ES2595930B1 - Compounds and their uses as haptens for S. aureus detection - Google Patents

Abstract

Compounds and their uses as haptens for the detection of S. aureus. # The present invention relates to compounds derived from the bacterial wall of Staphylococcus aureus for use as haptens, its applications and methods for the detection of S. aureus, as well as to the antibodies and antisera generated for use.

Description

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Compounds and their uses as haptens for S. aureus detection

DESCRIPTION

The present invention relates to compounds derived from the bacterial wall of Staphylococcus aureus for use as haptens. In addition, the invention relates to haptens and their conjugates useful for obtaining antibodies and / or for detecting said bacteria. Therefore, the present invention can be included in the field of developing new technologies for clinical medicine.

STATE OF THE TECHNIQUE

Staphylococcus aureus is a pathogen of great virulence and one of the main microorganisms responsible for infectious diseases in developed countries. S. aureus causes nosocomial diseases, respiratory tract infections, endocarditis and soft tissue infections. When the infection passes into the bloodstream, the pathogen identification time is a key factor in acting and ensuring patient survival. The eradication of the diseases caused by this pathogen is hindered by the multidrug resistance to antibiotics that it presents and at the same time by the lack of fast and specific techniques to identify the cause of the disease.

In addition, S. aureus also poses a problem in food safety since said pathogen can be present in the flora of a healthy individual and when the necessary hygiene measures are not taken it can be a food contaminant and a direct source of infection for the being. human.

At present, cell culture is the reference technique for the identification of pathogenic microorganisms but this technique has the disadvantage that the waiting time ranges between 24-72 hours (J Clin Microbiol 2012, 50, 195), which is too high when the patient's life is at stake. On the other hand, methods of detection of S. aureus walls by immunochemical tests using antibodies developed from the immunization of semisynthetic forms of S. aureus peptidoglycan have also been described.

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Peptidoglycan (PG) is a non-crystalline macromolecule, which makes it difficult to determine the three-dimensional structure at high resolution, but also various experiments of nuclear magnetic resonance (NMR), high performance liquid chromatography and mass spectrometry (HPLC-MS ) and microscopy have elucidated much of this structure. In general, the PG consists of repeated units of disaccharide units, W-acetylglucosamine (GlcNAc) and W-acetyl-muramic acid (MurNAc) entwined by peptide chains that alternate amino acids D- and L-. The S. aureus PG has a stalk peptide ([Ala-DGlu (& 1) -NH2] [(& 1) -Lys-DAla-DAla]) entwined with a pentaglycile (Gly) 5 bridge that joins on one side through of the group £ -NH2 of the Lys located in the third position of a peptide stalk and on the other through the carbonyl group of the group DAla located in the fourth position of another peptide stalk (J. Amer. Chem. Soc2009, 131, 7023 -7030; J. Bacteriol. 2004, 186, 5978).

Some authors in the past have dealt with the production of antibodies against particular peptide sequences of the PG of S. aureus. In 1977, Seidl and Schleifer (European JB / ochem1977, 74 (2), 353) developed an agglutination test of latex with antibodies produced against a five-glycine synthetic peptide conjugated to human seroalbumine and Wergel et al. (JImmunolMeth1987, 104, 57) developed an enzyme-linked immunosorbent assay (ELISA) in sandwich format with monoclonal antibodies of the IgM type produced from oligopeptide (Gly) 5 immunization in its free form. More recently, Sandhu et al. (Analyst2012, 137 (5), 1130) reported the preparation of antibodies against a semisynthetic precursor of peptidoglycan used as an immunogen, which contains a peptidic part that contains the pentaglycine chain plus bovine seroalbumine N-acetylmuramic acid (BSA) . All these works cited have demonstrated the possibility of generating antibodies against the S. aureus PG but none provide data regarding the detectability of the bacterium in terms of colony forming units (CFU) and also does not show the performance in clinical samples. Therefore, a method is necessary where the capacity of such detection is demonstrated in infections caused by S. aureus with low analysis times and with good sensitivity.

DESCRIPTION OF THE INVENTION

In the present invention haptens derived from the bacterial wall peptidoglycan of S. aureus useful for the generation of antibodies against said invention are described.

bacteria and useful for the detection of it. A method of detecting S. aureus by antibodies generated by inoculation of the immunogens obtained from the bioconjugation of the haptens of the present invention to a protein is also described; This method significantly decreases the time of analysis and is easy to implement.

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In a first aspect, the present invention relates to a compound of general formula (I):

image 1

where:

(I)

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R1 is selected from OH and NHR ’; where R ’is selected from H or (C1-C4) alkyl;

n is an integer selected from 0 to 1; m is an integer selected from 0 to 7;

X1 and X2 are each independently selected from D-Glu, D-Gln, D-y-Glu and D-y-Gln;

Z and Z 'are each independently selected from an amino acid, which may be L or D, selected from the list consisting of Gly (glycine), Ala (alanine), Leu (leucine), Ile (isoleucine) or Val (valine); p and q are each independently an integer selected from 0 to 1;

Y and Y ’are each independently selected from R4, COR4 and Cys (cystel), optionally the cysteine may contain a thiol protecting group;

R2 and R3 are independently selected from H, (C1-C4) alkyl and CO (C1-C2) alkyl;

r, s, t or v are, independently, an integer selected from 0 to 1; R4 is a (C1-C5) alkyl, substituted with a group -SH, -halogen, -CECH, -N3 or -COR5;

R5 is selected from H or OR ’’; where R 'is selected from H or (C1-C4) alkyl;

with the condition that: if r is 1, s is 0 and if s is 1, r is 0, and

if Y or Y ’is selected from R4 or COR4, then t or v, respectively, is 0.

Hereinafter we will refer to this as the "compound of the invention".

In a preferred embodiment of the compound of the invention p and q are 0.

In another preferred embodiment of the compound of the invention, m is from 3 to 6 and more preferably m is 5.

In another preferred embodiment of the compound of the invention, n is 0 and / or R1 is NHR ’and R’ is selected from H or methyl.

In another preferred embodiment of the compound of the invention, n is 1 and / or R1 is OH.

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In another preferred embodiment of the compound of the invention, X1 and / or X2 are D-Gln or D-y-Gln.

In another preferred embodiment of the compound of the invention, X1 and X2 are the same.

D-Glu or D-glutamic acid is the group:

vAAA

\

image2

D-Gln or D-glutamine is the group:

\ AAA

image3

D-y-Glu or D-y-glutamic acid or D-iGlu is the group:

v / V \ A

image4

D-y-Gln or D-y-glutamine or D-yGlu-NH2 or D-iGln is the group:

iAAA

\

image5

In another preferred embodiment of the compound of the invention Y or Y ’is Cys, optionally substituted by a thiol protecting group.

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When Y or Y 'is Cys, this amino acid may be free or protected by a thiol protecting group, such as and not limited to -alkyl (Ci-C6), -COalkyl (Ci-Cs), -CH2Oalkyl (Ci- Cs), -COC6H5, -CONHalkyl (Ci-Cs), -COOCH3Ph, preferably the thiol protecting group is -COCH3.

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In another preferred embodiment, t and / or v are 1, more preferably R2 and / or R3 are CO (C1-C2) alkyl, and more preferably COCH3. More preferably R2 and R3 are CO (C1-C2) alkyl, and more preferably COCH3.

In another preferred embodiment, the compound of the invention is selected from:

.. (

HS) = 0 HN

H2N HN O

v °

HN

image6

NH '—NH

image7

“Vo

HN

...

H, N

NH °

/? nh

1 'u

HN

HN

image8

NH

and ^ N

Or h

NH,

PSau3

9nesd

image9

pnesd

image10

IV0 £ 6 969 Z SR

5

V °

HN

Or NH

H, N

image11

HN

NH '—NH

“V ° o ^ hn-Vnh

H, N v-

> 1,

ML

'—I

PSau7

V = o

HN

C> NH

"L.

HN

OR

NH NH

^ N> 0 O H

image12

NH,

Or ^

NH

/ .. (

HS OR HN

OR

Or NH

"'V.O

HN

OR

image13

NH NH

O O ^ x nh

H2N

HN

\ l

OR

O. NH

OR

h2n

image14

OR

HN

tHN-Ji

OR

image15

NH

N

OH

NH

OR

nh2

PSau8

By "alkyl (CrC4)" refers, in the present invention, to saturated, linear or branched hydrocarbon chains having 1 to 4 carbon atoms, for example, methyl, ethyl, n-propyl, / -propyl, n -butyl, tert-butyl or sec-butyl. Preferably the alkyl group has between 1 and 2 carbon atoms or (C1-C2) alkyl, more preferably it is a methyl.

By "substituted (C1-C5) alkyl" refers, in the present invention, to saturated, linear or branched hydrocarbon chains, having 1 to 5 atoms of

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carbon, for example, methyl, ethyl, n-propyl, / -propyl, n-butyl, tert-butyl, sec-butyl, pentyl, etc., which is substituted by at least one substituent selected from the list consisting of -SH, -halogen, -CECH, -N3, -CHO, -COOH, -COOalkyl (C1-C4).

A second aspect of the invention relates to the use of the compound of formula (I) of the first aspect of the invention as hapten (hereinafter "hapten of the invention").

The term "hapten", as used in the present invention, refers to a molecule that by itself is not capable of generating an immune response in an animal and needs to be attached to a macromolecule to generate an immune response. In the present invention, the hapten is selected from a compound of formula (I) of the first aspect of the present invention.

In a preferred embodiment, the hapten can be selected from the list consisting of PSau3, PSau4, PSau6 and PSau8.

The term "antigen" refers to a molecule, such as a peptide, a carbohydrate, a glycolipid, a glycoproteln or a molecule that is recognized and binds to an antibody. The part of the antigen that is the binding target of the antibody corresponds to the antigenic determinant In the context of the present invention, the antigen refers to S. aureus, or an analyte derived from S. aureus, that is, to a peptide part of the chemical structure of the bacterial wall peptidoglycan of S. aureus or a hapten of the invention conjugated with a macromolecule.

A third aspect of the invention relates to a conjugate (hereafter conjugate of the invention) comprising at least one hapten selected from a compound of formula (I) of the first aspect of the invention (the hapten of the invention) together with at least one component selected from the list consisting of: a macromolecule, such as a protein or a fragment thereof, an oligonucleotide or a polysaccharide; a detectable label or a support, in particular the support can be a polymer or an inorganic material.

The term "conjugate" refers in the present invention to the complex formed by the covalent union of a hapten selected from a compound of formula (I) and a second component selected from the group formed by a macromolecule, such

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as a protein or a fragment thereof, an oligonucleotide or a polysaccharide; a detectable label or a support, such as a polymer or an inorganic material. In particular, it refers to the hapten-macromolecule complex. Methods for obtaining hapten-macromolecule conjugates are known to those skilled in the art.

The term "immunogen" refers to a conjugate according to the invention capable of generating an immune response of antibody generation, that is, a conjugate formed by a hapten of the invention, selected from a compound of formula (I), and a second component comprising a macromolecule that confers antigenicity, said macromolecule being responsible for the immunogenic character of the conjugate.

The macromolecules in the present invention can be proteins or fragments thereof, oligonucleotides or polysaccharides. Useful proteins have a mass greater than 10 kDA, preferably greater than 15 kDa. When the conjugates of the invention are used to immunize an animal, said proteins must come from an animal species other than the species to be immunized. Useful proteins include, without limitation, seroalbumins of various species, for example bovine (BSA), rabbit (RSA); mollusk hemocyanins, for example crab hemocyanin (HCH) or barnacle hemocyanin (KLH); horseradish peroxidase (HRP), ovoalbumine (OVA), conalbumine (CONA), thyroglobulin and fibrinogen, as! as fragments of said proteins that confer antigenicity, etc. Preferably the protein is selected from the list consisting of horseshoe crab hemocyanin (HCH), bovine serum albumin (BSA), ovalbumin (OVA) and conalbumine (CONA). More preferably BSA and HCH.

In the conjugates of the invention between a hapten and a protein, the proteins are linked to the hapten covalently by means of the amino acids that are accessible on their surface, preferably those amino acids with nucleophilic side chains. The reactant amino acid of the proteins is selected from the list consisting of, but not limited to, cystel, serine, tyrosine and lysine; preferably it is lysine. The procedures for achieving conjugation of haptens to other carrier molecules depend on the functional group present in the hapten molecule in question. The stability and solubility of the hapten must also be taken into account.

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The term "oligonucleotide" refers to a nucleic acid of at least 4, preferably at least 10, preferably at least 15, preferably not more than 100 nucleotides. Both oligonucleotides formed by conventional nucleotides linked by conventional phosphodiester bonds and variants thereof including modifications to the purine or pyrimidine moieties and modifications to the ribose or deoxyribose moieties are contemplated.

In a particular embodiment of the conjugate of the invention, the hapten and the macromolecule are linked by a crosslinking agent.

In a preferred embodiment of the present invention, the hapten of the invention, selected from a compound of formula (I), is conjugated to the macromolecule through the group Y or Y ’.

In a particular embodiment the conjugate of the invention is formed by the hapten and a protein.

When the Y or Y 'group of the hapten is a cystel, the -SH group is conjugated to proteins with maleimides, or by activation of the proteins with haloacetyls (such as iodoacetyl).

When the Y or Y 'group of the hapten contains carboxylic acid (-COOH), the mixed anhydride method, the carbodiimide (CDI) method or the ester-W-hydroxysuccinimide method (among others) can be used for conjugation. NHS) (the latter also known as the active ester method).

When the Y or Y 'group of the hapten contains an aldehldo (-CHO), it can be transformed into a carboxyl group by forming O- (carboxymethyl) oximes. The reaction is carried out by treating the hapten with O- (carboxymethyl) hydroxylamine. The reaction of the carboxylic acid formed with the protein is continued by one of the methods mentioned above. Haptens that have formyl groups can also be directly coupled through the formation of Schiff bases, which are transformed into amines by reduction with sodium borohydride.

When the Y or Y 'group contains a halogen, the conjugation is performed on a previously modified protein or peptide so that it has reactive thiol groups.

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All these methods of conjugation are well known in the state of the art and are described in a summary manner in Guerra et al. 2003 (Guerra M., Morris H. 2003. Cuban Journal of Qulmica, vol. XV, n ° 2). These methods are shown here for guidance purposes and not in a limiting sense, since other methods of conjugation known to those skilled in the art can be used.

In another particular embodiment, the hapten of the invention, selected from a compound of formula (I), may be linked to a labeling agent, as a marker for detection. In a particular alternative embodiment, the hapten may be attached to a support, such as a polymer or an inorganic material.

The term "support", as used in the present invention, refers to any solid material to which the components of the invention, in particular the antibodies, haptens or conjugates of the invention, are physically bound, thus being immobilized. Any of a wide variety of solid supports can be used in the immunoassays of the present invention.The materials suitable for solid support are synthetic such as polystyrene, polyvinyl chloride, polyamide or other synthetic polymers, natural polymers such as cellulose, as well as polymers natural derivatives such as cellulose acetate or nitrocellulose, and glass, especially glass fibers.The support can take the form of spheres, sticks, tubes, and microassay or microtiter plates.Sheet-like structures such as paper strips, plates small and membranes are also suitable.The surface of the supports can be permeable and impermeable to aqueous solutions Additional inorganic solid materials suitable for use in the present invention include, without limitation, silicone, glass, quartz, ceramic, metals and their oxides, silicon, silicates, silicides, nitrides, amorphous silicon carbide as well as any other suitable material for microfabrication. or microlithograph. Additional organic solid supports suitable for use in the present invention include, without limitation polymers such as polyimide, acrylate, polymethylmethacrylate, polystyrene or nitrocellulose.

The term "detectable label" or "labeling agent" refers to a molecular label that allows the detection, localization and / or identification of the molecule to which it is attached, by procedures and equipment suitable for detection, either by means of spectroscopic, photoqulmic means , biochemical, immunochemical or

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Chemicals Examples of detectable labels for the labeling of compounds include, but are not limited to radioactive isootopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophors, enzymes and combinations thereof. Methods for marking and gliding for the choice of suitable markings for different purposes are known to the person skilled in the art.

In another particular embodiment of any of the above, the conjugate of the invention has an immunogenic character, that is, it induces the formation of antibodies.

Thus, a fourth aspect of the invention relates to the use of a conjugate of the third aspect of the invention comprising a hapten of the invention, selected from a compound of general formula (I), and a macromolecule, which confers antigenicity for obtaining of antibodies.

The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain a binding site of antigen binding specifically (immunoreacts). with an antigen, such as, for example, a protein. As is known to one skilled in the art, there are 5 isotypes or major classes of immunoglobulins: immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin E (IgE) . The term "antibody" comprises monoclonal antibodies, or intact polyclonal antibodies, or fragments thereof; and includes human, humanized and non-human origin antibodies. In the context of this invention the term "antibody" refers to the immunoglobulin that the animal or a hybrid cell has synthesized specifically against the immunogen of the invention.

A fifth aspect of the invention relates to an antibody (hereinafter "antibody of the invention") obtained by immunization with the conjugate of the invention, this antibody specifically recognizes the hapten of the conjugate of the third aspect of the present invention, to S. aureus and / or an analyte derived from S. aureus. Preferably the antibody is monoclonal or polyclonal.

In a preferred embodiment, the antibody of the invention is linked to a labeling agent that allows its localization and / or identification, by means of spectroscopic, photochemical, biochemical, immunochemical or chemical means.

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"Monoclonal antibodies" are homogeneous populations of identical antibodies, produced by a hybrid cell resulting from the fusion of a clone of B lymphocytes descending from a single and single stem cell and a tumor plasma cell, which are directed against a single site or determinant antigenic The method of obtaining the monoclonal antibodies of the invention can be carried out according to conventional methods, known in the state of the art. Basically, the method consists in immunizing an animal with a conjugate according to the invention comprising a hapten, selected from a compound of formula (I), and a macromolecule that confers immunogenicity, and subsequently extracting cells from the spleen of the immunized animal, which are fused with myeloma cells in the presence of a fusion inducer, such as PEG-1500 by standard procedures (Harlow D and Lane D. Antibodies: a laboratory manual. 1988. Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York). The hybridomas are selected and subcloned by dilution. The clones suitable for expansion constitute a hybridoma cell line. Subsequently, said hybridoma cell line is cultured in a culture medium suitable for hybridoma cells to produce antibodies and secrete them into the medium, and the culture medium supernatant containing the produced monoclonal antibodies is subsequently collected. Optionally, said antibodies may be purified by conventional means, such as affinity chromatography, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis or dialysis. Therefore, in one embodiment of the invention the obtaining of antibodies comprises the administration of a conjugate of the invention and the collection of tissue cells of said animal capable of producing said antibodies.

"Polyclonal antibodies" are heterogeneous populations of antibodies that recognize several epltopos of the same antigen, that is to say against different antigenic determinants. To obtain polyclonal antibodies against a hapten of the invention, selected from a compound of formula (I), a non-human animal is immunized with a conjugate of the invention according to methods known to those skilled in the art. Once an acceptable antibody titer is obtained, the animal is exanguinated and the antiserum containing the seric antibodies formed by said animal is collected.

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In a preferred embodiment the conjugate of the invention used for the production of antibodies is a hapten of the invention, preferably PSau3, PSau4, PSau6 or PSau8, conjugated to HCH.

A sixth aspect of the invention relates to an antiserum comprising the antibody of the fifth aspect of the present invention.

The term "antiserum" refers to a serum obtained after immunization of an animal with an immunogen. The antiserum comprises specific antibodies of said immunogen generated after the immune response produced in the animal. In the context of the present invention, the immunogen is The conjugate of the invention and the antiserum comprises specific antibodies generated against the conjugate of the invention, the antibodies of the invention.

A seventh aspect of the invention refers to the use of the hapten of the second aspect of the invention, selected from a compound of formula (I), or of the conjugate of the third aspect of the invention, or of the antibody of the fifth aspect of the invention or antiserum of the sixth aspect of the invention for the detection of S. aureus in a biological sample isolated from a subject.

An eighth aspect of the invention refers to the use of the hapten of the second aspect of the invention, selected from a compound of formula (I), or of the conjugate of the third aspect of the invention, or of the antibody of the fifth aspect of the invention or antiserum of the sixth aspect of the invention for the detection of S. aureus in a food sample.

A ninth aspect of the present invention relates to a method for detecting S. aureus or an analyte derived from S. aureus in a sample (hereinafter "method of the invention") comprising the use of an antibody of the fifth aspect of the invention or of a fragment thereof capable of binding to the antigen or of an antiserum of the sixth aspect of the invention comprising the above antibody and the detection of the binding of said antibody to S. aureus, preferably also comprising the use of the conjugate of the invention.

The term "sample", as used in the present invention, refers to the sample to be analyzed by the method of the invention, capable of containing the analyte

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to be detected, in this invention, S. aureus analyte or an analyte derived from this bacterium is understood. Any type of sample can be analyzed according to the method of the invention. Thus, the analysis of isolated biological samples of both animal and human origin is contemplated, either for clinical purposes (for example, to detect infections caused by the presence of S. aureus and establish a therapeutic method to combat it) or in the field of food safety for the analysis of contaminants in food samples or environmental safety such as water, soil or surface, or samples from cell cultures.

By "analyte derived from S. aureus" is meant in this invention a chemical compound that maintains a peptidic part of the chemical structure of the peptidoglycan of the bacterial wall of S. aureus, which can be specifically recognized by the antibodies of the invention In a preferred embodiment the analyte derived from S. aureus peptidoglycan is a compound of formula (I), and in a more preferred embodiment it is a compound of formula (I) selected from PSau5 and PSau.7 These analytes are used as the standard sample to make the calibration line in the ELISA test.

In a preferred embodiment, the biological sample can be, for example, without limitation, blood, serum, plasma, saliva, urine, sputum, bronchoalveolar lavages (BAL) and bronchoaspirates (BAS). In a more preferred embodiment, the sample in which infections caused by S. aureus is detected is a BAL or BAS sample.

Therefore, the method of the invention comprises at least the following steps:

(a) contacting the sample to be analyzed with the antibody of the invention or a fragment thereof for the time necessary for binding (or incubation),

(b) identify the formation of the immunocomplexes formed with said antibody and / or measure the amount of said immunocomplexes.

Additionally, said method may also require a conjugate according to the invention. Therefore, in a particular embodiment, in addition to the antibody of the invention in step (a) a conjugate is used; wherein said conjugate comprises a hapten of the invention, selected from a compound of formula (I), and a

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second component that can be a macromolecule, a detectable labeling agent or a support, such as a polymer or an inorganic material.

The term "detection method" refers to a method that allows to establish whether a given sample comprises or does not comprise S. aureus or an analyte derived from S. aureus with adequate sensitivity and specificity. Typical detection desensibility intervals may be between approximately 20% and approximately 90% of the maximum signal The sensitivity of a detection method is given by the probability that the detection is positive in a sample containing the compound to be detected The sensitivity, expressed as percent, it represents the percentage of cases of positive detection with respect to the total of cases in which the compound to be detected is present, the greater the sensitivity, the greater the number of positive cases and the lower the number of false negatives.The specificity of a diagnostic method is given by the probability that the detection is negative in a sample that does not contain the compound to be detected. Ificity, expressed as a percentage, represents the percentage of cases of negative detection with respect to the total cases in which the compound to be detected is absent. The greater the specificity, the lower the probability of obtaining a false positive.

The term "immunoassay" or "immunochemical analysis technique" is an immunochemical method of analysis in which an antibody that specifically binds to an antigen is used. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, direct and / or quantify the antigen. Immunoassays include, but are not limited to, immunological techniques such as ELISA (Enzyme-Linked Immunosorbent Assay), Western blot, RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (Double Antibody Sandwich-ELISA), Immunoaffinity chromatography, strip or lateral flow tests (lateral-flow immunoassay), immunoprecipitation, dot-blot, radio immunoassay, flow cytometry, immunocytochemical and immunohistochemical techniques, techniques based on the use of biomarker biochips, biosensors or microarrays that include specific antibodies or tests based on colloidal precipitation in formats such as “dipsticks.” Among other immunoassays are also immunosensors whose principle of transduction can be optical, electrochemical, mass or thermometric. Likewise, immunosorbents are also included or extraction systems by

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immunoaffinity, which allow selective extraction of the analyte into a complex mixture. These systems are usually biohforid materials, the result of the stable binding of the antibody to a solid support (polymer, inorganic material, metal particles, etc.), and which are used for the separation or extraction of the analyte from the rest of the matrix components. . Such formats may be heterogeneous or homogeneous, sequential or simultaneous, competitive or non-competitive.

In a preferred embodiment of the method of the invention, the detection is performed by an immunochemical technique. Preferably the immunochemical technique is an ELISA type assay. Different types of ELISA are known, such as direct ELISA, indirect ELISA or sandwich ELISA, competitive or non-competitive. Methods included in the present invention are those ELISA assays in which the conjugate of the invention is anchored to the support, as well as those ELISA assays in which it is anchored to the support is the antibody of the invention. More preferably, the detection method is an indirect competitive ELISA.

The term "indirect competitive ELISA" refers to an assay in which the sample to be analyzed is added together, capable of containing a specific concentration of S. aureus or a derived analyte, and a specific antibody according to the invention recognized by S. aureus , where the analyte present in the sample will compete for its binding to the antibody with a conjugate immobilized on a solid support.When the sample contains the analyte there are no antibodies available to react with the immobilized conjugates and therefore the reaction produced by the labeling agent It is not very intense, while when there is no analyte present in the sample, a large amount of antibodies binds to the conjugate immobilized in the solid phase. The term "direct competitive ELISA" is similar and characterizes the assay in which the plate is immobilized of ELISA the antibody or specific antiserum and the sample to be analyzed is mixed with a hapten conjugate with a labeling agent of Tectable As in the case of the indirect test both compounds compete for the antibodies and the signal resulting from the test is inversely proportional to the amount of analyte present in the sample.

In a particular embodiment, the indirect competitive ELISA assay comprises the following steps:

to. immobilize on a solid support a hapten or the conjugate of the invention, preferably a conjugate comprising a hapten of the invention and

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a macromolecule or a conjugate comprising the hapten of the invention and a polymer;

b. remove hapten or non-immobilized conjugate,

C. add the sample to be analyzed and a first anti-PSau antibody obtained by immunizing an animal with a conjugate of the invention, or an antiserum containing the antibody, to the solid support of section (a) and incubating,

d. remove the first antibody and sample not bound to the conjugate or hapten,

and. adding a second antibody conjugated with a detectable labeling agent, said second antibody recognizing the first antibody that is bound in step (c), and incubating,

F. remove the second antibody not bound to the first antibody,

g. Identify and / or measure the amount of complex obtained according to section (e) with a composition containing a chromogenic, fluorogenic and / or chemiluminescent indicator substrate.

In the first stage of the method of the invention [step (a)] a hapten or a conjugate comprising a hapten of the invention and a macromolecule, preferably a proteme, or a conjugate comprising a hapten of The invention and a polymer. Any of a wide variety of solid supports can be employed in the immunochemical techniques of the present invention as described above. In the context of the present embodiment, the solid support are plates for the analysis of ELISA type immunoassays. The immobilization of the conjugate on the surface of a support such as polystyrene plastic is directed by the chemistry of the surface. The immobilization capacity of the conjugates that in this case act as competing antigens depends on many factors, such as time and temperature. The immobilization stage of the hapten or conjugate may include blocking support spaces that have not been occupied by said conjugates. The blocking is carried out by means of protemes or detergents, preferably non-ionic detergents. In order to reduce unstable interactions, the immobilized conjugate must comprise a macromolecule different from that used in the conjugate used as an immunogen. In a preferred embodiment the conjugate immobilized at this stage comprises a hapten of the invention and a macromolecule, preferably BSA.

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The components may be immobilized to the support by covalent bonds or non-covalent bonds such as hydrogen bonds, hydrophobic interactions or ionic bonds. A general review of suitable microarrays and supports has been described in Shalon et al. (Genome Res 1996, 6, 639). Alternatively, it is possible to use supports activated by epoxy groups, vinyl sulfonic groups, active ester groups, aldehldo groups, carboxyl groups, amino groups, thiol groups, isothiocyanate groups and the like. In the event that the support is activated by epoxy groups, these groups include 3-glisidoxypropyltrimethoxysilane (GTMS), 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, 3-glisidoxypropylmethyldiethoxysilane, 3- glisidoxypropyl triethoxysilane and the like.

Step (b) of the method consists in removing the hapten or the non-immobilized conjugate. Typically, this is done by washing, usually between 1 and 10 washes, preferably 2 to 5 washes. The washes are intended to eliminate conjugates that have not been immobilized, so that everything that is detected is specific and desired.

The third stage of the method of the invention [step (c)] consists in adding the analytical sample capable of containing the analyte to be detected and / or quantified, that is, S. aureus or a derived analyte, and a first anti-antibody. -Sau according to the invention on the solid support of section (a) and incubate. This stage is the competitive stage of the essay.

By "first anti-PSau antibody" is meant an antibody according to the invention, generated against a conjugate comprising a hapten of the invention and a macromolecule that confers antigenicity, preferably against a conjugate comprising the hapten of the invention and the HCH protein The S. aureus or the derived analyte present in the sample to be analyzed and the hapten or the conjugate immobilized on the solid support of step (a) compete for the binding of the first anti-PSau antibody.The hapten of the immobilized conjugate of the step (a) and the hapten of the conjugate used to obtain the antibodies from step (c) may or may not be the same.

Stage (c) is the crucial stage of the trial. In a preferred embodiment step (c) has a duration between 10 minutes and 1 hour, preferably 30 minutes. This test has been shown to tolerate a wide range of ionic forces and

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pH values The inventors have optimized the buffer used in the competitive stage of the test [step (c)]. In an embodiment of the invention, step (c) is performed in the presence of a buffer having the following characteristics:

i. a concentration of non-ionic detergent between 0% and 0.1% preferably

0.05%;

ii. a pH between 5.5 and 9.5, preferably 7.5;

iii. a conductivity between 1.5 and 52.2 mS / cm, preferably 1.5 mS / cm.

The fourth stage of the method of the invention [step (d)] is to remove the first antibody not bound to the hapten or immobilized conjugate. This unbound antibody will have bound to the analyte. Typically, this is done by washing under the same conditions as step (b).

The next step of the method of the invention [step (e)] is to add a second antibody conjugated with a detectable labeling agent, said second antibody recognizing the first antibody and incubating. Said second antibody is conjugated with a compound capable of reacting with some substrate, so that a chromogenic, fluorogenic and / or chemiluminescent detection is derived therefrom. This compound can bind to the antibody directly or through another component. The second antibody may be a natural immunoglobulin isolated from a non-human species and different from the species used to produce the first antibody (e.g., murine anti-IgG antibody, goat anti-IgG antibody, goat anti-IgM antibody) or It can be produced recombinantly or synthetically. It can be an immunoglobulin or an immunoglobulin fragment (for example, FAb, F [Ab] 2). As desired, other binding molecules may be employed together with or instead of such second antibodies. In a preferred embodiment, the second antibody is conjugated to an enzyme, preferably peroxidase, more preferably HRP. Suitable conditions for binding of the second antibody to the first antibody are known to those skilled in the art.

Next, step (f) takes place, which consists in eliminating the second antibody substituted to the first antibody. Typically, this is done by washing under the same conditions as in steps (b) and (d).

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Finally, the last step (g) consists of the identification and / or measurement of the amount of the complex obtained according to section (e) with a composition containing a chromogenic, fluorogenic and / or chemiluminescent indicator substrate. An indicator substrate is a substance capable of reacting with the labeling agent of the second antibody resulting in a chromophore, fluorescent or chemiluminescent compound that can be detected by methods known in the state of the art. Suitable indicator substrates for carrying out the present invention are known to those skilled in the art and accessible from various commercial sources. Preferably, the indicator substrate is chromogenic, such as 3,3 ', 5,5'-tetramethylbenzidine (TMB), acidoazino-bis (3-ethylbenzothiazoline 6- sulfonic) or phenylenediamine, without limitation of using other substrates such as chemical markers (for example, colloidal gold markers, latex balls). When chromogenic substrates are used, the presence of S. aureus or an analyte derived in the sample is evidenced by the appearance of a color whose intensity is inversely proportional to the number of analyte molecules bound to the first anti-PSau antibody added in the stage ( c) and its quantification can be carried out, for example, by means of a spectrophotometer. If a fluorogenic substrate is used the detection and quantification is carried out by means of fluorimetry, and if the substrate is chemiluminescent, the signal can be quantified by means of a luminometer. In a particular embodiment of the invention, the enzyme marker is horseradish peroxidase (HRP), the substrate is chromogenic and the reaction is enzymatic. In this case, the reaction is inhibited after a while since the substrate is added and for this the optimal operating conditions of the enzyme being used are changed.

The method can also comprise a calibration line prepared with known concentrations of analytes derived from S. aureus (among them PSau5 and / or PSau7 can be used) which allows quantifying the concentration of S. aureus detected in a sample.

A tenth aspect of the present invention relates to a kit or device for the detection of S. aureus or an analyte derived in a sample comprising at least one hapten of the invention, a conjugate of the third aspect of the invention, an antibody of the fifth aspect of the invention or an antiserum of the sixth aspect of the invention, or combinations thereof. Preferably the hapten, conjugate or antibody is immobilized on a solid support. Even more

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preferably the kit also comprises an antibody against the anti-PSau antibodies according to the invention.

A tenth aspect of the present invention relates to the use of the kit or device of the tenth aspect of the invention for the detection of S. aureus or an analyte derived in a biological sample or in a food sample.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Standard calibration curves for the ELISA PSau3-MP-BSA / As291. 1A: Calibration curve obtained with the commercial S. aureus PG in PBT.1B: Calibration curves of the PSau5 and PSau7 peptide epltopos in PBT, PSau7 in PBST-D, the assay buffer for deglycosylated samples and samples of BAL and BAS deglycosylated. Measures of N = 2 or 3 days, where each day the measurement is made in triplicate in three wells.

FIG. 2 Effects of the different physical-chemical parameters of the PSau3-MP-BSA / As291 immunoassay. Experiments were performed using PG standards in PBST. 2A: pH effect of the assay buffer; 2B: Effect of the ionic strength of the assay buffer; 3C: Percentage effect of Tween 20 in the assay buffer; 2D: Effect of pre-incubation time between antibody and analyte; 2E: Time effect of the competition stage. The results were obtained from the calibration of the calibration curves performed under the different conditions shown. Calibration curves were performed in triplicate in the wells of the plate for each of the concentrations. The IC50 values are expressed in p, g mL-1. Legend: ■ Amax; A. IC50.

FIG. 3 Evaluation of the matrix effect of the deglycosylation extract in the ELISA PSau3-MP-BSA / As291.

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FIG. 4 Immunoequivalents of PSau7 measured before and after the deglycosylation treatment of commercial PG prepared at different concentrations.

FIG. 5 Immunochemical response of S. aureus CECT 5190 with respect to P. aeruginosa CECT 110 and E. coli K12 CECT 433 before and after the deglycosylation treatment. All suspensions were prepared at a concentration of 107 CFU mL-1.

FIG 6 Samples of S. aureusCECT5190 doped at different densities in PBS, BAL and BAS. * Indicates the detection limit of the deglycosylated samples in PBS.

EXAMPLES

The invention will be illustrated below by tests carried out by the inventors, which shows the effectiveness of the product of the invention.

The compounds of formula (I) can be prepared following different methods known to any person skilled in the field of organic synthesis, in particular by the general procedures presented in the following schemes. The starting materials for the preparative methods are commercially available or can be prepared by methods described in the literature.

Material and methods:

A. CHEMISTRY

General methods and instrumentation. For the synthesis of the PSau3-PSau8 peptides. The crude peptides were purified using a semi-preparative method RP-HPLC Water 2487 and double wavelength detector XBridge BEH 130 and column C18, 5 pm, 10x100 mm with a flow rate of 16 mL min-1 in a gradient of 0-50 for 10 min, water eluents with 1% TFA and MeCN with 1% TFA.

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Synthesis of peptides:

The PSau3, PSau4, PSau5, PSau6, PSau7 and PSau8 peptides were manually synthesized in polypropylene syringes equipped with porous polyethylene discs using the appropriate polymeric support in each case. PSau3, PSau4, PSau5 and PSau6 were synthesized in 2-chloro trityl resin (2-CTC, 600 mg; 1 mmol g-1) and H-Rink Amide ChemMatrix® resin (600 mg, 0.53 mmol g-1) It was used for the synthesis of PSau7 and PSau8. Solvents and soluble reagents were removed by suction. The PSau peptides were synthesized using an Fmoc / tBu / Alloc strategy, using in all cases Fmoc-L-Lys (Alloc) -OH for the L-Lys in the third position of the first stem peptide and Fmoc-L-Lys (Boc ) for another L-Lyslocalized in the second peptide stem. Fmoc- DGlu-NH2 was used for PSau5, PSau6, PSau7 and PSau8 and Fmoc-DGln (Trt) -OH for PSau3, PSau4.Fmoc-LCys (Trt) was used to incorporate the LCys into these four peptides.

For PSau3, PSau4, PSau5 and PSau6, prior to the introduction of the first amino acid to the 2-CTC resin, it was washed with DMF (3 x 5 mL x 1 min) and CH2Cl2 (3 x 5 mL x 1 min). Fmoc-DAla-OH (1 equiv.) And DIEA (10 equiv.) In CH2Cl2 was added to the resin, and the mixture was stirred for 55 min at 25oC. The resin was blocked by adding MeOH for 10 min at 25 ° C, then washed with DMF (3 x 5 mL x 1 mL) and CH2Cl2 (3 x 5 mL x 1 min). For PSau7 and PSauS the introduction of the first amino acid (DAla) was achieved using standard blocking conditions.

In all PSau peptides, the sequence of the first stem peptide was synthesized and acetyl in the N-terminal position. Then, the Alloc group was removed and the necessary amino acids were introduced to synthesize the pentaglycyl bridge and the second stem peptide. Finally, the Fmoc group was removed from the ^ -terminal position of the second stem peptide and the acetylated amine. The coupling reactions were performed with the corresponding Fmoc-amino acid (3 equiv.) And DIC (3 equiv.) In DMF for 1 h. The Fmoc group was removed by treatment with a mixture of piperidine-DMF (1: 4 v / v; 2 x 5 min). The Kaiser colorimetric assay was used for the detection of amino acid incorporation. When necessary the re-coupling was performed with HBTU (3 equiv), HOAT (3 equiv) and DIEA (4 equiv) in DMF for 40 min.

The acetylations were carried out with acetic anhydride (10 equiv), DIEA (10 equiv) in DCM for 15 min. The Alloc group was removed with phenylsilane (10 equiv),

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Tetrakistriphenylphosphine Pd zero (0.1 equiv) in DCM (3 x 15 min). The cleavage of the resin peptides was carried out with a mixture of TFA-H2O-TIS (95: 2.5: 2.5) for 1 hour. After this time the TFA was removed by evaporation and the peptides were precipitated by the addition of cold anhydrous diethyl ether, centrifuged and the supernatant discarded. The solidose dissolved in H2O-MeCN (1: 1) and lyophilized. Peptides were characterized by HPLC and HPLC-MS.

The crude PSau peptides were purified by reverse phase semi-preparative HPLC chromatography using a linear gradient from 0% B to 50% B in 10 min, at a flow of 16 mL min-1 and UV detection at 220 nm. The elution system was A: H2O: TFA (99: 1, v / v) and B: CH3CN (99: 1), v / v).

PSau3: 62 mg, 98.3% purity (determined by HPLC at 220 nm). Theoretical mass for C54H87N19O18S: 1358.48; Found by LC-MS (ES): 1359.0 [M + H] +

PSau4: 62 mg, 99.9% purity (determined by HPLC at 220 nm). Theoretical mass for C54H87N19O18S: 1358.48; Found by LC-MS (ES): 1359.0 [M + H] +

PSau5: 35 mg, 96.2% purity (determined by HPLC at 220 nm). Theoretical mass for C54H87N19O18S: 1358.48; Found by LC-MS (ES): 1359.0 [M + H] +

PSau6: 5 mg, 99.9% purity (determined by HPLC at 220 nm). Theoretical mass for C54H87N19O18S: 1358.48; found by LC-MS (ES): 1360.18 [M + H] + and 644.77 [M + 2H] 2+

PSau7: 38 mg; 99.9% purity (determined by HPLC at 220 nm). Theoretical mass for C48H82N18O17: 1183.28; found by LC-MS (ES): 1184.65 [M + H] + and 593.04 [M + 2H] 2+

PSau8: 47 mg, 99.9% purity (determined by HPLC at 220 nm). Theoretical mass for C51H87N19O18S: 1286.42; found by LC-MS (ES): 1287.69 [M + H] + and 644.5 [M + 2H] 2+

B. IMMUNOCHEMISTRY

General methods and Instrumentation. The MALDI-TOF / TOF-MS (flight time mass spectrometer of ionization matrix assisted laser desorption) used to characterize protelna conjugates was AUTOFLEX (Bruker-

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Daltonics, Bremen, Germany) equipped with SmartBeam laser operating at 200 Hz. The pH and conductivity of all buffers and solutions were measured with a pH meter pH 540 GLP and a conductimeter LF 340, respectively (WTW, Weilheim, Germany ). The purification of protelnas was performed by dialysis using regenerated cellulose tubular membrane with a cut line of a molecular weight of 12,000-14,000 (CelluSep T-series membranes; Membrane filtration products Inc., Seguin, USA) or by molecular exclusion chromatograph (HiTrap® Sephadex® G-25 superfine column; Amersham Biosciences). Polystyrene microtiter plates were purchased from Nunc (Maxisorp, Roskilde, Denmark). The washing steps were carried out using an ELx405 HT microplate washer (BioTek, Vinooski, Vermont, USA). Absorbances were read using a SpectamaxPlus (Molecular Devices, Sunnyvale, CA) in a single wavelength mode of 450 nm. Competitive format curves were analyzed with a four-parameter logistic equation using SoftmaxPro v2.6 software (Molecular Devices), and GraphPad Prism® (GraphPad Sofware Inc., San Diego, CA). The standard curve was adjusted to a logistic equation of four parameters according to the formula Y = [(AB) / 1 - (x / C) AD] + B, where A is the maximum signal, B is the minimum of the signal , C is the one that produces 50% of the maximum concentration signal, and D is the slope at the point of inflection of the curve.

Tampons The PBS is 10 mM phosphate buffer in a 140 mM NaCl solution, and the pH is 7.5. PBST is PBS with 0.05% Tween 20. PBT is PBST without NaCl. PBST-D is a blank aqueous phase extraction solution resulting from chemical deglycosylation treatment, performed as described below, diluted 1 / 3.5 with PBT. The upholstery buffer is 50 mM carbonate-bicarbonate buffer pH 9.6. Citrate buffer is a 40 mM solution of sodium citrate pH 5.5. The substrate solution contains 0.01% TMB (3.3 ', 5,5'-tetramethylbenzidine) and 0.004% H2O2 in citrate buffer. The borate buffer is 0.2 M boric acid / sodium borate pH 8.7.

Reagents and immunoreactive. The peptides PSau3, PSau4, PSau5, PSau6, PSau7 and PSau8 were synthesized as described in the chemical section. Staphylococcus aureus peptidoglycan (77140) and rabbit anti-peroxidase IgG (A8275) were purchased from Sigma-Aldrich. Staphylococcus aureus CECT 5190, Escherichia coli K12 CECT 433 and Pseudomonas aeruginosa CECT 110 were supplied by the Protelnas Production Platform (PPP, Applied Microbiology Group,

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Autonomous University of Barcelona) previously inactivated by heat at 100oC. The samples were suspended in PBS and stored at -40 ° C.

Synthesis of bioconjugates. Prothena-peptide hapten bioconjugates with an LCys residue were synthesized using N-succinimidyl-3- maleimidyl propanoate (N-SMP) or N-succinimidyl iodoacetate (N-SIA) as heterobifunctional spacer arms, using the general procedure described then, to obtain the corresponding malimidopropyl peptide (peptide-MP) or peptide-iodoacetyl (peptide-IA-conjugates of protelna). PSau6 and PSau8 were coupled to HCH (horseshoe crab hemocyanin) and BSA (bovine serum albumin), while PSau3 and PSau4 only coupled to BSA. The bioconjugation of the haptens PSau3, PSau4, PSau6 and PSau8 was carried out through a two-step procedure:

Step 1: Activation of proteins. A solution of N-SMP (1.6 mol, 0.43 mg) or N-SIA (9.22 mol, 2.61 mg) in dry DMF (200 pL) was added dropwise to a solution of the protein (HCH or BSA, 10 mg each) in borate buffer (1.8 mL). The mixture was stirred 4 h at room temperature, and then purified by molecular size exclusion using borate buffer as eluent. The eluted fractions (0.5 mL) of the corresponding MP-protein or IA-protein conjugates were collected in Eppendorf tubes, and those that tested positive in the Bradford protein test were combined (3 mL). A part of this solution was reserved for MALDI-TOF-MS analysis, and the rest was used for conjugation as described in step 2.

Step 2: Bioconjugacion. Haptens PSau3 (2.17 mg, 1.6 mmol), PSau4 (2.17 mg, 1.6 mmol), PSau6 (6.26 mg, 4.6 mmol) and PSau8 (5.93 mg, 4, 6 mmol) were dissolved in 1: 1 ACN: H2O MilliQ (200 pL) and each solution was added dropwise to the corresponding purified activated protein solutions (10 mg; see step 1). PSau3 and PSau4 were conjugated to MP-protelna and PSau6 and PSau8 were conjugated to IA-protelna through the SH group. The mixture was kept under slight stirring for 4 h at room temperature to obtain the corresponding conjugate PSau3-MP-protelna and PSau4-MP-protelna or PSau6-CH2CO-protelna and PSau8-CH2CO-protelna. For the PSau6-CH2CO-protelna and PSau8-CH2CO-protelna conjugates, the blocking of the iodine atoms that did not react was carried out by adding a cystel solution (80 mM, 200 pL) and stirring the mixture for 2 h more at room temperature. Finally, the conjugates were purified

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by dialysis against 0.5 mM PBS (4 * 5 L) and MilliQ water (1 * 5 L), they were lyophilized and stored at -80 ° C. Working allcuotas were prepared at 1 mg mL-1 in PBS and kept at 4 ° C.

Hapten density analysis. The densities of the BSA conjugates were calculated by MALDI-TOF / TOF-MS by comparing the molecular weight of the native protein with the corresponding MP-or IA-protein and with the peptide-MP-protein or peptide-CH2CO-protein conjugate. The MALDI spectra were obtained by mixing the hydroxycinmic-trans-3,5-dimethoxy-4 matrix (2 pL, 10 mg mL-1 in CH3CN / H20 70:30, 0.1% formic acid, freshly prepared ) and the conjugate or protein solution (2 pL, 10 mg mL-1 in CH3CN / H2O 70:30, 0.1% TFA) (table 1).

Table 1. Hapten densities of the peptide-protelna conjugates.

 immunoreactive

 d-haptenoa% conjugation

 PSau3-MP-BSA

 2 5-6

 PSau4-MP-BSA

 2 5-6

 PSau6-CH2CO-BSA

 6 20-17

 PSau8-CH2CO-BSA

 9 26-30

The analyzes were performed using MALDI-TOF / TOF-MS. Moles of hapten per mole of protein. b Conjugation is calculated based on the assumption that the BSA has between 30-35 free lysine groups.

Polyclonal antibodies. Four white New Zealand female rabbits weighing 1-2 kg were immunized according to the immunization protocol previously reported (Anal. Chim. Acta 1997, 347 (1-2), 139-147). The antiserum obtained from immunization with PSau6-CH2CO-HCH were labeled As288 and As289 and those immunized with PSau8-CH2CO-HCH as As290 and 291. The evolution of antibody titers was evaluated by measuring the binding of dilutions in series of microtiter plate antisera coated with the homologous conjugate (PSau6-CH2CO-BSA and PSau8-CH2CO-BSA). After obtaining an acceptable antibody titer, the rabbits bled and the blood was collected in vacutainer tubes provided with a serum separation gel. The antiserum

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15 min at 4000 rpm was obtained by centrifugation and stored at -80 ° C in the presence of 0.02% NaN3. Work aliquots were stored at 4 ° C.

Deglycosylation treatment. The chemical deglycosylation of peptidoglycan (PG) from commercial preparations or bacterial suspensions was carried out with trifluoromethanesulfonic acid (TFMSA). PG (1 mg), PSau7 (1 mg) or bacterial samples (S. aureus, E. coli and P. aeruginosa; 1 mL of 109 UFCmL-1 of lyophilized bacteria) were suspended in a 2: 1 TFMSA solution: anisole (0.5 mL) and stirred for 1 hour at room temperature. Subsequently, diethyl ether (1.5 mL) was added dropwise to the suspension and then the mixture was added to a 1: 1 solution of pyridine: Milli-Q water (2.5 mL). The aqueous phase was washed with diethyl ether (2.5 mL x 3) and dried using a nitrogen flow for 10 min.

Samples of bronchoalvelolar (BAL) and bronchoaspirate (BAS) washes. BAL or

BAS of 10 patients diagnosed as having no S. aureus infection were treated with dithiothreitol (DTT) to hydrolyse their mucolrtic structure. The BAL or BAS samples were weighed (50 mg) and a 0.1% w / v DTT solution (200 p, L) was added and stirred for 30 min. Then, 200 p, L of PBS was added and the mixture was filtered with a 48 p, nylon filter and centrifuged (10 min, 4000 G). The supernatant was divided into aliquots and stored at -40 ° C. A representative white sample of BAL or BAS was prepared by mixing equal volumes of BAL or BAS with samples from 10 individuals diagnosed as having no S. aureus, respectively.

Matrix effect studies

Deglycosylation treatment. Non-specific interferences produced by the type of sample analyzed were determined.

to. White A white aqueous extract, prepared using the deglycosylation procedure in the absence of a sample, was used to prepare the PSau7 PBST-D calibration curves and was used to compare the response in the ELISA, with respect to the same curve prepared in PBST,

b. BAL or BAS: the calibration curves in BAL or BAS were prepared and evaluated by ELISA to compare the parallelism with the standard curve prepared in PBST-D. (See Figure 1B and Table 3)

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Non-deglycosylated doped samples: a. Samples prepared in buffer. Be

PBS dopo at various concentrations of PSau7, PG and S. aureus and the immunoreactive equivalents of PSau7 were measured in the PSau7 PBT reference curve by ELISA (Figures 4 and 6 and Table 4).

Deglycosylated doped samples: a. Samples prepared in buffer: PBS was doped at various concentrations of PSau7, PG, S. aureus, P. aeruginosa or E. coli. They were deglycosylated as described above and the immunoreactive equivalents of PSau7 were measured in the PSau7 PBST-D reference curve by ELISA using the procedure described for deglycosylated samples (Table 4 and Figure 4 for PG; Figure 5 P. aeruginosa and E. coli) b. BAL or BAS doped samples. BAL or BAS was doped at various concentrations of S. aureus and measured in the PSau7 PBST-D ELISA. (Figure 6 for S. aureus doped in PBS, BAL or BAS).

Indirect competitive ELISA: The appropriate concentrations of antisera (As288-As291) and bioconjugates (PSau3-MP-BSA, PSau4-MP-BSA, PSau6-CH2CO-BSA or PSau8-CH2CO-BSA) were selected to establish the tests ELISA after a 2D titration plate assay that was performed as described (Anal. Chim Acta 1997, 347 (1-2), 139) (table 2). The following quantitative procedure was established with the best As / bioconjugate combination:

The microtiter plates were upholstered with PSau3-MP-BSA (0.125 mg mL-1 in upholstery buffer, 100 pLcillo-1) 4 hours at 25 ° C. Then, the plates were washed with PBST (4 x 300 pL well-1) and standard peptide solutions (PSau5 or PSau7, from 1,500 nM to 0 nM in PBT) or PG (200 pg mL-1 to 0 were added) pg mL-1 in PBST) or samples (bacterial suspensions), previously pre-incubated with the antiserum (As291, 1/6000 diluted in PBT) for 30 min at room temperature, (100 pL well-1). After 30 min at room temperature, the plates were washed as before and the anti-IgG-HRP solution (1/6000 in PBST, 100 pL well-1) was added and incubated for an additional 30 min. After another wash cycle, the substrate solution (0.01% TMB (3.3 ', 5,5'-tetramethylbenzidine) and 0.004% H2O2 in citrate buffer) (100 pL well-1) was added, and The enzymatic reaction was stopped after 30 min at room temperature with H2SO44N (50 pL well-1). Absorbances were measured at 450 nm.

When the samples to be measured previously have been treated with TFMSA, the upholstery of the microplates was performed with PSau3-MP-BSA (1.5 mg mL-1 in upholstery buffer, 100 pL well-1) and the samples or solutions PSau7 standard (3350 nM to 0 nM in PBST-D, 90 pL well-1) previously pre-incubated with As291 5 (1/500 diluted in PBT, 10 pL well-1).

Table 2: Immunochemical parameters of the indirect competitive ELISA using PG solutions as standards.

 Immunization Hapten

 Test Absmax Absmin IC50 (pg mL-1) Pending R2

 As288 / PSau4-MP-BSA 1,033 0.225 3.08 -1.32 0.990

 As288 / PSai / 3-M P-BSA 1,055 0.251 2.63 -1.75 0.982

 As288 / PSai / 8-CH2CO-BSA 0.922 0.331 4.91 -1.06 0.993

 PSau6

 As288 / PSau6-CH2CO-BSA 0.936 0.208 2.58 -1.52 0.984

 As289 / PSau4- MP-BSA

 0.820 0.208 2.58 -1.21 0.977

 As289 / PSai / 3- MP-BSA 1,343 0,226 1.23 -0.92 0.999

 As289 / PSai / 8-CH2CO-BSA 0.926 0.263 1.68 -0.98 0.995

 As289 / PSau6-CH2CO-BSA 1,167 0,325 2.26 -1.15 0.995

 As290 / PSau4- MP-BSA 0.718 0.154 2.07 -1.73 0.977

 As290 / PSai / 3- MP-BSA 1,125 0,221 2.69 -1.66 0.999

 As290 / PSai / 6-CH2CO-BSA 1,441 0,239 3.44 -1.32 0.995

 PSau8

 As290 / PSau8-CH2CO-BSA 1,661 0.296 6.61 -1.19.995

 As291 / PSau4- MP-BSA

 0.806 0.149 1.20 -1.31 0.987

 As291 / PSau3- MP-BSA 1,340 0,165 1.59 -1.38 0.993

 As291 / PSat / 6-CH2CO-BSA 1,445 0.274 6.66 -1.02 0.997

 As291 / PSau8-CH2CO-BSA 1,790 0,299 12.43 -0.78 0.991

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Table 3 ELISA parameters developed based on microplate, using different analytes (PSau5, PSau7 and PG) and different conditions (PBT and PBST-D).

 Analyte>

 PSau5 PSau7 PGa

 Test conditions>

 PBTb PBTC PBST-DCd BAL Desglic.c, d BAS Desglic.c, d PBTb

 Amax

 0.926 ± 0.010 0.926 ± 0.010 1.609 ± 0.039 CO O +1 CD CT> CD 1.612 ± 0.061 0.752 ± 0.02

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 Amin

 0.059 ± 0.006 0.059 ± 0.006 O O OO CD 1+ O O -P ^ CSJ O o '+1 CD CD 0.152 ± 0.056 0.078 ± 0.019

 Pending

 -1.14 ± 0.06 -1.14 ± 0.06 -1.26 ± 0.09 -1.17 ± 0.14 -1.27 ± 0.22 -1.15 ± 0.13

 1 IC50, ng mL-

 0.86 ± 0.03 0.85 ± 0.06 7.62 ± 0.54 18.92 ± 1.55 14.98 ± 2.20 1002 ± 920

 nM or nmolKg -1

 0.69 ± 0.02e 0.72 ± 0.05e 6.42 ± 0.46e 128.10 ± 10.75f 101.20 ± 14.93f -

 _OD, ng mL- 1

 0.11 ± 0.01 0.13 ± 0.01 1.68 ± 0.11 1.13 ± 0.43 1.11 ± 0.22 120 ± 90

 nM or nmolKg-1

 0.10 ± 0.01e 0.11 ± 0.01e 1.37 ± 0.09e 19.08 ± 7.35f 18.8 ± 3.7g -

 R2

 0.998 ± 0.001 0.999 ± 0.001 0.931 ± 0.154 0.958 ± 0.126 0.878 ± 0.21 0.995 ± 0.004

aThe molecular weight of PG is not known. The tests were performed using three replication wells under the conditions shown in the table. PBT indicates that the immunoassay protocol was used for non-deglycosylated samples, while PBST-D indicates that the assay be performed under the conditions of deglycosylated samples. bThe values shown are the average and the standard deviation of the parameters of the calibration curve obtained in two different days. cThe values shown are the average and the standard deviation of the parameters of the calibration curve obtained in three different days. dA multiplication factor of 2.5 has been applied to the IC50 and LOD values corresponding to the deglycosylation treatment. eThe values are expressed in nM. fThe values are expressed in nmoles of PSau7Kg-1 BAL. g Values are expressed in nmoles of PSau7Kg-1 BAS.

RESULTS

In order to mimic a part of the Peptidoglycan (PG) of the bacterial wall of S. aureus, two epltopos of peptides corresponding to the structures PSau5 and PSau7 were synthesized that serve as analytes in the process of optimization of the method of detection of S. aureus . PSau5, contains the DAla-DAla sequence of the bacterial wall peptidoglycan of S. aureus, and PSau7 contains a single DAla residue. For the synthesis of these peptides, both carboxy and amino terminal residues were amidated (-CONH2 and CH3CONH-, respectively). Only in the case of the second residue DAla of the PSau5 the carboxy terminal group was left free.

The immunization haptens PSau6 and PSau8 were designed to mimic and maximize the recognition of these epltopos by placing an LCys residue at the amino terminus, next to one of the LAla residues that are linked to the PG MurNAc residues.

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Two other peptide haptens (PSau3 and PSau4), different from the immunization haptens, were designed and synthesized by introducing slight modifications with respect to the PSau5 peptide epltopo, to be used as competitors and to improve the detection capacity of the assay. PSau3 is very similar to the immunization hapten PSau6 except for the DGln residue that replaces D-yGlu-NH2, which is present in the PG structure and in PSau6. Therefore, as can be seen in PSau3, DGln forms a normal amide bond with the neighboring amino acid through the a-COOH group, unlike D-yGlu-NH2 in PSau6, which is linked through the group and -COOH, as in the actual structure of the S. aureus PG. PSau4 also has this modification, but also, the Cys residue is located on the opposite side leaving the terminal DAla-DAla more accessible for antibody recognition. A collection of four competing protelna-peptide bioconjugates with different degrees of heterology was obtained (PSau4-MP-BSA> PSau3-MP-BSA> PSau6-CH2CO-BSA «PSau8-CH2CO-BSA) for the development of competitive immunoassay.

The affinity of the antisera for the different bioconjugates was tested in titration experiments. All antisera showed greater affinity for homologous conjugates. Subsequently, the antiserum / bioconjugate combinations that showed good titers were tested for their ability to recognize the S. aureus PG. As a result, sixteen immunochemical tests with similar detectabilities were obtained (Table 2). Both immunization haptens PSau6 and PSau8 were found to obtain good immunoassays, indicating that both peptide epltopos are equally accessible for recognition of the antibody in the PG. However, although the hapten heterology did not play an important role for PSau6 antibodies, for those against PSau8, the detectability was slightly better using heterologous competitors (Table 2). PSau3-BSA / As291 was selected for further studies, since which was one of the combinations that show a better value of IC50 and relation

Absmin / AbsMax.

Study physical-chemical parameters in the trial

In order to evaluate the effect of physical-chemical parameters in the assay and improve the detectability of the S. aureus cell wall, parameters such as pH (5.5 to 9.5), ionic strength (1 , 5 to 52.2 mS cm -1), the percentage of Tween 20 (0-0.1%) of the assay buffer as other parameters such as the

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length of the competitive stage (10-60 min) or the introduction of a pre-incubation phase (0-60 min) of the analyte with the antiserum. As can be seen in Figure 2, the assay is stable between pH values of 5.5 and 8.5, low ionic strength values improve the detectability of the assay and the effect that Tween20 does not significantly affect the assay (Figure 2A, 2B and 2C). On the other hand, the introduction of a pre-incubation stage and increasing the time of the competition stage, improves the detectability of the test (Figure 2D and 2E). Therefore, a 15 min pre-incubation was established followed by a competitive stage 30 min, using a test buffer with an ionic force of 1.5 mS cm -1. Under these conditions, the ELISA is capable of reaching an LOD of 120 ± 90 ng ml-1 («3 ngpocillo-1; N = 2 days) with an IC50 of 1002 ± 920 ng mL-1 (N = 2 days) of PG (Figure 2A and Table 3), and the total test time is 2.5 h, using only 25 pL of sample.

Establishment of an immunochemical assay of ELISA type for the detection of peptide epitopes in the bacterial wall of S. aureus

Due to the lack of standardization of commercial preparations of PG, which are different depending on the procedure used for their extraction from the cell wall, in studies aimed at the determination of bacterial preparations it was decided to use the PSau7 or PSau5 epitopes as standards of quantification. Contrary to commercial preparations, these structures are well defined and chemically characterized, which provides more reproducible results that confer greater reliability to the assay.

As can be seen in Figure 1B and Table 3, the epitopes of selected peptide (PSau5 and PSau7) were recognized to a degree comparable with similar IC50 values of around 0.7 nM (0.8 ng mL-1). . PSau7 was chosen to calibrate the assay, expressing the results as equivalent immunoreactivity of PSau7 (equiv IR PSau7). On the other hand, the assay could be used to characterize the different batches of PG as a function of their immunochemical response in the assay, expressed as IR equivs of PSau7.

Deglycosylation of peptidoglycan to increase the detectability of the ELISA method

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The objective was to develop a diagnostic immunochemical tool capable of detecting infections caused by S. aureus without the need for pre-enrichment of the sample.

Initially, by measuring suspensions of serial dilutions prepared in PBS, it was observed that under these conditions only those prepared with cell densities greater than 107 CFU mL "1 can be detected effectively. Although this detectability is close to other tests reported by immunochemical measurements of S. aureus (Diagn Microbiol Infect Dis 2013, 75 (1), 28-36), this detectability is insufficient for diagnostic purposes and for this reason other authors have developed amplification methods and other techniques to improve sensitivity.

To increase the detectability of the assay, the inventors established that the peptide epltopos were more accessible to the antibody by breaking or deglycosylating the PG cell wall. Bacterial cell wall cleavage methods have been extensively studied to elucidate the PG structure or even to develop new antimicrobial agents but have not been described to increase assay detectability. The method of deglycosylation used was based on the chemical hydrolysis with TFMSA, although other methods described in the literature could have been used.The TFMSA is not dependent on mutations, the peptide amide bond is not affected and only breaks the bonds of the glycan fractions.

Taking into account that the TFMSA treatment could alter not only the PG, but also the physical-chemical characteristics of the resulting sample, in the first instance, the potential for non-specific interference caused by this treatment in the PSau3-MP ELISA was evaluated. BSA / As291. For this purpose, the deglycosylation treatment was performed as described in the experimental section but in the absence of a matrix, and the final solution was used to prepare the calibration curves. As can be seen in Figures 3A and 3B, the signal was completely inhibited in this medium. The extract had to be diluted at least 80 times in order to have a usable assay, substantially decreasing the detectability. Instead, it was decided to re-adjust the concentration of immune reagents in this deglycosylation medium, carrying out

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2D experiments using the TFMSA extract diluted 1/4 in PBT (pH 5.5 and a conductivity of 28 mS cm-1).

Figure 1B shows the calibration curve recorded under these conditions and Table 1 (column titled PBST-D) shows the parameters extracted from the adjustment of the calibration curve to an equation of four parameters. The LOD for PSau7 in the matrix was 1.37nM, a higher value than in the assay buffer (Figure 1B), but good enough to continue with our studies.

The robustness of the trial, evaluated by executing the calibration curves of PSau7 for several days (N = 3 days) showed that the assay is reproducible. Thus, in the value of IC50, the coefficient of variation (% CV) is 7.1%, while in the LOD it is 6.7%. (Figure 1B and Table 3, column titled PBST-D).

Recovery of PSau7 in the treatment of deglycosylation

On the other hand, the recovery of deglycosylation was evaluated by doping samples at known concentrations of PSau7, obtaining recoveries close to 100%. These results indicate that peptide epitopes are not altered by treatment, since the immunoreactivity equivalents (IR equiv.) Measured before and after treatment do not differ significantly (Table 4).

Table 4. Recovery of chemical deglycosylation.

 Measured, nM (% recovery)

 doped, nM

 PBST PBST-D

 22

 19.5 ± 0.88 (88) 21.1 ± 0.99 (96)

 2. 3

 25.1 ± 1.05 (109) 26.5 ± 0.98 (115)

Samples doped with PSau7y were measured by ELISA before and after deglycosylation treatment. The results shown are the average and the corresponding standard deviation of three doped samples at the same concentration, each measured in triplicate in three wells of the plate.

Samples of commercial preparations of PG and various bacterial suspensions (S. aureus, P. aeruginosa and E. Coli, 108-10 CFU mL-1) were deglycosylated following the

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The procedure described above and the IR equiv of PSau7 were measured before and after the treatment. The treatment allowed to significantly increase the immunochemical signal in the commercial PG preparation (Figure 4) and in samples of cultures of S. aureus (Figure 6), which indicates that the deglycosylation process efficiently increases the release of epltopos from peptides that react with the antibodies. Through this deglycosylation procedure, S. aureus can be detected in concentrations up to 104 CFU mL-1 without pre-enrichment of the cell culture (t-Test p <0.05 (p = 0.04) with respect to the S. aureus cell concentration of 0 CFU mL-1). In contrast, the samples of E. coli and P. aeruginosa show no response, either before or after treatment with TFMSA, at concentrations up to 108 and 107 CFU mL-1 (Figure 5), which demonstrates the specificity of the immunochemical method. that is presented here.

Evaluation in real samples

The incidence and importance of S. Aureus in the airways led to the evaluation of the functionality of this immunochemical method in BAL and BAS samples. Thus, a mixture demonstrates BAL or BAS obtained from patients not infected with S. aureus were used to evaluate the performance of the trial.

After a simple treatment of BAL and BAS with DTT followed by the deglycosylation treatment, it was seen that under these conditions the test continues to work well. The deglycosylated BAL and BAS samples showed a similar response compared to the PSEA7 PBST-D ELISA curve (Figure 1B) without the need for another treatment to minimize the matrix effect.

The specific peptide fraction of the PG of S. aureus can be detected in BAL and BAS samples with an LOD of 19.08 ± 7.35 nmoles Kg-1 (N = 3 days) and 18.8 ± 3.70 nmoles Kg-1 (N = 3 days), respectively.

BAL and BAS samples doped with S. aureus were prepared at different concentrations. Samples were treated as before and then titrated with the PSEA7 PBST-D ELISA reference curve. As can be seen in Figure 6, the immunochemical response of the BAL and BAS samples was very similar to that obtained from the S. aureus samples doped in PBST, except for the fact that the detectability achieved was lower ( 105 BAS and 106 for BAL), but still useful

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for the diagnosis, taking into account that the ELISA described does not need cell pre-enrichment culture steps to achieve this detectability.

In qulstic fibrosis (CF), bronchitis and pneumonia of patients have often reported bacterial loads around 106 CFU mL "1. As an example, Corne et al. (J Clin Microbiol 2005, 43, 3491) in one study With patients with pneumonia I report concentrations> 104 CFU mL "1 of S. aureus from BAL cultures and concentrations> 107 CFU mL" 1 from BAS cultures of patients with pneumonia. In patients with CF, S. aureus It is one of the first pathogenic agents that colonize respiratory vlas and bacterial loads reach up to 108 CFU mL "1 in the sputum (J Clin Microbiol 2002, 40, 2300).

In the present invention, a detection method based on a competitive ELISA is presented for the first time, using antibodies specific to the S. aureus PG, capable of detecting this microorganism at levels of 104 CFU mL "1, without pre-enrichment in cell culture Documents of the state of the art that addressed the development of antibodies against S. aureus PG do not show sufficient results to demonstrate the possibility of detecting bacteria in clinical samples, and often only show the data related to the detectability of the preparations of PG or cell wall.

In this regard, the strategy used in this document to generate antibodies against specific synthetic epltopos of the PG has allowed the creation of an immunochemical assay with better detectability and higher sampling performance capabilities, significantly reducing the test time and the volume of Sample needed. Thus, while this ELISA is able to detect 3 ng (120 ng mL "1) of PG (without deglycosylation) with only 50 pl and 2.5 h, the latex agglutination test reported by Seidl and Schleife (European J. Biochem1977, 74, 353 "363) detects 10 ng using 1 mL of sample (the total test volume is 11 mL) after 14 h (2 hours at 56oC), and Wergeland et al. (J Immunol Methods1987, 104, 57 "63) developed a sandwich ELISA reaching an LOD of 200 ng mL" 1 after a total test time of approximately 21 h. More recently, Sandhu et al. (Analyst 2012, 137, 1130 "1136) have reported a 4h ELISA based on the detection of PG previously adsorbed the well plates that reach a detectable value that is two orders of magnitude greater than the assay described here.

In addition, the ELISA described here detects S. aureus directly (without cell culture or pre-enrichment) in clinical samples at cell densities, which can be useful for diagnosis in almost 2.5 hours (1 h of sample treatment and 1.5 h for the test). On the other hand, the test is easy to perform, has a capacity for a high sample analysis and needs very small sample volumes.

Claims (33)

5 1. Compound of general formula (I) image 1 image2 image3 rT'1 (I) image4 2 where: R1 is selected from OH and NHR ’; where R 'is selected from H or alkyl (Ci-C4); n is an integer selected from 0 to 1; m is an integer selected from 1 to 7; 5 10 fifteen twenty 25 30 35 Xi and X2 are each independently selected from D-Glu, D-GIn, D-y-Glu and D-y-GIn; Z and Z ’are each independently selected from an amino acid selected from the list consisting of Gly, Ala, Leu, Ile and Val; p and q are each independently an integer selected from 0 to 1; Y and Y ’are each independently selected from R4, COR4 and Cys, optionally the cysteine may contain a thiol protecting group; R2 and R3 are independently selected from H, (C1-C4) alkyl and CO (C1-C2) alkyl; r, s, t or v are, independently, an integer selected from 0 to 1; R4 is an alkyl (CrC5), substituted with a group -SH, -halogen, -CECH, -N3 or -COR5; R5 is selected from H or OR ’’; where R ’’ is selected from H or (C1-C4) alkyl; with the condition of: if r is 1, s is 0, if s is 1, r is 0, and if Y or Y ’is selected from R4 or COR4 then t or v, respectively, is 0. 2. Compound according to claim 1, wherein p and q are 0. 3. Compound according to any of claims 1 or 2, wherein m is 5. 4. Compound according to any one of claims 1 to 3, wherein n is 0. 5. Compound according to any one of claims 1 to 4, wherein R1 is NHR "and R" is selected from H or methyl. 6. Compound according to any one of claims 1 to 3, wherein n is 1. 7. Compound according to the preceding claim, wherein R1 is OH. 8. Compound according to any of claims 1 to 7, wherein X1 and X2 are the same. 9. Compound according to any of claims 1 to 8, wherein X1 and / or X2 are D-Gln or D-y-GIn. 10 fifteen 10. Compound according to any of claims 1 to 9, wherein Y or Y ’are Cys. 11. Compound according to any one of claims 1 to 10, wherein R2 and / or R3 are the group -CO-C1-C2 alkyl 12. Compound according to the preceding claim, wherein R2 and / or R3 are the group -COCH3. 13. Compound according to claim 1 selected from NH AH /.V HS) = 0 HN H2N HN 0 = nn HN .. OH v ° HN 0 = ^ H2N HN OR HN .. HN -NH ^ already or HN- h2n Ah '2 "HN NH NH V_ NH2 NH ( cT'h PSau3 0 == ( NH /. ( HS) = or HN NH > 35, HN V-1 NH 0 = > 35 «- ^, nh ^ nh2 // Nl ^ NH ^ N> 0 Or h PSau4 Vc HN O, NH HN "V HN TO.. OH ° V NH / "" " HS) = 0 HN ... w HN O. NH H H image5 HN V TO v ° N -t O. NH H H image6 HN Vo -t NH '—NH o o HN hT \ —N 0 = O NH -to HN <7 ^ -TO \ —NH Vs N image7 NH '—NH2 or H NH '—NH h O O 'HN - \ _ Vo HN 0 = ^ 0 „NH M H2N) Vo HN NH qVhn Vo yes -t- image8 0 = 0, NH _F H, N> H, N NH '—NH O o HN ^ f ^ NH ^ —- NH PSau5 or NH N — NH2 NH - or H PSau6 image9 , NH ^ nh, V_nh ^ N> 0 Or h PSau7 Y 5 10 fifteen twenty 25 / .. ( HS) = O HN image10 "VO H? N Vc image11 C 14. Compound according to the preceding claim, wherein the compound is PSau3, PSau4, PSau6 or PSau8. 15. Use of the compound according to any of claims 1 to 14 as hapten. 16. Conjugate comprising at least one hapten selected from a compound of formula (I) according to any one of claims 1 to 14 together with at least a second component selected from the list consisting of: a macromolecule, preferably a protein or a fragment thereof, an oligonucleotide or a polysaccharide; a detectable label and a support. 17. Conjugated according to claim 16, wherein the second component is selected from a macromolecule, preferably a protein. 18. Conjugated according to any of claims 16 or 17 wherein the macromolecule is selected from the list consisting of: horseshoe crab hemocyanin (HCH), barnacle hemocyanin (KLH), bovine serum albumin (BSA), horseradish peroxidase ( HRP), ovoalbumine (OVA), and conalbumine (CONA), preferably HCH and BSA. 19. Use of a conjugate according to any of claims 17 or 18 for obtaining antibodies. 5 10 fifteen twenty 25 30 35 20. Antibody obtained by immunization with a conjugate defined in any of claims 17 or 18. 21. Antibody according to claim 20 wherein the antibody is monoclonal or polyclonal. 22. Antibody according to any of claims 20 or 21 wherein the antibody is also bound to a labeling agent. 23. Antiserum comprising the antibody according to any of claims 20 to 22. 24. Use of the hapten selected from a compound of formula (I) according to any of claims 1 to 14, or of the conjugate according to any of claims 16 to 18, or of the antibody according to any of claims 20 to 22 or of the antiserum according to Claim 23 for the detection of S. aureus or an analyte derived in a sample. 25. Use according to the preceding claim, wherein the sample is selected from an isolated biological sample of a subject, a food or environmental sample. 26. In vitro method to detect S. aureus or an analyte derived from S. aureus in a sample comprising the use of the antibody described in any of claims 20 to 22 or the antiserum according to claim 23 and the detection of the binding of said antibody or antiserum to S. aureus or an analyte derived from S. aureus. 27. Method according to claim 26 which further comprises the use of a conjugate according to any of claims 16 to 18. 28. Method according to any of claims 26 or 27 wherein the detection is performed by an immunochemical technique. 29. Method according to claim 28 wherein the immunochemical technique is an ELISA, preferably indirect competitive ELISA. 30. Method according to any one of claims 26 to 29 wherein the sample is selected from a biological sample isolated from a subject, a food or environmental sample. 5 31. Method according to claim 30, wherein the isolated biological sample is Select from the list consisting of: blood, serum, plasma, saliva, urine, sputum, BAL and BAS. 32. Kit or device comprising at least one hapten selected from a Compound of formula (I) according to any one of claims 1 to 14, or a conjugate according to any of claims 16 to 18, or an antibody according to any of claims 19 to 21 or an antiserum according to claim 22 or any combination thereof. 15 33. Kit or device according to claim 32 wherein the hapten, the conjugate or the antibodies are immobilized on a solid support. 34. Kit or device according to any of claims 32 or 33, comprising at least one conjugate and an antibody. twenty 35. Use of the kit or device according to any of claims 32 to 34 for the detection of S. aureus in an isolated, food or environmental biological sample.