Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
  • C12N5/12—Fused cells, e.g. hybridomas
  • C12N5/16—Animal cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0003—Invertebrate antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/22—Testing for sterility conditions
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
  • G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
  • G01N2333/4353—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from nematodes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/10—Detection of antigens from microorganism in sample from host
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

• This invention relates to the field of diagnosis, prevention and therapy of human and animal infections by species of the Anisakis genus. This invention also relates to the detection of Anisakis antigens in food.
• the genus Anisakis (family Anisakidae) includes nematode parasites that belong to several related species. Through the use of morphological and genetic / molecular markers, 8 species of Anisakis are currently recognized, including: a) the twin species Anisakis simplex "in the strict sense” (Rudolphi, 1809), A. pegreffi (Campana-Rouget and Biocca , 1955) and A. simplex C (Nascetti et al., 1986, Mattiucci et al., 2002), formerly included in the A. simplex complex; b) A.
• Anisakis species reach sexual maturity in the stomach of cetaceans and, less frequently, in pinpipeds. These mammals can be infected by ingestion of a) paratenic hosts, that is, fish (mainly teleost) and cephalopods (mainly squid), which house the third larval phase of the parasite (L3), and b) by ingestion of krill, as intermediate, which It also houses L3 larvae (for example, whales).
• Anisakis L3 larvae Like marine mammals, humans can also be infected by Anisakis L3 larvae by ingesting fish and raw squid carrying the parasite, which produces a clinical disease called anisakiosis or anisakiasis. Very often, several fish dishes are considered high risk for infection with Anisakis species. These include sushi and Japanese sashimi, sauteed or smoked German herring, Scandinavian gravlax, Hawaiian lomi-lomi, South American cebiche and Spanish pickled anchovies ("pickled anchovies").
• Anisakis L3 larvae When Anisakis L3 larvae infect humans, they often cause gastrointestinal symptoms that may be associated with allergic reactions of varying severity. Depending on the location of the larvae and the dominant symptoms, four main clinical forms of anisakiosis (gastric / gastroallergic, intestinal, extragastrointestinal and allergic) have been presented in humans. In the gastric (acute) form of anisakiosis, the larva penetrates the gastric wall and a clinical course characterized by acute epigastric pain, nausea and vomiting is observed a few hours after ingestion of fish containing live Anisakis L3 larvae. Endoscopic studies often reveal lesions consisting of punctate hemorrhages, petechiae and erosion at the site of penetration. In addition, allergic symptoms (i.e.
• urticaria / angioedema and sometimes anaphylaxis can also be observed in approximately 10% of cases.
• the combination of gastric infection and allergic symptoms was called "gastroallergic anisakiosis" (Daschner et al., 2000).
• Some patients may develop a subacute or chronic form whose clinical manifestations include epigastric pain, dyspepsia, vomiting and anorexia, which may persist for several months or even years.
• the larva In the intestinal form of the anisakiosis, the larva penetrates the intestinal wall, frequently at the level of the terminal ileum. In acute cases, the presence of abdominal pain is often observed (usually 24-48 hours after ingestion of contaminated fish), accompanied by nausea, vomiting, abdominal swelling, induration and rhythm deviation. bowel established with constipation or diarrhea.
• the macroscopic observation of infected tissues in case of intestinal anisakiosis reveals phlegmous lesions characterized by severe local edema, petechiae, hyperemia and diffuse inflammation of the serous and mesentery. These lesions can cause obstruction and proximal dilation of the intestine. In a significant number of patients, abdominal ascites fluid with a high eosinophil content can be found.
• subacute or chronic intestinal anisakiosis the granulomatous changes induced by the larvae lead to a thickening of the wall, luminal stenosis and chronic abdominal symptoms.
• allergic anisakiosis is reserved for patients who have allergic reactions after infection with Anisakis larvae, but without gastric or intestinal symptoms. Frequently, the first symptoms appear very soon, in the first hours after ingestion of contaminated fish and the clinical course follows the general pattern for type I allergic reactions. Its severity varies from a simple hives or angioedema to anaphylactic shock.
• the endoscopic examination is the preferable method to demonstrate the infection.
• This technique is also useful for extracting the larvae by means of the clamps associated with the endoscope, which leads to the healing of the patient.
• the preferable method to confirm the infection is to demonstrate the presence of specific antibodies in the serum of the patient. The latter is also the preferred method of demonstrating previous Anisakis infections in asymptomatic individuals.
• IgE antibodies are the most relevant since: 1) they are involved in the type I allergic reactions observed during infections with Anisakis, and b) this is the class of most relevant immunoglobulins in terms of specificity among those that are present in serum of infected patients.
• the first methods for measuring serum anti-Anisakis antibodies included latex agglutination (Akao and Yoshimura, 1989); double immunodiffusion and immunoelectrophoresis (Petithory et al., 1986; Tsuji, 1990), indirect hemagglutination (Asaishi et al., 1989; Tsuji, 1989); and complement fixation assay (Oshima, 1972; Tsuji, 1989). All these methods have two main drawbacks: a) they lack sufficient sensitivity and specificity and b) with these techniques it is not possible to determine IgE (reaginic) antibodies.
• anti-Anisakis IgE determinations could be performed in sera of patients infected with the introduction of other immunoassays, for example, radio allergy tests (Desowitz et al., 1985; Yamamoto et al., 1990), linked immunosorbent assays to enzymes (ELISA) (Rodero et al. 2002), fluorescent enzyme immunoassays (UniCap FEIA, Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden) and immunoblotting (Del Pozo et al., 1996; Garc ⁇ a et al., 1997), but also There were problems of sensitivity and / or specificity with these methods.
• radio allergy tests (Desowitz et al., 1985; Yamamoto et al., 1990), linked immunosorbent assays to enzymes (ELISA) (Rodero et al. 2002), fluorescent enzyme immunoassays (UniCap FEIA, Pharmacia & Upjohn Diagnostics AB, Uppsal
• the main difficulty in developing useful serological methods for the diagnosis of human Anisakiosis lies in obtaining parasite antigens (in our case allergens) that on the one hand (i) are recognized by IgE antibodies present in the sera of all those infected patients, and (ii) that the entire molecule (that is, all epitopes present in the selected allergen) be specific to the Anisakis genus. Otherwise, low sensitivity and / or cross reactions that lead to false positives would be obtained.
• Anisakis spp. cultured in vitro (Poggensee et al., 1989; Baeza et al., 2004), or using monoclonal antibodies to capture specific target antigens from crude L3 larval extracts of Anisakis (Yagihashi et al., 1990;
• the present invention provides sequences of peptides, polypeptides and proteins that maintain the antigenicity of the native Anisakis molecules from which they are derived, as well as the nucleic acids encoding said sequences and antibodies or fragments thereof specific for said peptides, polypeptides. or proteins
• the peptides, polypeptides and proteins described, as well as the antibodies or fragments thereof specific fronts to said peptides, polypeptides and proteins are useful for developing immunoassays for the serodiagnosis of human and animal anisakiosis, lacking the drawbacks of the methods previously presented .
• a first aspect of the present invention provides an isolated nucleic acid sequence (hereinafter referred to as the nucleic acid sequence of the invention) selected from the following group:
• a nucleic acid comprising SEQ ID N 0 1 b.
• a nucleic acid fragment of SEQ ID N 0 1 comprising SEQ ID N 0 15 or 17 d.
• N 0 1 encoding a contiguous stretch of 10 or more amino acids of SEQ ID N 0 2 capable of being recognized by anti-Anisakis antibodies.
• F. A nucleic acid comprising a fragment of SEQ ID N 0 1 that encodes a contiguous stretch of 12 or more amino acids of SEQ ID N 0 2 capable of being recognized by anti-anisakis antibodies.
• g. A nucleotide sequence having an identity of at least 70% with any of the nucleic acid sequences described in sections (a) to (f).
• the nucleotide sequence has an identity of at least 80% with any of the nucleic acid sequences previously mentioned in sections (a) to (f) and, in an even more preferred embodiment, said nucleotide sequence has an identity of at least 90% with any of the nucleic acid sequences previously mentioned in sections (a) to (f).
• a second aspect of the present invention provides an isolated polypeptide, peptide or protein (hereinafter referred to as the amino acid sequence of the invention) selected from the following group:
• a fragment of the SEQ ID N 0 2 comprising a contiguous span of 8 or more amino acids capable of being recognized by anti-Anisakis antibodies.
• d A fragment of the SEQ ID N 0 2 comprising a contiguous stretch of 10 or more amino acids capable of being recognized by anti-Anisakis antibodies.
• a fragment of the SEQ ID N 0 2 comprising a contiguous stretch of 12 or more amino acids capable of being recognized by anti-Anisakis antibodies.
• F An amino acid sequence having an identity of at least 70% with any of the amino acid sequences described in sections (a) to (e) at this point.
• the amino acid sequence has an identity of at least 80% with any of the amino acid sequences previously described in sections (a) to (e) at this point and, in an even more embodiment preferred, said amino acid sequence has an identity of at least 90% with any of the amino acid sequences previously described in sections (a) to (e) at this point.
• All these peptides, polypeptides or proteins can be used to overcome the main difficulty of developing useful serological methods for the diagnosis of human anisakiosis, by providing specific parasite antigens of the Anisakis genus and which are recognized by the IgE antibodies present in the sera of All patients infected by Anisakis.
• a preferred embodiment of the invention refers to an amino acid sequence that can be recognized by anti-Anisakis antibodies and that comprises a fragment with a contiguous section of at least 8 or more amino acids and with an identity of at least 70% with Any fragment of SEQ ID N 0 2, selected from the following amino acid sequences:
• amino acid sequence of the invention is produced, but not limited to any of the following techniques: to. Recombinant techniques b. Chemical synthesis and c. Substantial purification from nematodes of the Anisakidae family
• a fourth aspect of the invention refers to a peptide, polypeptide or protein comprising two or more of the amino acid sequences of the invention.
• a fifth aspect of the invention relates to a synthetic or recombinant nucleic acid molecule comprising a nucleic acid sequence encoding one or more of the amino acid sequences of the invention.
• a sixth aspect of the invention refers to an isolated nucleic acid or amino acid sequence of the invention, labeled with a signaling molecule.
• said marker molecule is selected, but not limited to any of the signaling molecules of the following group:
• the marking molecule is selected, but not limited to any of the signaling molecules of the following group: to. Biotin or its derivatives b. Nitrophenol derivatives c. Colloidal Gold d. Latex e. Fluorescein
• a seventh aspect of the invention relates to an antibody or fragment thereof (hereinafter referred to as the antibody of the invention) capable of interacting with any of the amino acid sequences of the invention. Also part of the present invention are nucleic acid sequences capable of encoding any of the antibodies of the invention.
• said antibody of the invention is the antibody produced by the hybridoma cell line with the deposit number DSM ACC2793 deposited on June 15, 2006 in the "German
• a further embodiment refers to the hybridoma cell line with deposit number DSM ACC2793 with deposit date June 15, 2006 deposited in the DSMZ institution.
• a further embodiment of the invention relates to the antigen recognition sequences of any of the antibodies of the invention, particularly the antigen recognition sequences of the UA3 antibody.
• Another aspect of the invention provides an isolated amino acid sequence of the invention, wherein said amino acid sequence is chemically fused or coupled to an additional peptide, polypeptide or protein.
• Another aspect of the invention relates to an expression vector or expression system (hereinafter referred to as the expression system of the invention) comprising a nucleotide sequence of the invention.
• Another aspect of the invention provides an isolated prokaryotic or eukaryotic host cell (hereinafter referred to as the host cell of the invention) transformed or transfected with a nucleic acid sequence of the invention or with an expression vector or system of the invention.
• the host cell of the invention transformed or transfected with a nucleic acid sequence of the invention or with an expression vector or system of the invention.
• An eighth aspect of the invention relates to a method for producing a recombinant peptide, polypeptide or protein encoded by a nucleic acid sequence of the invention, wherein said method comprises culturing a host cell of the invention under conditions such that it is expressed and expressed. produce said nucleotide sequence and then isolate said peptide, polypeptide or protein.
• a ninth aspect of the invention relates to an in vitro method for detecting antibodies against Anisakis spp. in an isolated biological sample, preferably serum, comprising: a. contacting a biological sample with an amino acid sequence of the invention, under conditions sufficient to form an immunological complex between said polypeptide and the antibodies of the sample, and b. detect said immune complex.
• the in vitro method is an immunoassay or an enzymatic immunoassay.
• a tenth aspect of the invention refers to an in vitro method for detecting Anisakis spp. in a sample, preferably a biological sample, or a food extract, comprising: a. contacting the biological sample or food extract with an antibody of the invention, under conditions sufficient to form an immune complex between said antibody and the antigens of the sample and b. detect said immune complex.
• the antibody of the invention that is used to detect antigens of Anisakis spp. In an in vitro sample it is the UA3 antibody.
• a preferred aspect of the invention refers to an in vivo method to diagnose or predict allergy to Anisakis spp. in a subject, which comprises subjecting the subject to the peptide, polypeptide or protein of the invention and controlling the reaction of the subject.
• kit of the invention suitable for the detection of Anisakis spp. in a biological sample or a food extract, comprising: a) at least one antibody of the invention or its fragments and b) a means of detection for the immune complex. It is also a preferred aspect of the present invention a kit suitable for the detection of antibodies anti-Anisakis spp. in a biological sample, preferably serum, comprising: a) at least one peptide, polypeptide or protein of the invention and b) a means of detection for the immune complex.
• the detection means used in the kit of the invention consists of labeled secondary antibodies capable of recognizing the formation of the immune complex.
• the detection means used in the kit of the invention is a labeled secondary reagent capable of recognizing the formation of the immune complex.
• Another aspect of the invention relates to the use of an amino acid sequence of the invention for the manufacture of a pharmaceutical composition for the treatment of any type of allergic reaction to
• Anisakis spp. A preferred embodiment of the invention refers to the pharmaceutical composition as is.
• nucleic acid or “nucleic acid molecule” refers to any nucleotide polymer composed of two or more subunits that are deoxyribonucleotides or ribonucleotides, linked together by phosphodiester bridges.
• Nucleic acids or “nucleic acid molecules” include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides and fragments generated by polymerase chain reaction (PCR) or by other methods such as ligation, cleavage, action of endonucleases and exonuclease action.
• nucleotide means a monomeric unit of DNA or RNA that contains a sugar moiety (pentose), a phosphate and a nitrogen heterocyclic base.
• the four DNA bases are adenine ("A”), guanine (“G”), cytosine ("C”) and thymine (“T”).
• the four bases of RNA are A, G, C and uracil ("U”).
• an "isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated into the genomic DNA of an organism. Said nucleic acid molecule can be separated from the genomic DNA of a cell, can be produced using recombinant DNA technology (eg, PCR amplification, cloning, etc.) or can be chemically synthesized. The isolated nucleic acid molecule can be obtained from its natural source as a complete gene or a part thereof capable of forming a stable hybrid with that gene. The nucleic acid molecule can be single stranded or double stranded.
• primer refers to a natural or synthetic oligonucleotide (preferably ranging from 15 to 40 nucleotides in length) that can hybridize with a complementary DNA or RNA template to form a duplex between the primer and the mold.
• a primer is a single stranded oligodeoxyribonucleotide, and serves as a starting point for the synthesis of nucleic acid by a polymerase after hybridization with a DNA or RNA chain.
• nucleotide sequence encoding refers to a nucleic acid sequence that directs the expression of a specific peptide, polypeptide or protein.
• the nucleic acid sequences comprise the sequence of the DNA chain that is transcribed into RNA and also the sequence of RNA that is translated into a protein.
• the nucleic acid sequences of the present invention include both full length nucleic acid sequences and truncated sequences (which do not have full length) derived from the full length protein.
• the variants are also included of the nucleotide sequence encoding the same peptide, polypeptide or protein as the native sequence, which can be constructed to provide a codon preference in a specific host cell.
• complement refers to the concept of an inverse correspondence between regions of two polynucleotide chains or between two nucleotides through the formation of base pairs. Consequently, a “complementary base sequence” refers to a polynucleotide chain in which all bases can form base pairs with a base sequence in another polynucleotide chain.
• an adenine nucleotide can form base pairs with thymine (A-T) or uracil (A-U), while a cytosine nucleotide can form base pairs with guanine (C-G).
• an "expression vector” refers to a set that is capable of directing the expression of a sequence or gene of interest.
• the expression vector can be any DNA or RNA expression vector capable of expressing Anisakis spp. Antigens, such as a plasmid or a phage.
• the expression vector according to the present invention is a plasmid (for example, pQE 31 and pTARGET).
• plasmid refers to any autonomous circular DNA molecule capable of replicating in a cell independently of chromosomal DNA, and includes both types of expression and types of non-expression.
• the term “recombinant” refers to a compound DNA molecule prepared outside living cells by artificially binding natural or synthetic DNA fragments to molecules of
• recombinant also refers herein to a peptide, polypeptide or protein that is expressed using a recombinant DNA molecule.
• polypeptide and "protein” are used interchangeably herein to designate a linear sequence of amino acids connected to each other by amide bonds between the alpha-amino group of an amino acid and an alpha-carboxy group of the adjacent amino acid.
• Polypeptides with a length of twenty-five amino acids or less are considered “peptides.”
• a polypeptide that is encoded by a cistron is considered a “protein.”
• fragment of the amino acid sequence means a portion of contiguous amino acids of the specified peptide, polypeptide, protein or amino acid sequence.
• an "isolated peptide, polypeptide or protein” is a peptide, polypeptide or protein that is essentially free of cellular contaminants, such as lipids, carbohydrates or other impurities associated with the polypeptide in nature.
• An isolated peptide, polypeptide or protein can be purified from its natural environment by conventional chromatographic techniques. Isolated peptides, polypeptides and proteins can also be obtained by recombinant methods, or by chemical synthesis.
• a "substantially purified polypeptide” is obtained when it is separated from at least 50%, more preferably at least 75% and even more preferably at least 90% from other polypeptides with which it naturally coexists in a cell, tissue or organism
• amino acid As used herein, the terms "amino acid" and
• amino acids refer to all naturally occurring L-alpha-amino acids or his rests.
• Table 1 shows the three letter code and the one letter code (recommended by the IUPAC-IUB Biochemical Nomenclature Commission) for the designation of natural amino acids:
• mammal refers to any animal classified as a mammal, including humans, mice, rats, marine mammals, and domestic and farm animals.
• the mammal in this document is the human being.
• the term “antibody” refers to glycoproteins of the family of immunoglobulins, characterized in that they have antigen binding properties, and are produced by plasma cells.
• the term “antibody” includes both monoclonal and polyclonal antibodies that belong to any class of antibodies, for example, IgG, IgD, IgM, IgA, IgE or derivatives thereof.
• the term “monoclonal antibody” refers to an antibody composition that has a population of homogeneous antibodies. It is not intended to be limited to a particular source of the antibody or to the form in which it is produced, and includes antibodies and fragments thereof produced by cell fusion or by recombinant techniques.
• the term “antigen” refers to any molecule or agent capable of inducing the production of specific antibodies when introduced into an immunocompetent host. As used herein, the term “antigen” also refers to any molecule or agent capable of being recognized by a specific antibody. Typically, peptides, polypeptides, proteins, carbohydrates, lipids, nucleic acids or combinations of these molecules can be recognized as antigens. These antigens may also contain attached organic and inorganic chemical groups.
• epitope refers to the portion of an antigen that is recognized by an antibody (monoclonal or polyclonal) or by the receptor with antigen specificity of a cell.
• the antigen is a peptide, polypeptide or a protein
• its epitopes can be fragments of at least 5 amino acids of the primary amino acid sequence, but also regions exposed on the surface of the mature folded protein (three-dimensional tertiary structure) composed of 5 or more amino acids
• epitopes can be classified as B cell epitopes and T cell epitopes based on the types of immune response they cause.
• biological sample applies to any biological tissue or fluid sample that contains one or more of the nucleic acids, antibodies, peptides, polypeptides or proteins. of the present invention.
• biological sample also applies to any biological tissue or fluid sample that can be used as a source for the determination of anti-antibody antibodies.
• samples include, but are not limited to, isolated tissues, blood, serum, plasma, cerebrospinal fluid, ascites fluid, synovial fluid, saliva, urine or feces.
• Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.
• a biological sample can be obtained from any eukaryotic organism, and preferably from a mammal such as a rat, mouse, rabbit, marine mammals, or a human being.
• the term "food sample” refers to any sample obtained from the wide range of edible materials. These samples include, but are not limited to, pieces of raw, smoked and cooked fish and foods that have fish materials in their composition. The term “food sample” also includes foods for children's nutrition.
• anti-Anisakis antibodies refers to any class or subclass of antibodies present in a sample capable of recognizing at least one epitope that is present in the antigens of any of the species of the Anisakis genus.
• the expression includes antibodies produced during natural or experimental infections of humans and animals with the parasite, as well as monoclonal and polyclonal antibodies produced after the immunization of humans or animals with recombinant or synthetic antigens not purified or substantially purified, obtained from species of the genus Anisakis.
• the expression also includes antibodies and antigen binding fragments produced by recombinant techniques, chemical synthesis or equivalent techniques.
• percent identity refers to the percentage of amino acids or nucleotides that occupy the same relative position when two amino acid sequences, or two nucleic acid sequences are aligned side by side.
• a suitable method to calculate the percentage of sequence identity between two nucleotide or amino acid sequences is to use the William Pearson lalign program that applies the Huang and Miller algorithm, published in Adv. Appl. Math (1991) 12: 337-
• the term "immunoassay” refers to a method of detecting an analyte in a sample by means of an antigen-antibody reaction.
• the analytes can be small molecules (haptens) or large molecules, such as many plasma proteins. Very often, the analyte is an antibody that can be detected by means of the specific binding between the antibody and a ligand.
• enzyme immunoassay EIA refers to a broad type of immunoassay, characterized in that at least one of the reagents is labeled with an enzyme. EIA-linked enzyme-linked immunosorbent assays (ELISAs) of which there are a number of variants are typical.
• a "pharmaceutical composition” refers to a chemical or biological composition that is suitable for administration to a mammalian individual.
• a pharmaceutical composition comprises an amount of a pharmacologically effective active agent and a pharmaceutically acceptable carrier. Examples of carriers and suitable pharmaceutical formulations are described in Remington's Pharmaceutical Sciences, 20 edition, Mack Publishing
• a pharmaceutical composition can be formulated specifically for administration by different routes including, but not limited to, oral, parenteral, intravenous, intraarterial, subcutaneous, intranasal, sublingual and the like.
• the expression "diagnostic method” refers to a method for the identification of a pathological state in an animal or in an individual.
• the diagnosis can often be made using an immunoassay (see above) that allows the detection of the antigens released by the pathogen, or of the antibodies induced in the host by said pathogen.
• the diagnostic methods of the present invention include in vitro tests (such as an ELISA test, or histamine release test of basophils) and in vivo tests such as skin tests, for example, but not limited to, the prick in the skin ⁇ prick-test), intradermorreactions, or epicutaneous tests (for example, the patch test), which are frequently used for the diagnosis of allergies.
• diagnosis methods also includes provocation tests, consisting of the controlled and gradual administration of the substance suspected of causing an allergic condition through different routes: oral, conjunctival, nasal, bronchial, etc., to check your tolerance
• provocation tests consisting of the controlled and gradual administration of the substance suspected of causing an allergic condition through different routes: oral, conjunctival, nasal, bronchial, etc.
• sequence repetitions refers to repetitive motifs in the sequence of a given protein or of a nucleic acid molecule. Sequence repetitions in the polypeptide sequences of the present invention were performed using the RADAR program (see EXAMPLE N 0 12).
• EXAMPLE 1 mRNA isolation and construction of an expression library from Anisakis simplex. Larvae of Anisakis simplex were manually extracted in the larval phase L3 (L3) of the viscera and peritoneal cavity of the bacaladilla
• mRNA Total messenger RNA
• the Anisakis simplex poly (A) + RNA was first converted to double-stranded cDNA, then ligated to the lambda-ZAP expression vector and the vector was packaged using Gigapack III packaging extracts. E. coli strain XL1-Blue MRF 'was used as host. The resulting library was amplified, and contained approximately 1.3 x 10 10 pfu / ml and a
• the A. simplex cDNA library was subjected to immunological screening using UA3 monoclonal antibodies. Following the recommendations provided in the ZAP-cDNA Gigapack III GoId Cloning Kit (Stratagene), XL1-Blue MRF 'host cells were grown in LB broth with supplements, centrifuged at 1000 xg, and the sediment was resuspended in magnesium sulfate 10 mM. To this suspension he was added an aliquot of Ie recombinant phages and the mixture was incubated 15 minutes at 37 0 C to allow the phage bind to the cells.
• the cell suspension with attached phages was mixed with melted NZY agar (cooled to about 55 0 C), and the mixture was spread evenly on a plate of 150 mm diameter agar containing freshly poured NZY. After a time of incubation from approximately 3 h to 42 0 C to allow phage plaques to begin to form, the expression of recombinant proteins was induced by coating the plates with nitrocellulose filters previously impregnated with 10 mM isopropyl thio-beta-D-galactoside (IPTG) in water ( Schleicher & Schuell, Dassel, Germany).
• IPTG isopropyl thio-beta-D-galactoside
• the plates were then incubated at 37 0 C for 3.5 h, after which were carefully removed Io nitrocellulose filters containing phage adsorbed plates and blocked with 3% BSA in TBS (50 mM Tris-HCl , 150 mM NaCl, pH 7.5) at 37 0 C for 30 minutes and then incubated with monoclonal antibodies (mAb) IgG1 / kappa UA3 (1: 10,000 dilution in TBS) at 4 0 C overnight.
• TBS 50 mM Tris-HCl , 150 mM NaCl, pH 7.5
• Nitrocellulose filters were washed in TBS containing 0.05% Tween 20 (TBS-T), and incubated for 2 h at room temperature with goat anti-rabbit IgG conjugated to alkaline phosphatase at 1: 10,000 (Pierce, Rockford , IL, USA). After washing again, immune complexes were visualized using NBT (tetrazolium nitro blue) and BCIP (p-toluidine-5-bromo-4-chloro-3-indolyl phosphate phosphate) (Sigma Chemical Corp., St Louis, MO , USES).
• NBT tetrazolium nitro blue
• BCIP p-toluidine-5-bromo-4-chloro-3-indolyl phosphate phosphate
• the cleaved pBluescript phagemid obtained packaged as phage particles filamentous, was used to infect SOLR host cells. Colonies of transfected SOLR cells containing the double-stranded pBluescript phagemid with the cloned DNA insert of interest were obtained by allowing the cells to grow on Luria-Bertani (LB) agar plates containing ampicillin (100 micrograms / ml). Individual bacterial colonies were grown in LB-ampicillin broth and pBluescript phagemid DNA was purified using a Perfectprep Plasmid Maxi kit (Eppendorf).
• nucleotide sequence described comprises a 3288 nucleotide sequence, represented by the sequence of SEQ ID N 0 : 1, and encodes a deduced amino acid sequence of 1096 amino acids represented by SEQ ID N 0 : 2.
• EXAMPLE 2 Subcloning of a fragment of the cDNA sequence (SEQ ID N 0 : 1) of Anisakis simplex in the vector pQE-31.
• an internal recombinant DNA fragment (SEQ ID N 0 : 15) corresponding to nucleotide positions 1303 and 2139 of SEQ ID N 0 1, which encodes a 279 amino acid polypeptide sequence (SEQ ID N 0 : 16), in the expression vector pQE-31 using a QIAexpress Type IV kit (Qiagen, IZASA SA, Barcelona, Spain) according to the manufacturer's instructions, and using the restriction sites Sal I and Hind III at the multiple cloning site of pQE-31.
• a QIAexpress Type IV kit Qiagen, IZASA SA, Barcelona, Spain
• the sequence of the cDNA fragment corresponding to SEQ ID N 0 : 15 was amplified by PCR, and the ends of the sequence were modified using the recombinant pBluescript phagemid as a template and a set of direct primers (5 ' SEQ ID N 0 21 3 ') and inverse (5' SEQ ID N 0 22 3 '), which contained restriction sites Salt I and Hind III.
• This modification by PCR provided specific nucleotide sequences for the restriction enzymes Sal I and Hind III so that the recombinant insert had cohesive ends with respect to the digested plasmid.
• the PCR conditions for the amplification and modification of the sequence of the DNA fragment corresponding to SEQ ID N 0 : 15 comprised: a cycle of denaturation at 94 0 C for 4 minutes followed by 35 cycles of (30 s of denaturation at 94 0 C; 30 s hybridization at 55 0 C; and one minute elongation at 72 0 C), and a final elongation cycle of 72 0 C for 7 minutes.
• the amplified Anisakis recombinant DNA was purified using a QiaQuick gel purification kit (Qiagen).
• one microgram of the plasmid DNA and 2 micrograms of the DNA encoding the fragment to be inserted were digested with the restriction enzymes Sal I and Hind III, according to the buffer and incubation conditions recommended by the manufacturer (Invitrogen SA, Barcelona, Spain).
• both the digested vector and the DNA insert were purified with the kit QiaQuick Gel purification kit (Qiagen) and ligated in a 1: 3 between the vector and the insert, using T4 DNA ligase at 16 0 C for one night.
• SEQ ID N 0 16 of Anisakis (central portion) and two additional amino acid sequences (tails): SEQ ID N 0 23 and SEQ ID N 0
• 6 x Histidine in the amino portion of the encoded polypeptide may facilitate the purification of recombinant polypeptides, but other vectors and purification methods may also be used as appropriate.
• E. coli M 15 [pREP4] cells with the recombinant pQE-31 vector was performed according to the instructions provided in the manual (The QIAexpresionist: A handbook for high-level expression and purification of 6xHis-tagged proteins) from the manufacturer (Qiagen). According to this protocol, an aliquot of cells were incubated 15 M competent previously prepared and an aliquot of the mixture of ligation on ice for 20 minutes and then incubated at 42 0 C for 90 minutes. After such incubations, the cells were resuspended in Psi broth and incubated for 90 minutes at 37 ° C.
• the M15 transformed cells were thawed and grown at 37 0 C in LB broth supplemented with kanamycin and ampicillin, with orbital stirring (200 rpm) until an OD 6 oo 0 ,5. After the expression of the polypeptide by adding 1 mM IPTG she was induced, and the cells were incubated for an additional 4 h at 37 0 C with the same stirring conditions. Subsequently, the cells were centrifuged at 5000 xg and stored frozen at -3O 0 C until the purification of the expressed polypeptide, as described in Example 5. Under these culture conditions, M15 cells produce the recombinant polypeptide of SEQ ID N 0 : 4 precipitate in the form of inclusion bodies.
• the inclusion bodies of the sedimented M15 cells were purified according to the following steps: a) the bacterial sediment was resuspended in 15 ml of B-PER reagent ( Pierce, Rockford, IL, USA) and stirred gently for 10 minutes; b) the sample was centrifuged at 15,000 xg and the supernatant, which contained soluble proteins, was discarded; c) the sediment containing the inclusion bodies was resuspended in another 15 ml of B-PER and lysozyme was added to the final cell suspension at a final concentration of 200 micrograms (Sigma-Aldrich, Madrid, Spain) and incubated at RT for 5 minutes; d) the suspension was diluted with 100 ml of a 1: 10 dilution of B-PER and the cells were resuspended using a vortex shaker; e) the cell suspension was centrifuged at 15,000 xg for 15 minutes
• the resin suspension was loaded on an empty column, and washed with two volumes of the loading buffer; i) the retained polypeptides were eluted with a solution containing Tris. 0.15 M Cl, pH 10.5, containing 250 mM imidazole, and stored frozen at -3O 0 C until use.
• EXAMPLE 6 Subcloning of the fragment of the Anisakis simplex cDNA sequence of SEQ ID N 0 : 17 into the vector pTARGET and transformation of competent JM109 cells.
• an internal fragment of 834 amino acids (SEQ ID N 0 : 17) corresponding to nucleotide positions 1306-2139 of SEQ ID NO: 1, which encodes the amino acid sequence of the polypeptide represented by SEQ ID N 0 : 18, was subcloned into the vector pTARGET (Promega).
• SEQ ID N 0 : 17 was amplified by means of PCR using a set of direct (5 'SEQ ID N 0 25 3') and reverse primers (5 'SEQ ID N 0 26 3') and the transformed vector pQE-31 of EXAMPLE 3 as a mold.
• the PCR conditions were: one cycle of denaturation at 94 0 C for 5 minutes, followed by 35 cycles of (45 seconds of denaturation at 94 0 C; 45 seconds of hybridization at 55 0 C; and 1.5 minutes of elongation at 72 0 C), and a final elongation cycle of 10 minutes at 72 0 C.
• the PCR products were analyzed on a 2% agarose gel and then cloned into the pTARGET vector.
• the ligation of pTARGET and the Anisakis insert, and the transformation of competent JM109 cells were performed as indicated in the instructions for use of the product. Positive JM109 transformants were selected by selection of white colonies using LB agar plates supplied with ampicillin / IPTG / X-Gal as described by the manufacturer.
• the subcloning of the cDNA sequence corresponding to SEQ ID N 0 : 17 in the pTARGET vector originated a plasmid ORF of 846 nucleotides (SEQ ID N 0 : 5) that codes for the sequence of 282 amino acids of SEQ ID N 0 : 6, which includes the Anisakis polypeptide of SEQ ID N 0 : 17, and an additional sequence of 4 amino acids (SEQ ID N 0 : 24) located in the carboxyl terminal portion of SEQ ID N 0 : 17.
• EXAMPLE 7 Isolation of recombinant plasmid DNA from J M 109 cells and vaccination of mice with plasmid DNA
• a positive colony of JM109 cells was grown in LB-ampicillin broth, centrifuged at 15,000 x g for 15 minutes and the sediment was used for the extraction of plasmid DNA using the Plasmid Maxi kit (Qiagen).
• a total of 50 microliters (concentration of one microgram per microliter) of plasmid DNA without endotoxins was inoculated into the quadriceps muscle of the two legs of Balb / c mice 10-20 weeks old. Inoculations were performed using an insulin syringe as described by Leiro et al. (2002).
• An immunoassay according to the present invention can be any immunoassay capable of detecting the presence of anti-Anisakis antibodies in any biological fluid from an individual or animal species.
• types of immunoassays include liquid phase and solid phase immunoassays, and competitive and non-competitive immunoassays in a direct or indirect format.
• the measurement of the levels of specific anti-Anisakis antibodies in a biological fluid can be carried out by ELISA methods, such as the ELISA methods described in this example.
• Serum samples The serum samples used in the ELISA methods of this example were obtained from uninfected individuals (healthy blood donors) and from individuals infected with the Anisakis simplex parasite. The sera were considered positive when they were obtained from individuals who had a recent history of having eaten raw fish and the presence of the parasite could be confirmed by endoscopic examination.
• a capture ELISA method (UA3-ELISA) based on the capture of O-deglycosylated antigens of Anisakis spp. specific for immobilized UA3 monoclonal antibodies.
• the O-deglycosylated antigen used in this immunoassay was obtained as previously described, and is based on the treatment of whole Anisakis antigens with 0.02 M NaOH at 5O 0 C for 16 h, and further neutralization of the pH with HCI.
• FITC was carried out according to the method of Liddel and Cryer, 1991); g) aspiration and washing 3 times with 200 microliters / well of PBS-T; h) aspiration, and incubation with 100 microliters of a 1: 1,500 dilution of rabbit anti-FITC antibodies conjugated to peroxidase (Dako, Barcelona, Spain); i) aspiration, and washing with 200 microliters per well of PBS-T; j) aspiration, and incubation with peroxidase substrate (Sigma Fast OPD, Sigma); k) reading of optical densities (OD) at 492 nm in multi-well reader (Molecular Devices, Sunnyvale, CA).
• the recombinant polypeptide of SEQ ID N 0 : 4 was used to perform an indirect ELISA to measure anti-Anisakis antibodies in sera of individuals infected with Anisakis.
• an ELISA method for the determination of IgE antibodies in human serum samples is described, but other classes (IgA, IgG, IgM) and subclasses (IgAI, lgA2, IgGI, lgG2, lgG3 and lgG4) of antibodies can also be measured changing the specificity of secondary anti-human antibodies.
• the immunoassay can also be adapted for antibody determinations in biological fluids of other animal species (eg, other mammals, birds or fish) by choosing the appropriate secondary anti-species antibodies.
• the method for indirect ELISA (rUA3-ELISA) of this example was performed according to the following steps: a) coupling of the ELISA plates with the recombinant antigen of Anisakis of Ia SEQ ID N 0: 4 in 0.1 M Tris.CI, pH 10.5; b) aspiration, and then washing the ELISA plate with PBS three times; c) aspiration and , after blocking ELISA wells for 1 h at 37 0 C with a solution of Tris buffered saline (TBS) containing skim milk powder and 3% Tween-20 0.2% (TBS -TM); d) aspiration and , after incubation of the plates for 1, 5 37 0 C with 100 microliters undiluted human serum; e) washed three times with 200 microliters per well of TBS containing 0.2% Tween-20 (TBS-T); f) incubation with 100 microliters of a 1: 2500 dilution of
• Table I shows the comparison of the IgE values obtained for 22 positive serum samples from patients infected with the Anisakis simplex nematode, tested by the capture ELISA (UA3-ELISA) and indirect ELISA (rUA3-ELISA) methods described in Example 8 of the present invention.
• the table shows the average absorbance values, measured at 492 nm, for each serum.
• EXAMPLE 9 Production of antibodies in mice transfected with the recombinant pT ARGET vector encoding the polypeptide of SEQ ID N 0 : 6
• blood was extracted from mice transfected with recombinant pTARGET encoding the polypeptide of SEQ ID N 0 : 6 (see EXAMPLE 7) with anesthesia, and the serum obtained was tested in indirect ELISA to determine the presence of IgGI antibodies reactive with the polypeptide of SEQ ID N 0 : 4 immobilized in the ELISA plate.
• the ELISA method was the same as described in EXAMPLE 8b, with the exception that peroxidase-labeled mouse goat anti-lgG antibodies (Caltag Laboratories, Burlingame, CA, USA; 1: 3000 dilution) were used instead of anti-reagent reagents -Human side.
• mouse sera were tested at a 1: 100 dilution.
• Table II shows the response of IgGI antibodies obtained in BALB / c mice after vaccination (parenterally) with 100 micrograms / mouse of the recombinant p-TARGET plasmid containing the Anisakis simplex DNA insert encoding the polypeptide of SEQ ID N 0 : 6. Blood extraction from mice was done one month after immunization.
• the table shows the mean absorbance values ⁇ SD for each serum, measured at 492 nm (three independent determinations).
• mapping of the peptides and polypeptides of the present invention with a biomolecule or detectable chemical molecule is useful for purposes such as in vivo and in vitro diagnosis and laboratory research.
• markers and methods for marking known to those skilled in the art (for example, Kessler (Ed). Nonradiactive labeling and detection of biomolecules. Springer-Verlag, Berlin 1992); Walker (Ed). The protein protocols handbook. Humana Press, Totowa, New Jersey (1996)).
• markers include enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds and chromogenic substances including colored particles such as colloidal gold and latex particles.
• the peptides and polypeptides of the present invention are radiolabeled with, but not limited to, 32 P, 14 C, 3H, 35 S, 125 I or 131 I.
• the labeled peptides can be detected by methods such as scintillation counting , gamma ray spectrometry or autoradiography.
• the peptides and polypeptides of the present invention can be labeled with the firefly luciferin.
• the bioluminescent compound can be covalently bound to the peptide or polypeptide selected by conventional methods, and the labeled peptide or polypeptide can be detected when an enzyme, for example, luciferase, catalyzes a reaction with ATP that causes the bioluminescent molecule to emit photons of light.
• an enzyme for example, luciferase
• Fluorogens are also widely used as markers. Examples of fluorogens include fluorescein and derivatives, rhodamine, Texas Red, phycoerythrin, phycocyanin and others. Generally, the fluorogen molecules are detected by a fluorescence detector.
• the peptides and polypeptides of the present invention can be labeled with biotin and captured on a solid support by a specific biotin ligand such as avidin or streptavidin.
• a specific biotin ligand such as avidin or streptavidin.
• the peptides and polypeptides of the present invention can be labeled with enzymes (for example, peroxidase, alkaline phosphatase and other enzymes that provide a chromogenic or fluorogenic reaction after the addition of the appropriate substrate).
• enzymes for example, peroxidase, alkaline phosphatase and other enzymes that provide a chromogenic or fluorogenic reaction after the addition of the appropriate substrate.
• Peptides and polypeptides labeled with selected enzymes can be used, for example, to detect specific antibodies that recognize said peptides or polypeptides in a capture ELISA method, where the antibody to be detected is first captured by another antibody immobilized on a solid support and the peptide or labeled polypeptide, plus the appropriate substrate, are used to reveal that the antibody-polypeptide binding took place.
• the peptides and polypeptides of the present invention can be labeled with colloidal gold for use in lateral flow immunochromatography assays, in accordance with methods well known to those skilled in the art.
• methods for producing gold particles of various sizes and for the conjugation of gold with proteins are described in: Dykstra (Ed). A manual of applied techniques for biological electron microscopy. Plenum Press New York
• the marking molecule can be magnetic microparticles and the system immobilized a magnet.
• EXAMPLE 11 Marking and applications of Anisakis simplex nucleic acid molecules of the present invention.
• Labeled nucleic acid molecules can be used as probes to detect complementary target sequences in a complex nucleic acid mixture by specific hybridization methods such as Southern, Northern blot, slot blot and dot blot transfer, in situ hybridization
• ISH ISH
• microarrays microarrays
• marking agents including, but not limited to: radioactive agents, fluorescent dyes, chemiluminescent agents; microparticles; enzymes; colorimetric markers (such as, for example, colorants, colloidal gold and the like); magnetic markers and haptens (such as, for example, biotin, dioxigenin (DIG) and others for which antisera or monoclonal antibodies are available).
• marking agents including, but not limited to: radioactive agents, fluorescent dyes, chemiluminescent agents; microparticles; enzymes; colorimetric markers (such as, for example, colorants, colloidal gold and the like); magnetic markers and haptens (such as, for example, biotin, dioxigenin (DIG) and others for which antisera or monoclonal antibodies are available).
• DIG dioxigenin
• the labeled probes of the present invention can be used as diagnostic tools, for example, for the detection of Anisakis spp. in infected human and / or animal tissues, and for research.
• the selected nucleic acid sequences of the present invention can be labeled with DIG by PCR amplification using DIG-labeled nucleotides (Boehringer-Mannheim, Germany).
• a nucleotide probe labeled with selected DIG can be obtained, corresponding to the positions of nucleotides 1490 and 1971 of SEQ ID N 0 : 1, using the transformed vector pQE-31 of the EXAMPLE 3 as a mold, and a set of direct priming (5 'SEQ ID N 0 : 27 3') and inverse (5 'SEQ ID N 0 : 28 3').
• the tissue samples to be analyzed obtained from human biopsies or from infected animals, can be fixed in 4% paraformaldehyde, and then analyzed to detect the presence of Anisakis spp. using ISH methods and the probe labeled with previous DIG.
• a detailed description of a method for performing the ISH of the present example has been described by Leiro et al. (2001), which was incorporated in this document as a reference.
• EXAMPLE 12 Analysis of synthetic peptide sequences that inhibit the binding of US3 monoclonal antibody with Anisakis simplex peptides and polypeptides of the present invention.
• peptide sequences are described that are recognized by the UA3 monoclonal antibody.
• Said peptide sequences were recognized as "repetitions" of amino acids by analyzing the amino acid sequence of SEQ ID N 0 : 2 using the RADAR program http://www.ebi.ac.uk/Radar/ in the EMBL-EBI (European Bioinformatics Institute).
• Selected sequences of 12 amino acids were chemically synthesized from these "repeats” and tested in competitive ELISA with respect to the inhibition of binding of monoclonal antibodies UA3 with the recombinant polypeptide of SEQ ID N 0 : 4, immobilized in the wells of an ELISA plate.
• the inhibitory activity of peptides corresponding to the N 0 : 7-11 amino acid sequences were tested in a capture ELISA, following the following typical steps: a) binding of the polypeptide of
• Table III shows the inhibition values obtained for the synthetic peptides of SEQ ID N 0 : 7-11 (tested at three concentrations) on the binding of the monoclonal antibody UA3 to the polypeptide of SEQ ID N 0 : 4 immobilized in a ELISA plate. The results were expressed as the percentage of inhibition for each dilution of peptide with respect to the control (without inhibition). ND: Not done. As was deduced from the results observed, the monoclonal antibody UA3 has a preference for peptides of SEQ ID N 0 : 7 and 8, which differs only in one amino acid.
• EXAMPLE 13 Importance of the synthetic amino acid sequences of SEQ ID N 0 : 7-11 as a target for anf / ' -Anisakis IgE antibodies present in sera of infected patients
• the inhibitory activity of the amino acid sequences (SEQ ID N 0 : 7-11) on the binding of human IgE anti-Anisakis antibodies to the polypeptide of SEQ ID N 0 : 4 in an indirect ELISA was tested.
• Table IV shows the inhibitory activity of a mixture of the synthetic peptides corresponding to SEQ ID N 0 : 7-11 on the binding of IgE antibodies present in sera of five infected patients to the immobilized SEQ ID N 0 4 polypeptide in ELISA plates.
• the peptide mixture was tested at a final concentration of 1.5 x 10 ⁇ 3 M for each peptide.
• the results were expressed as a percentage of inhibition of the peptide mixture with respect to the control (inhibited with a mixture of irrelevant peptides at the same final concentration).
• the sequences tested were targets for a range of 25-74 percent of all anti-Anisakis IgE antibodies present in the sera of infected patients.
• EXAMPLE 14 Characterization of Anisakis larval antigens in biological tissues
• the peptides or polypeptides of the present invention can be used to immunize a mammal (such as, for example, rabbits, sheep, goats, mice or rats) and the specific polyclonal antibodies obtained can be used to reveal the presence of larvae of Anisakis in infected tissues of humans or animals.
• the procedure for revealing Anisakis antigens in tissues with parasites can be performed following a conventional immunohistochemical method according to the following general steps: a) preparation of slides containing the sections of tissues included in paraffin; b) tissue blocking with TBS-TM or any other blocking reagent; c) washing stage; d) incubation with anti-polyclonal antibodies
• the immunization (in this example, the preferred tidal molecule is the enzyme peroxidase, but any other marker such as, for example, those indicated in EXAMPLE 10 of the present invention, for the mapping of secondary antibodies) can be used; g) washing step; h) incubation with the appropriate enzyme substrate (such as, for example H2O2 + DAB); i) observation under the microscope. In another preferred embodiment, the presence of Anisakis spp. Proteins can be revealed. which comprise the amino acid sequences of the present invention in infected tissues of humans or animals using the monoclonal antibody UA3 as the primary antibody, using conventional immunohistochemical methods.
• EXAMPLE 15 Detection of the presence of Anisakis antigens in food extracts using monoclonal antibodies and polyclonal antibodies of the present invention
• the AcMs eg, the Ac3 UA3
• the polyclonal antibodies obtained against the peptides, polypeptides or Proteins of the present invention can be used to design a capture ELISA method intended to detect the presence of Anisakis antigens in food extracts containing fish in its composition.
• the ELISA method captures for the determination of Anisakis antigens in a food extract can be carried out in accordance with the following steps: a) coupling of polyclonal anti-Annisakis antibodies (100 microliters / well; antibody concentration of 10 micrograms / ml) overnight; b) aspiration, and then washing the ELISA plate with PBS three times; c) aspiration and , after blocking ELISA wells for 1 h at 37 0 C with a PBS solution containing skim milk powder and 3% Tween-20 0.2% (PBS-TM); d) aspiration, and then overnight incubation at 4 0 C and orbital agitation, with 100 microliters of the corresponding food extract (supernatant obtained by homogenization of the sample in PBS and subsequent centrifugation at 3,000 g for 30 minutes); e) washed three times with 200 microliters per well of PBS containing 0.2% Tween-20 (PBS-T);
• Gastroallergic anisakiasis borderline between food allergy and parasitic disease-clinical and allergologic evaluation of 20 patients with confirmed acute parasitism by Anisakis simplex. Journal of Allergy and Clinical Immunology 105: 176-81.
• Garc ⁇ a M., Moneo, I., Aud ⁇ cana, M. T., Del Pozo, M. D., Mu ⁇ oz, D., Fernández, E., Diez, J., Etxenagusia, M.A., Ansotegui, IJ. ,

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