized animal and correspond to preferential responses whereas fragment 5 has a weaker response. Longer radiographic exposure times did not make it possible to demonstrate recognition of fragments 2 to 4 both by immunoprecipitation and by immunohybridization (immunoblot).
On a testé le profil de reconnaissance des sérums de malades contenant des anticorps anti-p53 The recognition profile of the sera of patients containing anti-p53 antibodies was tested.
(figure 1 et tableau 3). Les immuno hybridations réalisées ont clairement montré que la réponse immunitaire chez les patients est dirigée principalement contre des épitopes localisés dans les fragments 1 et 6 de la p53. Le sérum de certains patients reconnaît seulement le fragment 1 , mais aucun ne reconnaît uniquement le fragment 6 ce qui laisse supposer que la réponse primaire est dirigée principalement contre le fragment 1. Des résultats semblables sont obtenus par immunoprécipitation. De même que chez l'animal, il existe quelques variations dans la réponse immunitaire et quelques patients ont des anticorps dirigés contre les fragments 4 et 5.(Figure 1 and Table 3). The immuno hybridizations carried out have clearly shown that the immune response in patients is directed mainly against epitopes located in fragments 1 and 6 of p53. The serum of certain patients recognizes only fragment 1, but none recognizes only fragment 6, which suggests that the primary response is directed mainly against fragment 1. Similar results are obtained by immunoprecipitation. As in animals, there are some variations in the immune response and some patients have antibodies against fragments 4 and 5.
Aucun anticorps dirigé contre les fragments 2 et 3 n'a pu être détecté. Ce phénomène n'est pas spécifique au cancer du sein. Des résultats identiques ont été obtenus avec des sérums de patients ayant des carcinomes de l'ovaire ou du poumon (figure 1 et exemple 2). No antibodies against fragments 2 and 3 could be detected. This phenomenon is not specific to breast cancer. Identical results were obtained with sera from patients with ovarian or lung carcinomas (FIG. 1 and Example 2).
Ces résultats établissent clairement que la partie amino terminale de la p53 contient un ou plusieurs épitopes dominants impliqué dans la réponse immunitaire cellule B chez des patients présentant des cancers divers. La région carboxy terminale contient aussi un ou plusieurs épitopes immunodominant mais leur importance est moindre par rapport à la région amino terminale. These results clearly establish that the amino terminal part of p53 contains one or more dominant epitopes involved in the response. B cell immune system in patients with various cancers. The terminal carboxy region also contains one or more immunodominant epitopes, but their importance is less compared to the amino terminal region.
EXEMPLE 2:  EXAMPLE 2:
Mise en évidence des épitopes reconnus par les anticorps anti-p53 présents dans le sérum de malades atteints de divers types de cancer : analyse avec des peptides de synthèse .  Demonstration of epitopes recognized by anti-p53 antibodies present in the serum of patients suffering from various types of cancer: analysis with synthetic peptides.
1) MATERIELS ET METHODES  1) MATERIALS AND METHODS
Les plaques de microtitration utilisées sont des plaques en polychlorure de vinyle à fond plat, à 96 puits. Ref: M129B, Société Dynatech.  The microtiter plates used are 96-well flat bottom polyvinyl chloride plates. Ref: M129B, Dynatech company.
La streptavidine en poudre, référence (S4762) , Streptavidin powder, reference (S4762),
Société Sigma. Sigma company.
Peptides biotinilés synthétisés par la Société CAT (Angleterre)  Biotinilized peptides synthesized by the CAT Company (England)
Une série de peptides chevauchants (longueur de 15 acides aminés (aa) , chevauchement de 10 aa) couvrant l'intégralité de la protéine p53. On dispose actuellement d'une série de 77 peptides biotinylés correspondant à la totalité de la p53. Les 76 premiers peptides à partir de l'extrémité N-terminale ont une longueur de 15 acides aminés chacun et le 77ème peptide ne comprend que 13 acides aminés ( SEQ ID NO: 15).  A series of overlapping peptides (length of 15 amino acids (aa), overlap of 10 aa) covering the entire p53 protein. We currently have a series of 77 biotinylated peptides corresponding to all of p53. The first 76 peptides from the N-terminus are 15 amino acids in length each and the 77th peptide only comprises 13 amino acids (SEQ ID NO: 15).
2 peptides témoins ne correspondant pas à la protéine p53 sont utilisés comme témoin négatif.  2 control peptides not corresponding to the p53 protein are used as a negative control.
Tablette ABTS 50 mg; ref 1112422, Boehringer ABTS 50 mg tablet; ref 1112422, Boehringer
Tampon ABTS ref 1112597, Boehringer ABTS buffer ref 1112597, Boehringer
Anti human couplé à la peroxydase IgG-GAH; PDSOF, Silenus  Anti human coupled with IgG-GAH peroxidase; PDSOF, Silenus
Lait en poudre  Powdered milk
METHODES DU TEST ELISA PREPARATION DES PLAQUES DE MICROTITRATION. ELISA TEST METHODS PREPARATION OF MICROTITRATION PLATES.
100 μl de streptavidine ( concentration 5μg/ml dans l'eau distillée ) sont déposés dans les puits de la plaque.  100 μl of streptavidin (concentration 5 μg / ml in distilled water) are deposited in the wells of the plate.
Les plaques sont ensuite incubées à 37ºC dans une étuve sèche pendant 48 heures. Après cette étape de déshydratation, elles peuvent être emballées individuellement et stockées pendant plusieurs mois à 4°C.  The plates are then incubated at 37ºC in a dry oven for 48 hours. After this dehydration stage, they can be individually wrapped and stored for several months at 4 ° C.
LAVAGE :  WASHING :
Les plaques déshydratées sont lavées 5 fois avec du PBS ( phosphate buffer saline) contenant 0,05% Tween 20. Ce tampon PBS avec du Tween (PBS-T) sera utilisé tout le long de l'expérience comme solution de lavage. Les 5 lavages se font de manière usuelle en utilisant 200 μl de tampon PBS-T par puits.  The dehydrated plates are washed 5 times with PBS (phosphate buffered saline) containing 0.05% Tween 20. This PBS buffer with Tween (PBS-T) will be used throughout the experiment as a washing solution. The 5 washes are carried out in the usual way using 200 μl of PBS-T buffer per well.
Après le dernier lavage, la plaque est séchée sur un papier absorbant. Tous les lavages sont effectués de cette façon.  After the last wash, the plate is dried on absorbent paper. All washes are done this way.
SATURATION SATURATION
100 μl de solution de saturation sont ajoutés dans chaque puits.  100 μl of saturation solution are added to each well.
solution de saturation: tampon PBS  saturation solution: PBS buffer
0,2 % Tween 20  0.2% Tween 20
5% Lait  5% Milk
Les plaques sont ensuite incubées 1 heure à 37°C avec une légère agitation puis sont ensuite lavées comme indiqué précédemment.  The plates are then incubated for 1 hour at 37 ° C with gentle shaking and are then washed as indicated above.
ADDITION DES PEPTIDES BIOTYNILES ADDITION OF BIOTYNILIC PEPTIDES
50 μl de solution de peptides (125 nanogrammes de peptide dilué dans du tampon PBS contenant 0,1 % de Sérum Albumine Bovine et 0,02% d'azide de sodium) sont ajoutés dans chaque puits.  50 μl of peptide solution (125 nanograms of peptide diluted in PBS buffer containing 0.1% bovine serum albumin and 0.02% sodium azide) are added to each well.
Chaque puits reçoit un peptide différent correspondant à une région précise de la p53. Les plaques sont incubées 1 heure à 20ºC avec une légère agitation et sont ensuite lavées comme indiqué précédemment . Each well receives a different peptide corresponding to a specific region of p53. The plates are incubated for 1 hour at 20ºC with gentle shaking and are then washed as indicated above.
ADDITION DES SERUMS DES PATIENTS  ADDITION OF PATIENT SERUMS
Les sérums à tester sont dilués au 1/50 dans une solution de PBS contenant du lait à 5%.  The test sera are diluted 1/50 in a PBS solution containing 5% milk.
50 μl de sérum ainsi dilué sont ajoutés dans chaque puits de la plaque.  50 μl of serum thus diluted are added to each well of the plate.
Les plaques sont incubées 1 heure à 20°C avec une légère agitation et sont ensuite lavées comme indiqué précédemment.  The plates are incubated for 1 hour at 20 ° C. with gentle shaking and are then washed as indicated above.
INCUBATION AVEC L'ANTICORPS SECONDAIRE.  INCUBATION WITH SECONDARY ANTIBODY.
100 μl d'anticorps monoclonal anti- immunoglobuline humaine sont ajoutés dans chaque puits ( commercialisé par Silenus, anticorps dilué au 1/2500 dans du PBS 5%lait).  100 μl of human anti-immunoglobulin monoclonal antibody are added to each well (marketed by Silenus, antibody diluted to 1/2500 in PBS 5% milk).
Les plaques sont incubées 30 minutes à 37°C avec une légère agitation et sont ensuite lavées comme indiqué précédemment.  The plates are incubated for 30 minutes at 37 ° C with gentle shaking and are then washed as indicated above.
REVELATION. REVELATION.
100 μl de substrat (ABTS 1 mM, commercialisé par Boerhinger) sont ajoutés dans chaque puits.  100 μl of substrate (1 mM ABTS, marketed by Boerhinger) are added to each well.
Les plaques sont incubées à 20°C avec une légère agitation.  The plates are incubated at 20 ° C with gentle shaking.
Le résultat est lu dans un appareil de lecture The result is read in a reading device
ELISA (405 nm) 15 minutes, 30 minutes et 60 minutes après addition du substrat . ELISA (405 nm) 15 minutes, 30 minutes and 60 minutes after addition of the substrate.
2) RESULTATS OBTENUS  2) RESULTS OBTAINED
Dans l'exemple 1 deux phénomènes ont été mis en évidence.  In Example 1 two phenomena were highlighted.
i) La présence d'anticorps anti-p53 est retrouvée chez les patients ayant une forme agressive de cancer du sein (mauvais pronostic ).  i) The presence of anti-p53 antibodies is found in patients with an aggressive form of breast cancer (poor prognosis).
ii) Les anticorps anti-p53 reconnaissent principalement une région de 112 acides aminés situés dans la partie amino terminale de la p53 ainsi qu'une région carboxy terminale localisée entre les résidus 306-393. L'étude ne pouvait pas permettre de détailler plus précisément la région reconnue par les anticorps anti-p53. ii) The anti-p53 antibodies mainly recognize a region of 112 amino acids located in the amino terminal part of p53 as well as a carboxy terminal region located between residues 306-393. The study could not provide more detailed information on the region recognized by anti-p53 antibodies.
L'étude décrite dans cet exemple présente une étude très détaillée de ces régions.  The study described in this example presents a very detailed study of these regions.
Une analyse de plus de 1 000 sérums de patients atteints de divers types de cancers a été faite. 47 sérums positifs ont été choisis pour une étude très détaillée afin de déterminer la nature exacte des épitopes que reconnaissent les anticorps sériques.  More than 1,000 sera from patients with various types of cancer were analyzed. 47 positive sera were chosen for a very detailed study in order to determine the exact nature of the epitopes recognized by serum antibodies.
Ces 47 sérums contenant des anticorps anti-p53 ont été testés sur une série de 77 peptides couvrant l'intégralité de la protéine p53 humaine sauvage. La méthode utilisée est la détection ELISA .  These 47 sera containing anti-p53 antibodies were tested on a series of 77 peptides covering the entire wild-type human p53 protein. The method used is ELISA detection.
Des exemples de résultats obtenus sont décrits dans les figures 2 à 7.  Examples of the results obtained are described in Figures 2 to 7.
L'ensemble des résultats est résumé dans le tableau 5.  All the results are summarized in Table 5.
Dans ce tableau, les peptides donnant une réponse comprise entre 50 et 100 % de la valeur maximale de l'ELISA ont été indiqués en noir. Les peptides donnant une réponse comprise entre 20 et 50 % de la valeur maximale de l'ELISA ont été indiqués en gris.  In this table, the peptides giving a response of between 50 and 100% of the maximum value of the ELISA have been indicated in black. Peptides giving a response between 20 and 50% of the maximum ELISA value have been indicated in gray.
Les initiales des cancers étudiés sont les suivantes :  The initials of the cancers studied are as follows:
LC: cancer du poumon  LC: lung cancer
UC: cancer de la vessie  CU: bladder cancer
PA: cancer du pancréas  PA: pancreatic cancer
BC: cancer du sein  BC: breast cancer
BL: lymphome de Burkitt  BL: Burkitt's lymphoma
H : cancer du foie  H: liver cancer
OV: cancer de l'ovaire L : leucémie OV: ovarian cancer L: leukemia
TY: cancer de la thyroïde  TY: thyroid cancer
PR: cancer de la prostate.  PR: prostate cancer.
La figure 8 illustre les fréquences obtenues pour chacun de ces peptides.  FIG. 8 illustrates the frequencies obtained for each of these peptides.
Cet histogramme montre la fréquence de reconnaissance de chaque peptide par les 47 sérums étudiés (voir tableau 5). Il montre, par exemple, que 40% des sérums reconnaissent au moins le peptide 3 et que 63% reconnaissent au moins le peptide 4. 98% des sérums reconnaissent au moins l'un des 5 peptides de la région aminoterminale (N-ter) tandis que seulement 47% reconnaissent l'un des peptides de la région carboxy terminale (C-ter). Bien sur, 100% des sérums reconnaissent l'un des 13 peptides décrits dans ce tableau.  This histogram shows the frequency of recognition of each peptide by the 47 sera studied (see Table 5). It shows, for example, that 40% of the sera recognize at least peptide 3 and that 63% recognize at least peptide 4. 98% of the sera recognize at least one of the 5 peptides of the amino terminal region (N-ter) while only 47% recognize one of the peptides of the carboxy terminal region (C-ter). Of course, 100% of the sera recognize one of the 13 peptides described in this table.
L'ensemble de ces résultats montre que 98% des sérums de malades reconnaissent au moins l'un des épitopes présents dans les 5 peptides de la région amino terminale. 47% des sérums reconnaissent au moins un des peptides de la région carboxy terminale.  All of these results show that 98% of patient sera recognize at least one of the epitopes present in the 5 peptides of the terminal amino region. 47% of the sera recognize at least one of the peptides of the terminal carboxy region.
Il apparait donc que l'utilisation de ces peptides, amino ou carboxy terminaux, sont d'une importance majeure dans la possibilité de mettre au point un test ELISA pour mesurer la présence de ces anticorps dans le fluide biologique de patients atteints de cancers ou d'états précancéreux. It therefore appears that the use of these terminal amino or carboxy peptides are of major importance in the possibility of developing an ELISA test to measure the presence of these antibodies in the biological fluid of patients suffering from cancers or d 'precancerous states.
Figure imgf000022_0001
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Figure imgf000027_0001
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Figure imgf000032_0001
BIBLIOGRAPHIE
Figure imgf000028_0001
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Figure imgf000032_0001
BIBLIOGRAPHY
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C.A., Krausz T. and Lane D.P. P53 immunostaining as a marker of malignant disease in diagnostic cytopathology. Lancet, 338: 513, 1991. C.A., Krausz T. and Lane D.P. P53 immunostaining as a marker of malignant disease in diagnostic cytopathology. Lancet, 338: 513, 1991.
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StYrzbecher H.W., Ullrich S., Jenkins J. and May P. Evolutionary conservation of the biochemical properties of p53- specifie interaction of xenopus- laevis p53 with simian virus 40 large T-antigen and mammalian heat shock proteins- 70. J. Virol., 63: 3894-3901, 1989. StYrzbecher HW, Ullrich S., Jenkins J. and May P. Evolutionary conservation of the biochemical properties of p53- specifies interaction of xenopus- laevis p53 with simian virus 40 large T-antigen and mammalian heat shock proteins- 70. J. Virol. , 63: 3894-3901, 1989.
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LISTE DE SEQUENCES LIST OF SEQUENCES
(1) INFORMATION GENERALE: (1) GENERAL INFORMATION:
(i) DEPOSANT:  (i) DEPOSITOR:
(A) NOM: LABORATOIRES EUROBIO  (A) NAME: EUROBIO LABORATORIES
(B) RUE: 7, Avenue de Scandinavie (B) STREET: 7, Avenue de Scandinavie
(C) VILLE: LES ULIS CEDEX (C) CITY: LES ULIS CEDEX
(E) PAYS: FRANCE  (E) COUNTRY: FRANCE
(F) CODE POSTAL: 91953  (F) POSTAL CODE: 91953
(G) TELEPHONE: 69 07 94 77  (G) TELEPHONE: 69 07 94 77
(H) TELECOPIE: 69 07 95 34  (H) FAX: 69 07 95 34
(I) TELEX: 681 425 F  (I) TELEX: 681 425 F
(ii) TITRE DE L' INVENTION: FRAGMENTS DE LA  (ii) TITLE OF THE INVENTION: FRAGMENTS OF THE
PROTEINE P53 ET LEURS UTILISATIONS DANS LA DETECTION ET LE SUIVI D'ETATS PATHOLOGIQUES  PROTEIN P53 AND THEIR USES IN THE DETECTION AND MONITORING OF CONDITIONS
(iii) NOMBRE DE SEQUENCES: 15  (iii) NUMBER OF SEQUENCES: 15
(iv) FORME LISIBLE PAR ORDINATEUR:  (iv) COMPUTER-READABLE FORM:
(A) TYPE DE SUPPORT: Floppy disk (A) TYPE OF SUPPORT: Floppy disk
(B) ORDINATEUR: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) SYSTEME D' EXPLOITATION: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) LOGICIEL: PatentIn Release #1.0, Version #1.25 (OEB) (D) SOFTWARE: PatentIn Release # 1.0, Version # 1.25 (EPO)
(vi) DONNEES DE LA DEMANDE ANTERIEURE:  (vi) DATA FROM THE PREVIOUS APPLICATION:
(A) NUMERO DE DEPOT: FR 9213110  (A) DEPOSIT NUMBER: FR 9213110
(B) DATE DE DEPOT: 02-NOV-1992  (B) DEPOSIT DATE: 02-NOV-1992
(2) INFORMATION POUR LA SEQ ID NO: 1: (2) INFORMATION FOR SEQ ID NO: 1:
(i) CARACTERISTIQUES DE LA SEQUENCE;  (i) CHARACTERISTICS OF THE SEQUENCE;
(A) LONGUEUR: 25 acides aminés  (A) LENGTH: 25 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 1:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp 1 5 10 Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp 1 5 10
Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu  Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu
15 20 25 (2) INFORMATION POUR LA SEQ ID NO: 2: 15 20 25 (2) INFORMATION FOR SEQ ID NO: 2:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 20 acides aminés (A) LENGTH: 20 amino acids
(B) TYPE: acide aminé (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 2:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln 1 5 10 Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln 1 5 10
Trp Phe Thr Glu Asp Pro Gly Pro  Trp Phe Thr Glu Asp Pro Gly Pro
15 20  15 20
(2) INFORMATION POUR LA SEQ ID NO: 3:  (2) INFORMATION FOR SEQ ID NO: 3:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés  (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 3:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:
Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu 1 5 10 Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu 1 5 10
Trp Lys Leu  Trp Lys Leu
15  15
(2) INFORMATION POUR LA SEQ ID NO: 4:  (2) INFORMATION FOR SEQ ID NO: 4:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés  (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide (vi) ORIGINE: (ii) TYPE OF MOLECULE: peptide (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 4: Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro 1 5 10 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro 1 5 10
Glu Asn Asn  Glu Asn Asn
15  15
(2) INFORMATION POUR LA SEQ ID NO: 5:  (2) INFORMATION FOR SEQ ID NO: 5:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés  (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 5:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 1 5 10 Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 1 5 10
Ser Pro Leu  Ser Pro Leu
15  15
(2) INFORMATION POUR LA SEQ ID NO: 6:  (2) INFORMATION FOR SEQ ID NO: 6:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés  (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 6:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu GlnAsp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln
1 5 10 1 5 10
Trp Phe Thr  Trp Phe Thr
15 (2) INFORMATION POUR LA SEQ ID NO: 7: 15 (2) INFORMATION FOR SEQ ID NO: 7:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 7:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:
Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu AspSer Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp
1 5 10 1 5 10
Pro Gly Pro  Pro Gly Pro
15  15
(2) INFORMATION POUR LA SEQ ID NO: 8:  (2) INFORMATION FOR SEQ ID NO: 8:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés  (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 8:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys 1 5 10 Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys 1 5 10
Glu Pro Gly  Glu Pro Gly
15  15
(2) INFORMATION POUR LA SEQ ID NO: 9:  (2) INFORMATION FOR SEQ ID NO: 9:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés  (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE: (A) ORGANISME: Homo sapiens (vi) ORIGIN: (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 9:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:
Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly SerLys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser
1 5 10 1 5 10
Arg Ala His  Arg Ala His
15  15
(2) INFORMATION POUR LA SEQ ID NO: 10:  (2) INFORMATION FOR SEQ ID NO: 10:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés  (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 10:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:
Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser SerGly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser
1 5 10 1 5 10
His Leu Lys  His Leu Lys
15  15
(2) INFORMATION POUR LA SEQ ID NO: 11:  (2) INFORMATION FOR SEQ ID NO: 11:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés  (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 11:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser LysGly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys
1 5 10 1 5 10
Lys Gly Gln  Lys Gly Gln
15 (2) INFORMATION POUR LA SEQ ID NO: 12: 15 (2) INFORMATION FOR SEQ ID NO: 12:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 12:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:
Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser ThrSer Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr
1 5 10 1 5 10
Ser Arg His  Ser Arg His
15  15
(2) INFORMATION POUR LA SEQ ID NO: 13:  (2) INFORMATION FOR SEQ ID NO: 13:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés  (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(vi) ORIGINE:  (vi) ORIGIN:
(A) ORGANISME: Homo sapiens  (A) ORGANIZATION: Homo sapiens
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 13:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13:
Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys 1 5 10 Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys 1 5 10
Leu Met Phe  Leu Met Phe
15  15
(2) INFORMATION POUR LA SEQ ID NO: 14:  (2) INFORMATION FOR SEQ ID NO: 14:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 15 acides aminés  (A) LENGTH: 15 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide (xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 14:(ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14:
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr 1 5 10 Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr 1 5 10
Glu Gly Pro  Glu Gly Pro
15  15
(2) INFORMATION POUR LA SEQ ID NO: 15:  (2) INFORMATION FOR SEQ ID NO: 15:
(i) CARACTERISTIQUES DE LA SEQUENCE:  (i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEUR: 13 acides aminés  (A) LENGTH: 13 amino acids
(B) TYPE: acide aminé  (B) TYPE: amino acid
(C) NOMBRE DE BRINS: simple  (C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linéaire  (D) CONFIGURATION: linear
(ii) TYPE DE MOLECULE: peptide  (ii) TYPE OF MOLECULE: peptide
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 15:(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15:
Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser 1 5 10 Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser 1 5 10
Asp  Asp

Claims

REVENDICATIONS
1. Peptide, à l'exclusion de la protéine p53, présentant une réaction spécifique vis-à-vis d'anticorps anti-p53 présents dans les fluides biologiques de patients atteints d'un cancer ou précancéreux.  1. Peptide, excluding the p53 protein, exhibiting a specific reaction vis-à-vis anti-p53 antibodies present in the biological fluids of cancer patients or pre-cancer patients.
2. Peptide selon la revendication 1, caractérisé en ce que les anticorps sont ceux de patients atteints d'un cancer quel que soit son origine.  2. Peptide according to claim 1, characterized in that the antibodies are those of patients suffering from cancer whatever its origin.
3. Peptide selon l'une des revendications 1 et 2 , caractérisé en ce qu'il est compris dans la séquence des amino acides 1 à 112 de la protéine p53.  3. Peptide according to one of claims 1 and 2, characterized in that it is included in the sequence of amino acids 1 to 112 of the protein p53.
4. Peptide selon l'une des revendications 1 à 3, caractérisé en ce qu'il comprend en partie ou en totalité l'une des séquences suivantes :  4. Peptide according to one of claims 1 to 3, characterized in that it partly or wholly comprises one of the following sequences:
Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu (SEQ ID NO:1)  Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu (SEQ ID NO: 1)
Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Asp Asp Leu Met Leu Ser Pro Asp Asp Ile
Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro (SEQ ID NO : 2). Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro (SEQ ID NO: 2).
5. Peptide selon l'une des revendications 1 à 4, caractérisé en ce qu'il comprend en partie ou en totalité l'une des séquences suivantes :  5. Peptide according to one of claims 1 to 4, characterized in that it partly or wholly comprises one of the following sequences:
Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu (SEQ ID NO: 3 )  Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu (SEQ ID NO: 3)
Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn (SEQ ID NO :4)  Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn (SEQ ID NO: 4)
Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn
Val Leu Ser Pro Leu (SEQ ID NO: 5) Val Leu Ser Pro Leu (SEQ ID NO: 5)
Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr (SEQ NO : 6)  Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr (SEQ NO: 6)
Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro (SEQ ID NO: 7). Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro (SEQ ID NO: 7).
6. Peptide selon l'une des revendications 1 et 2, caractérisé en ce qu'il est compris dans la séquence des amino-acides 350 à 393 de la protéine p53. 6. Peptide according to one of claims 1 and 2, characterized in that it is included in the sequence of amino acids 350 to 393 of the protein p53.
7. Peptide selon l'une des revendications 1, 2 et 6, caractérisé en ce qu'il comprend en partie ou en totalité l'une des séquences suivantes :  7. Peptide according to one of claims 1, 2 and 6, characterized in that it partly or wholly comprises one of the following sequences:
Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly (SEQ ID NO : 8)  Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly (SEQ ID NO: 8)
Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly
Gly Ser Arg Ala His (SEQ ID NO : 9) Gly Ser Arg Ala His (SEQ ID NO: 9)
Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys ( SEQ ID NO: 10)  Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys (SEQ ID NO: 10)
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln ( SEQ ID NO: 11)  Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln (SEQ ID NO: 11)
Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His ( SEQ ID NO: 12)  Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His (SEQ ID NO: 12)
Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe (SEQ ID NO: 13)  Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe (SEQ ID NO: 13)
Ser Thr Ser Arg His Lys Lys Leu Met Phe Ser Thr Ser Arg His Lys Lys Leu Met Phe
Lys Thr Glu Gly Pro (SEQ ID NO: 14) Lys Thr Glu Gly Pro (SEQ ID NO: 14)
Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp (SEQ ID NO: 15).  Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp (SEQ ID NO: 15).
8. Séquence nucléotidique codant pour la synthèse d'un peptide selon l'une des revendications 1 à 7.  8. Nucleotide sequence coding for the synthesis of a peptide according to one of claims 1 to 7.
9. Utilisation d'un peptide selon l'une des revendications 1 à 7 pour la détection ou le suivi d'états pathologiques .  9. Use of a peptide according to one of claims 1 to 7 for the detection or monitoring of pathological states.
10. Utilisation selon la revendication 9 pour la détection ou le suivi de l'évolution d'états cancéreux et pré-cancéreux.  10. Use according to claim 9 for the detection or monitoring of the evolution of cancerous and pre-cancerous states.
11. Procédé pour la détermination de la présence ou pour le dosage d'anticorps dans un fluide ou tissu caractérisé en ce que: - on met en présence le fluide ou tissu et l'un des peptides selon l'une des revendications 1 à 7, et 11. Method for determining the presence or for the assay of antibodies in a fluid or tissue characterized in that: - the fluid or tissue and one of the peptides according to one of claims 1 to 7 are brought into contact, and
- on détermine la présence soit de l'anticorps soit du peptide soit du couple anticorps-peptide et on effectue le dosage correspondant .  - the presence of either the antibody or the peptide or of the antibody-peptide pair is determined and the corresponding assay is carried out.
12. Procédé selon la revendication 11, caractérisé en ce que les anticorps à doser ou dont on détermine la présence sont des anticorps représentatifs d'états pathologiques .  12. Method according to claim 11, characterized in that the antibodies to be assayed or the presence of which is determined are antibodies representative of pathological states.
13. Procédé selon l'une des revendications 11 et 12 , caractérisé en ce que ledit peptide est fixé sur une phase solide .  13. Method according to one of claims 11 and 12, characterized in that said peptide is fixed on a solid phase.
14. Procédé selon l'une des revendications 11 à 13, caractérisé en ce que les complexes formés sont révélés à l'aide d'un anticorps réagissant spécifiquement avec les anticorps à doser ou dont on détermine la présence .  14. Method according to one of claims 11 to 13, characterized in that the complexes formed are revealed using an antibody reacting specifically with the antibodies to be assayed or whose presence is determined.
15. Procédé selon la revendication 14, caractérisé en ce que l'anticorps permettant la révélation des complexes est conjugué à un marqueur .  15. The method of claim 14, characterized in that the antibody allowing the revealing of the complexes is conjugated to a marker.
16. Procédé selon la revendication 15, caractérisé en ce que le marqueur est un enzyme , une molécule chimioluminescente, bioluminescente, fluorescente ou radioactive .  16. The method of claim 15, characterized in that the marker is an enzyme, a chemiluminescent, bioluminescent, fluorescent or radioactive molecule.
17. Coffret pour le dosage et la détermination d'anticorps , caractérisé en ce qu'il comprend au moins :  17. Kit for the assay and determination of antibodies, characterized in that it comprises at least:
- une préparation d'un ou plusieurs peptides selon l'une des revendications 1 à 7, et  - a preparation of one or more peptides according to one of claims 1 to 7, and
- un moyen permettant la révélation de la réaction entre l'un de ces peptides et les anticorps à doser .  a means allowing the revelation of the reaction between one of these peptides and the antibodies to be assayed.
18. Coffret selon la revendication 17 , caractérisé en ce que le complexe peptide-anticorps formé lors de la réaction est révélé par un anticorps conjugue a un marqueur. 18. Box according to claim 17, characterized in that the peptide-antibody complex formed during the reaction is revealed by an antibody conjugates to a marker.
19. Coffret selon la revendication 18, caractérisé en ce que le marqueur est un enzyme, une molécule chimioluminescente, bioluminescente, fluorescente ou radioactive .  19. Box according to claim 18, characterized in that the marker is an enzyme, a chemiluminescent, bioluminescent, fluorescent or radioactive molecule.
PCT/FR1993/001082 1992-11-02 1993-11-02 P53 protein fragments and use thereof for detecting and monitoring diseased conditions WO1994010306A1 (en)

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JP6510784A JPH08502414A (en) 1992-11-02 1993-11-02 Use of p53 protein fragment and its detection and monitoring of disease
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FR9213110A FR2698367B1 (en) 1992-11-02 1992-11-02 Fragments of the p53 protein and their uses in the detection and monitoring of disease states.

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WO2017153336A1 (en) * 2016-03-07 2017-09-14 F. Hoffmann-La Roche Ag Detection of anti-p53 antibodies
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