DE4238806A1 - Methicillin-resistant staphylococcus aureus (MRSA)detection method - by detection of MRSA penicillin binding protein (PBP)2 antigen-antibody complex, and synthetic MRSA PBP2 peptides - Google Patents

Abstract

A detection method for methicillin-resistant Staphylococcus aureas (MRSA) or parts thereof in a sample using the disclosed peptides or derivs. thereof in immunological reactions with samples contg. antibodies against the MRSA and detection of an immunological complex. Also calimed is (a) MRSA-antibody detection in a sample using the disclosed and immobilised peptides, (b) isolated substantially pure MRSA-Penicillin binding protein (PBP) 2'-antigen, purified by affinity chromatography using a gel coupled to at least one of the peptides shown, (c) peptides of the sequences shown, (d) polyclonal and monoclonal antibodies reacting with MRSA-PBP2', (e) antibodies produced by hybridomas DM2-12, DM2-13, DM2-39, DM2-40, DM2-45, DM4-11, DM4-17, DM4-20, DM4-26, DM4-32, DM3-1. DM3-19 and DM3-26. USE/ADVANTAGE - Staphylococcus aureus causes dermatitis, pneumonia and organ failure. Since MRSA-infections are resistant to antibiotics and antimicrobial medicaments, for proper medication it first has to be established whether the disease is caused by MRSA, i.e. using the method above. Additionally MRSA is highly infectious and its detection is vital for appropriate precautions. In contrast to prior art methods using SDS-polyacrylamide gel electrophoresis for PBP2' in which the protein is often not pure and denatured thereby inhibiting the detection after cumbersome processing, the synthetically produced peptides in this method provide a simple and accurate MRSA-testa'-

Description

The invention relates to a synthetic peptide, the immuno chemically with an antibody (Ab) against MRSA (methicillin resistant Staphylococcus aureus) responds, and in particular a specific synthetic peptide that is immunochemical with Ab against MRSA-PBP2 ′ (methicillin-resistant Sta phylococcus aureus-penicillin binding protein 2 ′) reacts. These synthetic peptides can be used as immunogens become, which in animals including humans the Bil induction of anti-MRSA-Ab.

The invention further relates to a composition for du formation of anti-MRSA-Ab and a method of Induction of the formation of anti-MRSA-Ab.

The invention further relates to a method for detection and for measuring MRSA, MRSA-PBP2 ′ or anti-MRSA antibodies, that the use of said synthetic peptide, one polyclonal caused by this synthetic peptide or monoclonal antibody as well as MRSA-PBP2 ', which from MR Staphylococcus aureus strains using anti-MRSA-PBP2′-Ab has been cleaned. These procedures allow any human or animal MRSA infection ascertain.  

The invention relates to kits for the detection of MRSA or anti MRSA-Ab in a sample and for the diagnosis of MRSA infections.

The invention further relates to a method for separating and Purification of MRSA-PBP2 'from an MRSA lysate or cul turmedium using affinity chromatography using of that prepared with the synthetic peptide as an immunogen Antibody and essentially pure MRSA-PBP2 ', the has been obtained by this cleaning process.

Finally, the present invention relates to a mono clonal antibody which is used to react specifically with MRSA PBP2 'is capable, and a process for its preparation.

Reports of Staphylococcus aureus with methicillin resistance already existed in the early 1960s (M. P. Jevons, Br. Med. J., 1961). MRSA (Methicillin-resistant Staphylococcus aureus) is a Staphylococcus aureus with resistance to Β-lactam type drugs including penis cillin family, such as methicillin, and the cephem family. MRSA is a bacterium with multiple resistance to drugs known because its resistance is not only different antimicro bielle drugs including those of β-lactam type, but also from various other antibiotics sums up. MRSA has its ids with increasing number of cases tification found increasing attention since he was serious causes hospital infections (E.J. Benner and F.H. Kayser, Lancet, 1968; J.J. Klimek et al., Am. J. Med., 1976).

Staphylococcus aureus tends to have various disease types pen, such as dermatitis, pneumonia and organ failure which, in some cases, cause serious symptoms which eventually led to the death of the infected people and animals being able to lead. In view of the fact that numerous medicines substances for the treatment of MRSA, which both have no resi resistance to antimicrobial drugs as well as an anti biotic resistance, are not effective, what the Be of MRSA infections is extremely difficult  very first step in treatment is to determine whether the disease was caused by MRSA or Not. If an MRSA infection is the cause of the disease has been pointed out, it is essential to choose and do antimicrobial drugs and Antibiotics that are effective against MRSA. Staphylococcus aureus is also more widespread, mainly in surface areas and also other parts of live animals including man before, where such a distribution in the nasal cavity, in the anterior na not only in patients but also in the throat in numerous healthy people, the carrier of the pathogen have been determined. The same applies to MRSA, whereby any contact of an MRSA-free person with an MRSA carrier with the first named person an MRSA-In can cause infection. Because medical personnel as well MRSA carriers seriously questioned as infection transmitters comes, it is important to make every effort in Hospitals must be extremely clean from the start and periodic cleaning, so that on this Appropriate countermeasures can be taken. In view of the above, it is for important to sick people, effective in the presence of MRSA Ensure treatment while it is for healthy people important, for health maintenance, is risk keep an MRSA infection as low as possible.

So far, numerous studies on the drug Resistance mechanism made by MRSA. Drugs of the β- Lactam family such as methicillin, cloxacillin and cephalothin, couple with enzymes such as D-alanine carboxypeptidase and Pepti doglycan transpeptidase, used for the synthesis of bacteriel len cell walls are essential. By such a dome the enzyme is inactivated and thereby at the Synthesis of the bacterial cell wall hindered, so that the bacteria die off (D.J. Waxman and J.L. Strominger, Ann. Rev. Biochem., 1983). These enzymes are called Pe denoted nicillin binding proteins (PBP); in the case of Sta  Phylococcus aureus has been used to produce such proteins with 4 or 5 different molecular weights discovered: MG 79,000-87,000 (PBP1), 73,000-80,000 (PBP2), 70,000-75,000 (PBP3), 70,000 (PBP3) and 41,000-46,000 (PBP4) (N.H. Georgo papadakou et al., Antimicrob. Agents Chemother., 1986; N.H. Georgopapadakou and F. Y. Liu, Antimicrob. Agents Chemother., 1980; and A.W. Wyke, FEMS Microbial Lett., 1984). It's going on taken that the differences in number and mo molecular weight on different conditions of the gel electrophoresis and not on differences in each strains used in laboratories. It has been clarified that Staphylococcus aureus with penicillin resistance PBP with a molecular weight of 74,000-78,000 forms, whose affinity for drugs of the β-lactam family is lower than with PBP, which is due to common types of emp sensitive Staphylococcus aureus (D.F.J. Brown and P.E. Reynolds, FEBS Lett., 1980; N.H. Georgopapadakou et al., Antimicrob. Agents Chemother., 1982, M.V. Hayes et al., FEBS Microbial. Lett., 1981). This PBP is called PBP2 ', PBP2a or MRSA-PBP, which acts as a transpeptidase and has a lower level of affinity for drugs and from for this reason cell walls are synthesized and its activi maintained without it even in the presence of β- Lactam in one to kill sensitive Staphylococcus sufficient concentration is saturated (D.F.J. Brown and P.E. Reynolds, FEBS Lett. 1980; B.J. - Hartman and A. Tomasz, J. Bacteriol., 1984). Consequently There is also a constant in the presence of drugs MRSA propagation and to bring about various Disease types. There have been studies of other factors ren (besides PBP2 ′), which affect MRSA specificity, by guided. Several other factors were found however, PBP2 'has so far been considered the most important factor considered resistance (K. Murakami and A. To masz, J. Bacteriol., 1989). Methicillin is a semi-synthe Table β-lactam, which is not by β-lactamase (enzyme for cer setting of drugs of the β-lactam type) can be inactivated can. As for the position of the gene (mec gene) that ko for PBP2 ′  dated, concerns, it is known that this gene on the chromosome of Staphylococcus aureus is present (S. Cohen and H.M. Sweeney, J. Bacteriol., 1970; F.H. Kayser et al., Antimicrob. Agents Chemother., 1972, P.W. Stiffler et al., J. Bacteriol., 1973, J.-E. Sjoestroem et al., J. Bacteriol., 1975). His Position was determined on a chromosome map (S.A. Kuhl et al., J. Bacteriol., 1978). The position of Restriction site of the chromosome including the mec- Gens clarified (W.D. Beck et al., J. Bacteriol., 1986; K. Ubu kata et al., Antimicrob. Agents Chemother., 1989). In the An finally succeeded in vector cloning the mec gene (W.D. Beck et al., J. Bacteriol., 1986, M. Matsuhasi et al., J. Bacteriol., 1986). M.D. Song et al. (FEBS Letters. 1987) clarified a base sequence of a 4 Kb Re Striction enzyme Hind III fraction including the mec gene and showed that the promoter and that in the upward direction located region of the mec gene is similar to the gene for Coded plasmid-type Staphylococcus penicillinase.

It was over a strain of coagulase-negative staphylo coccus sp. reported methicillin resistance and simultaneously formed PBP2 ′ (H.F. Chambers, Antimicrob. Agents Chemother., 1982; K. Ubukata et al., Antimicrob. Agents Che mother., 1985) and that the mec gene is simultaneously a De termination factor acts (W. Tesch et al., Antimicrob. Agents Chemother., 1988; K. Ubukata et al., Antimicrob. Agents Che mother., 1990). Therefore, PBP2 'and mec gene are considered factors considered that the resistance of Staphylococcus sp. against antimicrobial agents such as methicillin, as well as others Antibiotics are involved.

There are currently 3 procedures (with rough classification) of MRSA antigen available in test samples. It deals is a procedure with a drug sensation sensitivity test, a procedure for determining one for MRSA specific antigen (mainly PBP2 ′) by antigen anti body response and a method of detecting an MRSA specific gene (predominantly mec gene) by a Southern  Blot analysis or a PCR method. A procedure under Ver SDS-PAGE uses a common method Purification of PBP2 ′, which is considered to be MRSA specific In the current application of SDS-PAGE is the Rein unit of the desired substance in numerous cases pregnant if there is a substance whose mo molecular weight close to that of the target substance. In the case of MRSA it is when using PBP2 'as the target substance knows that PBP2 'has a molecular weight that extremely is close to that of PBP2. Furthermore, the desired purpose not always achieved what is due to proteins denatured by SDS is due.

In addition, cleaning with SDS-PAGE has the disadvantage that Numerous steps are required to be a agreed to achieve yield. These are elaborate manual and time-consuming work steps necessary. When using the isoelectric focusing on substances with similar isoelectric points, it is also difficult to get one satisfactory separation or cleaning result aim.

The test for drug sensitivity enables a Be Judging whether a strain is an MRSA acts or not. For this, the MIC value (minimal inhibition concentration) with the disk diffusion process, the Mi co-dilution process or automated equipment certainly. This procedure requires incubation of the Strain in a culture medium. It is known that the Sensitivity of individual strains depending on the volume of the strain used, the pH of the culture medium, the NaCl concentration, the sucrose concentration, the Incuba tion temperature and the incubation period as well as depending varies from the question whether the bacterium already has a β- Lactam has come into contact or not (H.F. Chamber and C.J. Hackbarth, Antimicrob. Agents Chemother., 1987; B.J. Hartman and A. Thomasz, J. Bacteriol., 1984). PBP2 'is at a temperature of more than 42 ° C and a pH of we  less than 5.2 inactivated. Therefore, this procedure is not absolutely easy to handle. Furthermore, the Disk Diffu not always a clear decision to make fen, as these are often determined by the quality of the market available discs that are used for this purpose, be is influenced (Watanabe, Modern Medical Technology (Kensa To Gÿutsu), 1988; Sugano, Japanese Journal of Clinical Urology (Rinshou Hinyoukika), 1990). The American National Committee for Clinical Laboratory Standards (NCCLS) or the Japan Che motherapy Society use Muller-Hinton broth with a Zu Set of 2% salt as a culture medium for the senses and measure that Staphylococcus aureus is to be regarded as MRSA if the MIC value against oxacillin after 24 hours of cultivation at 35 ° C, 4 µg / ml or more wearing.

Lately, numerous studies have been done on a Detection method of an MRSA-specific gene (predominantly mec gene) using Southern blot analysis leads. As a probe for Southern blot analysis synthetic oligonucleotide (K. Hiramatsu et al., Anti microb. Agents Chemother., 1990) or one by splitting recombinant DNA obtained with a restriction enzyme Fragment (W. D. Bech et al., J. Bacteriol., 1986) after Mar used with a radioactive isotope. It's going on given that MRSA can be demonstrated by the application Precision of the mec gene when hybridizing the probe with the DNA of the tribe determined. However, it is about a tribe judges who shows no drug resistance, although he has mec genes; in other words, it doesn't form any PBP2 ', so that you pay special attention to this test result must give. When using the PCR method for after an MRSA-specific gene (predominantly mec gene) as with Southern blot analysis, a higher detection sensitivity to be expected as this procedure becomes a Gene amplification is capable (Minamiide et al., The 6th General Meeting of Japan Environmental Infectious Disease So ciety (Nippon Kankyou Kansenshou Gakkai Sokai), 1991). This  However, the method has the unrecoverable disadvantage that a Strain is classified as positive even if it does not PBP2 'forms and shows no drug resistance. What is the procedure for determining an MRSA-specific An tigens (mainly PBP2 ′) using the antigen anti body reaction, D.M. O′Hara and P.E. Reynolds, (FEBS Lett, 1987) that PBP2 ′ with the polyclonal Antibody derived from an immunized rabbit under Ver obtained from a partially purified antigen from MRSA has been proven. Because this antibody however, the antibodies in addition to an anti-PBP2 'antibody which contained antibodies against other types of antigens this antibody before its application on sensitive Sta phylococcus aureus an absorption pretreatment. In connection O'Hara et al. immunized to the aforementioned work. (FEMS Microbiol. Lett., 1989) a mouse using the same antigen as an immunogen and constituted a hybridoma by vertebral cells with the mouse myeloma cells ver melted. They received two monoclonal antibodies, namely 1/235 (isotype IgG2a) and 2 / 401.43 (isotype IgG1). they however, only used this antibody for Western Blot analysis. However, Western blot analysis is routine Not suitable test procedure, so that a strong There is a need to take a simple test procedure To provide use of such an antibody.

According to the invention is a convenient and simple method for the detection and / or measurement of MRSA-PBP2 ′ or anti MRSA-Ab, which is caused by MRSA infection, be provided. Furthermore, an expedient method for measuring Solution and diagnosis of MRSA infection in humans and animals ren provided.

According to the invention is used as a special means of detection and / or be a synthetic peptide for measuring MRSA provided that reacts immunochemically with anti-MRSA-Ab, in particular a synthetic peptide that immunochemically with anti-PBP2′-Ab reacts. Also provided are: a  specific antibody that is immunologically determined by the synthe table peptide has been formed; essentially pure MRSA-PBP2 'made from MRSA lysates and the like using the said antibody has been isolated and purified; as well as a simplified MRSA-specific procedure for follow-up white and / or for measuring MRSA in a sample that the Use of the synthetic, immunochemically with anti-MRSA-Ab reactive peptide, by this synthetic peptide formed MRSA-specific antibody or the ess chen pure MRSA-PBP2 'comprises. The invention further relates to a method for measuring PBP2 '.

The invention also relates to new synthetic MRSA related peptides that are superior Have specificity in MRSA detection, as well as MRSA-PBP2 ′, the from solutions containing MRSA, such as bacterial lysa ten, extracted and isolated.

The invention also relates to a detection method for anti-MRSA-Ab, the aforementioned synthetic peptides or MRSA-PBP2 'can be used directly. Subject of the he are also test packs or kits for the detection of MRSA or anti-MRSA-Ab in a test sample as well as diagnostic Tools for the detection of MRSA infections.

Furthermore, a method for the production of purified MRSA-PBP2 'from MRSA lysates or culture media provided by affinity chromatography, wherein An Antibodies that are immunologically recognized by the synthetic peptide Immunogen have been used. Furthermore concerns the invention also MRSA-PBP2 'itself, which is such a isolation process has been obtained.

The invention further relates to a composition for Induction of the formation of anti-MRSA antibodies which a effective amount of the new synthetic peptide or purified MRSA-PBP2 'contains. The subject of the invention is  a method of inducing the formation of an anti body that uses this composition.

A method for the detection of anti-MRSA-Ab provided the use of the antibody pers includes, with the Ab-inducing together composition containing the new synthetic peptide or MRSA-PBP2 'has been formed. In particular concerns the invention a detection method using a monoclonal antibody.

Finally, the invention also relates to monoclonal antibodies by (Ab), which leads to specific binding with MRSA-PBP2 ′ in the Are capable of producing hybridomas that secrete monoclonal secretions Munospecific with MRSA-PBP2 ′ reactive antibodies in the Are able, as well as a process for the formation of the mo noclonal antibodies and / or hybridomas.

The synthetic peptides and antigens according to the invention that as a valuable prophylactic and therapeutic effect viewed fabrics.

The invention relates to a method for the detection of MRSA or a fragment thereof, comprising the competitive Reaction of at least one peptide from the following group:

Gly-Ser-Lys-Lys-Phe-Glu-Lys-Gly-Met-Lys-Lys-Leu-Gly-Val-Gly-Glu-Asp-Ile-Pro-Ser-Asp-Tyr-Pro-Phe,
Pro-His-Leu-Leu-Lys-Asp-Thr-Lys-Asn-Lys-Val-Trp-Lys-Lys-Asn-Ile-Ile-Ser-Lys-Glu,
Met-Gln-Gln-Val-Val-Asn-Lys-Thr-His-Lys-Glu-Asp-Ile-Tyr-Arg-Ser-Tyr-Ala-Asn-Leu-Ile-Gly,
Ile-Gln-Asp-Arg-Lys-Ile-Lys-Lys-Val-Ser-Lys-Asn-Lys-Lys-Arg-Val-Asp-Ala-Gln-Tyr-Lys,
Met-Gln-Lys-Asp-Gln-Ser-Ile-His-Ile-Glu-Asn-Leu-Lys-Ser-Glu-Arg-Gly-Lys-Ile-Leu-Asp-Arg,
Met-Asp-Glu-Tyr-Leu-Ser-Asp-Phe-Ala-Lys-Lys-Phe-His-Leu- Thr-Thr-Asn-Glu-Thr-Glu-Ser-Arg-Asn-Tyr-Pro Leu-Gly,
Pro-Ile-Asn-Ser-Glu-Glu-Leu-Lys-Gln-Lys-Glu-Tyr-Lys-Gly-Tyr-Lys-Asp-Asp-Ala-Val-Ile-Gly-Lys or
His-Thr-Leu-Ile-Glu-Lys-Lys-Lys-Lys-Asp-Gly-Lys-Asp-Ile-Gln-Leu-Thr-Ile-Asp-Ala;

Derivatives thereof, which by addition of at least one Ami residues or by substitution of at least one amino nosacid residue with another amino acid residue or fragment have been obtained, and analogs thereof with less than 32 Amino acid residues containing at least part of it and for immunological reaction with an antibody against MRSA penicillin binding protein 2 ′ (MRSA-PBP2 ′) capable are;

Peptides of the formula:

X-Gly-Ser-Thr-Gln-Lys-Ile-Y,

in the X the meaning Tyr-Asn-Lys-Leu-Thr-Glu-Asp-Lys-Lys- Glu-Pro-Leu-Leu-Asn-Lys-Phe-Gln-Ile-Thr-Thr-Ser-Pro has or means a corresponding group in which one or more any amino acids from the N-terminal end of the peptide chain have been removed or replaced by substitution of minde least one amino acid residue by another amino acid residue has been obtained, -Y has the meaning -Leu-Thr-Ala-Met or means a group in which any one or more Amino acid residues from the C-terminal end of the peptide chain ent have been removed or by substitution of at least an amino acid residue thereof by another amino acid residue has been obtained;

Peptides of the formula:

X′-Lys-Ile-Asp-Gly-Lys-Gly-Trp-Gln-Lys-Asp-Lys-Ser-Trp- Gly-Gly-Tyr,

in the X ′ has the meaning Met-Ile-Gly-Leu-Asn-Asn-Lys-Thr-Leu-Asp-Asp-Lys-Thr-Ser-Tyr- or means a group in which one or more arbitrary amino acids from N-terminal end of the peptide chain have been removed or has been obtained by substitution of at least one amino acid residue thereof by another amino acid residue, derivatives thereof by addition of at least one amino acid residue or by substitution of at least one amino acid residue by another amino acid residue or a Fragment have been obtained, and analogues thereof with less than 32 amino acid residues, which contain at least a part thereof and are capable of immunological reaction with anti-MRSA-PBP2'-Ab;
with an antibody against MRSA in the presence of the sample to form the immunological complexes of the peptide and MRSA or its fragment in the sample with the anti-MRSA-Ab and the detection of the immunological complex formed or the reaction of the anti-MRSA formed by the peptide -Ab with the sample to form an immunological complex of anti-MRSA-Ab with MRSA or its fragment in the sample and the detection of the immunological complex formed.

The invention also relates to a method for detection of anti-MRSA antibody in a sample comprising the Immo bilization of at least one peptide from the following group:

Peptides of the formulas:

Gly-Ser-Lys-Lys-Phe-Glu-Lys-Gly-Met-Lys-Lys-Leu-Gly-Val-Gly-Glu-Asp-Ile-Pro-Ser-Asp-Tyr-Pro-Phe,
Pro-His-Leu-Leu-Lys-Asp-Thr-Lys-Asn-Lys-Val-Trp-Lys-Lys-Asn-Ile-Ile-Ser-Lys-Glu,
Met-Gln-Gln-Val-Val-Asn-Lys-Thr-His-Lys-Glu-Asp-Ile-Tyr-Arg-Ser-Tyr-Ala-Asn-Leu-Ile-Gly,
Ile-Gln-Asp-Arg-Lys-Ile-Lys-Lys-Val-Ser-Lys-Asn-Lys-Lys-Arg-Val-Asp-Ala-Gln-Tyr-Lys,
Met-Gln-Lys-Asp-Gln-Ser-Ile-His-Ile-Glu-Asn-Leu-Lys-Ser-Glu-Arg-Gly-Lys-Ile-Leu-Asp-Arg,
Met-Asp-Glu-Tyr-Leu-Ser-Asp-Phe-Ala-Lys-Lys-Phe-His-Leu- Thr-Thr-Asn-Glu-Thr-Glu-Ser-Arg-Asn-Tyr-Pro Leu-Gly,
Pro-Ile-Asn-Ser-Glu-Glu-Leu-Lys-Gln-Lys-Glu-Tyr-Lys-Gly-Tyr-Lys-Asp-Asp-Ala-Val-Ile-Gly-Lys or
His-Thr-Leu-Ile-Glu-Lys-Lys-Lys-Lys-Asp-Gly-Lys-Asp-Ile-Gln-Leu-Thr-Ile-Asp-Ala;

Derivatives thereof, which by addition of at least one Ami residues or by substitution of at least one amino nosacid residue with another amino acid residue or fragment have been obtained, and analogs thereof with less than 32 Amino acid residues containing at least part of it and for the immunological reaction with anti-MRSA-PBP2′-Ab in are able;

Peptides of the formula:

X-Gly-Ser-Thr-Gln-Lys-Ile-Y,

in the X the meaning Tyr-Asn-Lys-Leu-Thr-Glu-Asp-Lys-Lys- Glu-Pro-Leu-Leu-Asn-Lys-Phe-Gln-Ile-Thr-Thr-Ser-Pro has or means a corresponding group in which one or more any amino acids from the N-terminal end of the peptide chain have been removed or replaced by substitution of minde least one amino acid residue by another amino acid residue has been obtained, -Y has the meaning -Leu-Thr-Ala-Met or means a group in which any one or more Amino acid residues from the C-terminal end of the peptide chain ent have been removed or by substitution of at least an amino acid residue thereof by another amino acid residue has been obtained;

Peptides of the formula:

X ′ -Lys-Ile-Asp-Gly-Lys-Gly-Trp-Gln-Lys-Asp-Lys-Ser-Trp- Gly-Gly-Tyr,

in the X ′ has the meaning Met-Ile-Gly-Leu-Asn-Asn-Lys-Thr-Leu-Asp-Asp-Lys-Thr-Ser-Tyr- or means a group in which one or more arbitrary amino acids from N-terminal end of the peptide chain have been removed or has been obtained by substitution of at least one amino acid residue thereof by another amino acid residue, derivatives thereof by addition of at least one amino acid residue or by substitution of at least one amino acid residue by another amino acid residue or a Fragment have been obtained, and analogues thereof with less than 32 amino acid residues, which contain at least a part thereof and are capable of immunological reaction with anti-MRSA-PBP2'-Ab;
on a solid support, reaction of the immobilized peptide with the sample to form an immunological complex of the immobilized peptide with the anti-MRSA-Ab and detection of the immunological complex formed or competitive implementation of the anti-MRSA-Ab formed by the peptide with the sample with formation of both immunological complexes of anti-MRSA-Ab and detection of the immunological complex formed.

The invention is also isolated, in essence chen pure MRSA-PBP2'-antigen, which by affinity topography has been cleaned, including contacting a gel made with an antibody against at least one selected peptide is coupled to the following group:

Peptides of the formula:

Gly-Ser-Lys-Lys-Phe-Glu-Lys-Gly-Met-Lys-Lys-Leu-Gly-Val-Gly-Glu-Asp-Ile-Pro-Ser-Asp-Tyr-Pro-Phe,
Pro-His-Leu-Leu-Lys-Asp-Thr-Lys-Asn-Lys-Val-Trp-Lys-Lys-Asn-Ile-Ile-Ser-Lys-Glu,
Met-Gln-Gln-Val-Val-Asn-Lys-Thr-His-Lys-Glu-Asp-Ile-Tyr-Arg-Ser-Tyr-Ala-Asn-Leu-Ile-Gly,
Ile-Gln-Asp-Arg-Lys-Ile-Lys-Lys-Val-Ser-Lys-Asn-Lys-Lys-Arg-Val-Asp-Ala-Gln-Tyr-Lys,
Met-Gln-Lys-Asp-Gln-Ser-Ile-His-Ile-Glu-Asn-Leu-Lys-Ser-Glu-Arg-Gly-Lys-Ile-Leu-Asp-Arg,
Met-Asp-Glu-Tyr-Leu-Ser-Asp-Phe-Ala-Lys-Lys-Phe-His-Leu- Thr-Thr-Asn-Glu-Thr-Glu-Ser-Arg-Asn-Tyr-Pro Leu-Gly,
Pro-Ile-Asn-Ser-Glu-Glu-Leu-Lys-Gln-Lys-Glu-Tyr-Lys-Gly-Tyr-Lys-Asp-Asp-Ala-Val-Ile-Gly-Lys or
His-Thr-Leu-Ile-Glu-Lys-Lys-Lys-Lys-Asp-Gly-Lys-Asp-Ile-Gln-Leu-Thr-Ile-Asp-Ala;

Derivatives thereof, which by addition of at least one Ami residues or by substitution of at least one amino  nosacid residue with another amino acid residue or fragment have been obtained, and analogs thereof with less than 32 Amino acid residues containing at least part of it and for immunological reaction with an antibody against MRSA penicillin binding protein 2 ′ (MRSA-PBP2 ′) capable are;

Peptides of the formula:

X-Gly-Ser-Thr-Gln-Lys-Ile-Y,

in the X the meaning Tyr-Asn-Lys-Leu-Thr-Glu-Asp-Lys-Lys- Glu-Pro-Leu-Leu-Asn-Lys-Phe-Gln-Ile-Thr-Thr-Ser-Pro has or means a corresponding group in which one or more any amino acids from the N-terminal end of the peptide chain have been removed or replaced by substitution of minde least one amino acid residue by another amino acid residue has been obtained, -Y has the meaning -Leu-Thr-Ala-Met or means a group in which any one or more Amino acid residues from the C-terminal end of the peptide chain ent have been removed or by substitution of at least an amino acid residue thereof by another amino acid residue has been obtained;

Peptides of the formula:

X ′ -Lys-Ile-Asp-Gly-Lys-Gly-Trp-Gln-Lys-Asp-Lys-Ser-Trp- Gly-Gly-Tyr,

in the X ′ has the meaning Met-Ile-Gly-Leu-Asn-Asn-Lys-Thr-Leu-Asp-Asp-Lys-Thr-Ser-Tyr- or means a group in which one or more arbitrary amino acids from N-terminal end of the peptide chain have been removed or has been obtained by substitution of at least one amino acid residue thereof by another amino acid residue, derivatives thereof by addition of at least one amino acid residue or by substitution of at least one amino acid residue by another amino acid residue or a Fragment have been obtained, and analogues thereof with less than 32 amino acid residues, which contain at least a part thereof and are capable of immunological reaction with anti-MRSA-PBP2'-Ab;
with a culture medium or lysate of MRSA to bind an MRSA-PBP2'-component to the immobilized anti-MRSA-PBP2'-Ab and elution of MRSA-PBP2 'from the immunological complex.

The invention further relates to polyclonal or monoclonal Antibodies responsible for the specific reaction with MRSA-PBP2 ′ in are able.

The invention further relates to hybridomas which are used for Se cretion of the monoclonal, for specific implementation with MRSA-PBP2 'suitable antibodies are able, and ins following hybridomas in particular:

DM2-12, DM2-13, DM2-39, DM2-40, DM2-45, DM4-11, DM4-17, DM4-20, DM4-26, DM4-32, DM3-1, DM3-19 and DM3-26.

The invention further relates to peptides of the formula:

X-Gly-Ser-Thr-Gln-Lys-Ile-Y,

in the X the meaning Tyr-Asn-Lys-Leu-Thr-Glu-Asp-Lys-Lys- Glu-Pro-Leu-Leu-Asn-Lys-Phe-Gln-Ile-Thr-Thr-Ser-Pro has or means a group in which one or more arbitrary Ami residues removed from the N-terminal end of the peptide chain or by substitution of at least an amino acid residue by another amino acid residue is -Y has the meaning -Leu-Thr-Ala-Met or one Group means one or more of any amino acids have been removed from the C-terminal end of the peptide chain or by substituting at least one Amino acid residue obtained by another amino acid residue has been.

The invention further relates to peptides of the formula:

X′-Lys-Ile-Asp-Gly-Lys-Gly-Trp-Gln-Lys-Asp-Lys-Ser-Trp-Gly- Gly-Tyr,

in the X ′ the meaning Met-Ile-Gly-Leu-Asn-Asn-Lys-Thr-Leu- Asp-Asp-Lys-Thr-Ser-Tyr- or has a group that either by removing one or more of them  Amino acids obtained from the N-terminal end of the peptide chain or has been replaced by at least one amino acid residue by another amino acid residue leads, as well as derivatives thereof, which by addition of min at least one amino acid residue or by substitution of min at least one amino acid residue of it by another ami nosacid residue or a fragment have been obtained, and Ana loge of it, which contain at least a part of it and for immunological response with anti-MRSA-PBP2 'capable are.

The synthetically obtained peptide above can be used as MRSA Antigen can be used.

According to the invention, a correspondence to Peptide sequence (as an antigen that is particularly representative of MRSA is) that is part of MRSA-PBP2 ′ with 670 amino acids residues (M.D. Song et al., FEBS Letters, 1987) be taken.

Specific examples of peptide sequences include those with an optional fragment of the 32 amino acid sequence residues in the range from the tyrosine residue 382 to the methionine residue 413 on the C-terminal side of MRSA-PBP2 ′ and those with an optional fragment of the 31 amino acid sequence residues in the range from methionine residue 413 to tyrosine residue 443 as well as derivatives thereof, which can be obtained either by adding at least one amino acid residue or by substitution of at least one amino acid residue thereof by another Amino acid residue or a fragment have been obtained and the like

Particularly preferred examples are peptides of the following Formulas

These peptides can be according to the ver drive on the basis of the well-known Boc or Manufacture Fmoc process. But you can also go to ver various other conventional processes used for the Pep are known to be produced. For example Solid phase synthesis is possible, either manually or with a commercial automatic peptide synthesizer is carried out.

The peptide also has its fragments (e.g. 7 Ami nosacid residues or more less) an antigenic nature. Furthermore, the terminal group or even part of the Peptides are replaced by cysteine or tyrosine and the like, to couple the peptide with a carrier protein or with to enable a marker.

Furthermore, the provision of peptides is according to the invention of the following formulas:

Gly-Ser-Lys-Lys-Phe-Glu-Lys-Gly-Met-Lys-Lys-Leu-Gly-Val-Gly-Glu-Asp-Ile-Pro-Ser-Asp-Tyr-Pro-Phe,
Pro-His-Leu-Leu-Lys-Asp-Thr-Lys-Asn-Lys-Val-Trp-Lys-Lys-Asn-Ile-Ile-Ser-Lys-Glu,
Met-Gln-Gln-Val-Val-Asn-Lys-Thr-His-Lys-Glu-Asp-Ile-Tyr-Arg-Ser-Tyr-Ala-Asn-Leu-Ile-Gly,
Ile-Gln-Asp-Arg-Lys-Ile-Lys-Lys-Val-Ser-Lys-Asn-Lys-Lys-Arg-Val-Asp-Ala-Gln-Tyr-Lys,
Met-Gln-Lys-Asp-Gln-Ser-Ile-His-Ile-Glu-Asn-Leu-Lys-Ser-Glu-Arg-Gly-Lys-Ile-Leu-Asp-Arg,
Met-Asp-Glu-Tyr-Leu-Ser-Asp-Phe-Ala-Lys-Lys-Phe-His-Leu- Thr-Thr-Asn-Glu-Thr-Glu-Ser-Arg-Asn-Tyr-Pro Leu-Gly,
Pro-Ile-Asn-Ser-Glu-Glu-Leu-Lys-Gln-Lys-Glu-Tyr-Lys-Gly-Tyr-Lys-Asp-Asp-Ala-Val-Ile-Gly-Lys or
His-Thr-Leu-Ile-Glu-Lys-Lys-Lys-Lys-Asp-Gly-Lys-Asp-Ile-Gln-Leu-Thr-Ile-Asp-Ala, or
Gln-Asp-Lys-Gly-Met-Ala-Ser-Tyr-Asn-Ala-Lys-Ile-Ser-Gly- Lys-Val-Tyr-Asp-Glu-Leu-Tyr-Glu-Asn-Gly-Asn- Lys-Lys-Tyr-Asp-Ile-Asp-Glu;

as well as derivatives thereof, either by adding at least one amino acid residue or by substitution of at least one amino acid residue thereof with another ami nosacid residue or fragment have been obtained; and analogs of which with less than 32 amino acid residues containing at least a part of it is responsible for the immunological reaction with Ab against MRSA penicillin binding protein 2 ′ (MRSA-PBP2 ′) in are able.

These peptides can be made in the manner described above used and synthesized. Furthermore, these can be Modify peptides like the aforementioned peptides.

The following peptides are particularly preferred:

Gly-Ser-Lys-Lys-Phe-Glu-Lys-Gly-Met-Lys-Lys-Leu-Gly-Val-Gly-Glu-Asp-Ile-Pro-Ser-Asp-Tyr-Pro-Phe,
Pro-His-Leu-Leu-Lys-Asp-Thr-Lys-Asn-Lys-Val-Trp-Lys-Lys-Asn-Ile-Ile-Ser-Lys-Glu,
Met-Gln-Gln-Val-Val-Asn-Lys-Thr-His-Lys-Glu-Asp-Ile-Tyr-Arg-Ser-Tyr-Ala-Asn-Leu-Ile-Gly,
Ile-Gln-Asp-Arg-Lys-Ile-Lys-Lys-Val-Ser-Lys-Asn-Lys-Lys-Arg-Val-Asp-Ala-Gln-Tyr-Lys,
Met-Gln-Lys-Asp-Gln-Ser-Ile-His-Ile-Glu-Asn-Leu-Lys-Ser-Glu-Arg-Gly-Lys-Ile-Leu-Asp-Arg,
Met-Asp-Glu-Tyr-Leu-Ser-Asp-Phe-Ala-Lys-Lys-Phe-His-Leu- Thr-Thr-Asn-Glu-Thr-Glu-Ser-Arg-Asn-Tyr-Pro Leu-Gly,
Pro-Ile-Asn-Ser-Glu-Glu-Leu-Lys-Gln-Lys-Glu-Tyr-Lys-Gly-Tyr-Lys-Asp-Asp-Ala-Val-Ile-Gly-Lys or
His-Thr-Leu-Ile-Glu-Lys-Lys-Lys-Lys-Asp-Gly-Lys-Asp-Ile-Gln-Leu-Thr-Ile-Asp-Ala and the like

The peptides according to the invention are suitable for the detection of anti-MRSA-Ab in one sample. The peptides according to the invention can be contacted with the sample under conditions, un an immunological complex (antigen-antibody Complex; Ag-Ab complex) between the peptide and anti-MRSA-Ab can be formed in the sample. The formation of the Ag-Ab-Kom plexes shows the presence of anti-MRSA-Ab in the sample at. Such changes can be done with certain ge demonstrate and measure suitable measures.

In the aforementioned method, it is possible to use immunolo in addition measuring procedures including radioimmunoassays, Enzyme immunoassays and fluorescence immunoassays any ver apply procedures that involve an antigen-antibody Use reaction, e.g. B. turbidimetric method, aggluti nationwide process or Western blot process.

When using the peptides according to the invention, one can preferably the simpler method, e.g. B. a radioim munoassays or enzyme immunoassays.

The synthetic peptides according to the invention can each according to the type of test method with known markers to mark. You can use fluorescent radioisotopes Dyes, enzymes, chromogenic pigments, biotin, avidin, Gold colloid, magnetic particles and the like are marked. For example, the radioisotope ¹²⁵I using the Chloramine method used for marking without that the antigenic nature is impaired. Further the synthetic peptides can be mixed with different types of Carriers are coupled using known methods. In these cases, the synthetic peptide can also be a che be subjected to mixing modification. Examples of derar term supports are microtiter plates made of polystyrene or poly vinyl, glass tubes or glass beads, gelatin beads, Latex beads and supports for chromatography, such as Pa pier, cellulose and cellulose derivatives.  

When administering the synthetic peptides according to the invention Antibodies can be generated in animals, since the peptides Induce formation of specific antibodies. Doing so the synthetic peptide as an antigen in a first stage Carrier proteins such as bovine serum albumin (BSA), "keyhole limpet hemocyanin "(KLH) and ovalbumin (OVA) using crosslinking agents such as succinimidyl-4- (N-maleimidome ethyl) -cyclohexane-1-carboxylate (SMCC) and 1-ethyl-3- (3-dime thylaminopropyl) carbodiimide (ECDI) conjugated. Then will in the next step according to the invention the anti-synthesis pep tid-Ab by immunizing animals such as mice, rats, Ka rabbits and sheep, using the one above Obtained peptide-carrier protein conjugate prepared. Antibodies can be raised by immunizing animals with the synthetic peptide alone, even without carrier protein conjugate receive. Furthermore, the synthetic peptide or the syn thesepeptide-carrier protein conjugate with Freund's complete ad juvants or aluminum hydroxide gels and the like are mixed and to form an antibody-inducing composition be emulsified.

Antibody induction can be carried out according to conventional practice parenteral administration of such a composition Animals are carried out. In this case, preferably the administration sterilized with alcohol or the like wanted to avoid bacterial infections.

The peptide according to the invention can be according to a conventional Chen process for the production of monoclonal antibodies use by immunizing animals and then spleen cells isolated from the immunized animal and then this subject to a conventional cell fusion process.

For cell fusion, one can use an HVJ process, one PEG process, an electrical fusion process and the like. The monoclonal antibody according to the invention per can be obtained from the supernatant of a hybridoma cell culture  isolate and purify using this cell culture extension of conventional animal cell culture media with or without Serum in a culture flask or in a bioreactor for large-scale breeding has been preserved. Even the Ge recovery from an exudate obtained by in vivo breeding Vaccination of hybridoma cells in animals, such as mice, before forth pristan (2,6,10,14-tetramethylpentadecane) has been received.

The detection method for MRSA, MRSA-PBP2 ′ and their parti All antigens are guaranteed by using anti-MRSA-Ab or monoclonal anti-MRSA-Ab by immunizing animals obtained with the synthetic peptide of the invention wor are used.

It should therefore be emphasized that the use of the fiction according to monoclonal anti-MRSA-PBP2′-Ab a procedure for the after of MRSA, MRSA-PBP2 'and their partial antigens or guaranteed antigenic fragments.

In these cases it is preferred to use the antibody with the Contact sample under conditions such that anti-MRSA-Ab and MRSA or partial derived from Ag MRSA in the sample an immunological complex (Antigen-An antibody complex) can form. The formation of such Ag-Ab complexes show that MRSA-Ag is present in the sample be detected and measured by suitable methods can.

In addition to immuno, detection methods for this purpose come logical measurement methods including radioimmunoassays and enzyme immunoassays question all types of procedures that make use of an antigen-antibody reaction, e.g. B. turbi dimetric methods, agglutination methods and western Blot procedure.

Depending on the type of assay used, the antibody can also be used a known marking agent. The anti  body can be with radioactive isotopes, fluorescent Pigments, enzymes, chromogenic coloring substrates, bio Mark tin, avidin, gold colloid and magnetic particles. For example, a label with the radioactive iso top ¹²⁵I according to the chloramine T process without impairment the activity of the antibody. Furthermore, using the antigen with different carrier types known methods are conjugated. In such cases can occasionally be a chemical modification of the antibodies to be required. Carriers for such purposes are at for example microtiter plates made of polystyrene or polyvinyl, Glass tubes or glass beads, gelatin beads, latex beads gels or chromatography supports, such as paper, cellulose, Cellulose derivatives and silicon dioxide.

The by immunizing animals with the invention Anti-MRSA-Ab obtained in peptides are also suitable for extracting tion and purification of MRSA-PBP2 ′ from liquids with a MRSA content, such as MRSA lysate. The purification of MRSA-PBP2 ′ can be determined by affinity chromatography using a with anti-MRSA-Ab coupled gels.

The invention will be explained in more detail below with the aid of examples explained.

example 1

This example illustrates the production of synthetic Peptides.

Peptides of the formulas given below were found under Ver Model 431A Automatic Synthesizer Applied Biosystems Co. synthesized.

Commercial reagents such as. B. Fmoc- Amino acids and coupling agents from Applied Biosystems Co. used.

After the synthesis of the individual peptides using a solid phase method on a scale of 0.15 mmol, the peptide which was coupled to the resin was freed from protective groups and released by a conventional method, for example by treatment with anhydrous hydrogen fluoride, to release a crude one Cleaved peptide product. In the aforementioned case b), 56 mg of peptide were obtained. 40 mg of the crude peptide product was purified by reverse phase high performance liquid chromatography (RP-HPLC). The peptide produced under b) was obtained in the form of a white, flaky powder (7 mg). Peptide A was also obtained in the form of a white, flaky powder. The RP-HPLC diagrams for peptides a) and b) are shown in FIGS. 1 and 2, respectively.

Example 2

This example explains an EIA method for the detection of anti-MRSA-Ab using a synthetic peptide from Example 1.  

1) Manufacture on a microtiter plate for EIA immobilized synthetic peptide

First, the peptide was in 0.1 m carbonate buffer (pH 9.6) to form a solution with a concentration of 10 µg / ml dissolved. Then 0.1 ml of the peptide solution on the individual wells of a microtiter plate with 96 Wells (product from Flow) distributed. The microti terplatte was incubated overnight at room temperature, so that there is a thorough coupling of the peptide in the recess fung revealed. The peptide solution was then discarded. The Ver Wells were buffered three times with PBS (with phosphate physiological saline) with a content of 0.1% Tween 20 (polyoxyethylene sorbitan monolaurate). In the The next step was 0.1 ml of PBS containing 5% Rin serum albumin and 1% casein on each well distributed, after which a 1-hour incubation at 37 ° C to Blocking non-coupled areas in the recess connected. After another wash, the mic could titer plate can be used for the test. The on this Microtiter plate with immobilized peptide obtained in this way can last several months without losing its antigenic nature nate at -20 ° C.

2) Test procedure

Using the microtiter plate obtained above an EIA procedure was carried out.

One from a rabbit extracted with and from MRSA purified antigen had been immunized, serum obtained sample was made with PBS containing 5% bovine serum albu min diluted 1: 100 to form a solution. The resulting diluted serum sample (0.05 ml each) was in given the individual wells, after which a 1-hour incubation at 37 ° C (non-immunized Rabbit serum was used as a negative control). After washing, a 1: 1000-fold diluted solution of with Horseradish peroxidase labeled anti-rabbit IgG goat serum (product of Cappel Co.) in PBS with a content  on 5% bovine serum albumin as a secondary, enzyme-labeled Antibodies in an amount of 0.05 ml each given a deepening, after which there is a 1 hour Incuba tion at 37 ° C. After washing again, 0.1 ml o- Phenylenediamine (OPD) as a substrate in the presence of Hydrogen peroxide (20 mg OPD and 10 µg 35% hydrogen per oxide were dissolved in 50 ml 0.1 m citrate buffer with pH 5) added. During a standing time of 15 minutes in the room temperature resulted in decomposition of the substrate Effect of horseradish peroxidase with color development. By dropping 0.05 ml of 1N sulfuric acid into each The enzyme reaction was stopped and the wells Color development ended. The absorption at 492 nm was ge measure up. It was found that the synthetic peptide with MRSA Rabbit immune serum had responded (Table I).

Table I

Measurement results obtained in a similar manner using of the following peptides 6 ′ to 13 ′ are listed below:

Peptide (6 ′) with 25 amino acid residues with Cys before amino acid 1 of SEQ ID No. 6.
Peptide (7 ′) with 21 amino acid residues with Cys before amino acid 1 in SEQ ID No. 7.
Peptide (8 ′) with 22 amino acid residues, replacing amino acid 1, Met from SEQ ID No. 8 with Cys.
Peptide (9 ′) with 22 amino acid residues with Cys before amino acid 1 in SEQ ID No. 9.
Peptide (10 ′) with 22 amino acid residues, replacing amino acid 1, Met from SEQ ID No. 10 with Cys.
Peptide (11 ′) with 27 amino acid residues, replacing amino acid 1, Met from SEQ ID No. 11 with Cys.
Peptide (12 ′) with 24 amino acid residues with Cys before amino acid 1 in SEQ ID No. 12.
Peptide (13 ′) with 21 amino acid residues with Cys after amino acid 1 in SEQ ID No. 20.

Table II

Example 3

This example explains how to make an MRSA spec fish antibody using the according to Example 1 synthesized peptides.

1) Preparation of a conjugate from synthetic peptide and Carrier protein

According to Example 1, the synthetic peptide A) in which the methionine residue 413 in the MRSA-PBP2'-peptide by cysteine is and the synthetic peptide B), in which the Me thionine residue 413 MRSA-PBP2′-peptide is replaced by cysteine, produced.

100 ul dimethylformamide solution containing 2.54 mg Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, product of Pierce Co.) were used as cross-linking medium to 500 µl of a 0.5 m phosphate buffer solution (pH value 7.5) containing bovine serum albumin (BSA, crystalline, Given by Sigma Co.). After 30 minutes of change Free crosslinking agent from liquid mixture by gel filtration on a PD-10 G25 Se phadex® column (product of Pharmacia Co.) removed. The obtained mixture was with 100 ul 0.1 M phosphate buffer solution (pH 7.0) containing 17 mg of the synthetic Peptide A) or the synthetic peptide B) added. On finally, the reaction was carried out at room temperature for 3 hours. To the liquid mixture was terminated by gel film tration separately. A synthetic conjugate was obtained Peptide and carrier protein. The according to this example Coupling the synthetic peptides A) and B) with the carrier protein-made products are hereinafter referred to as A) -BSA or B) -BSA.

2) Antibody production

1 ml of a solution of the above products A) -BSA and B) -BSA containing 25 µg each of the synthetic peptide was with an equivalent volume of Freund's comm blended adjuvant mixed to form an emulsion. These Emulsion became subcutaneous once every 2 weeks to 6 weeks old female rabbits (Japanese, white) a total of 5 times inji graces. One week after the last administration it was Blood was collected and the antiserum pooled.  

3) Determination of antibody responsiveness

To measure the reactivity of the antisera against MRSA, An EIA procedure was carried out in the following way:

After recovering bacterial cells from MRSA cultures (Cultivation overnight at 37 ° C in a bacterial culture medium with shaking) by centrifugation the bacteria cells with Lysostaphin (product of Sigma Co.), a proteolytic enzyme, lysed, taking on one from 100 ml Culture medium obtained amount of bacteria 2 mg enzyme used were. An MRSA lysate was obtained. This MRSA lysate was in 0.1 m carbonate buffer solution (pH 9.6) in one concentration tration of 10 µg / ml dissolved. This solution was in a Vo lumen of 0.1 ml each on the wells of a micro titer plate with 96 wells (product of Flow Co.) distributed. This was followed by overnight at room temperature incubated for adequate coupling of the bacterial lysate to ensure in the wells. After removing the Ly The individual wells were sats three times with PBS containing 0.1% Tween 20 (polyoxyethylene sorbitan mono laurat) washed. Then 0.1 ml of PBS with a Content of 5% bovine serum albumin and 1% casein on the ver wells, followed by a 1 hour incubation at 37 ° C and blocking the non-coupled areas in of the recess joined to the expected non-specific Coupling from Ab during the EIA test to a minimum hold. After washing the wells again, the Mi Criteria plate used for the test.

After diluting the one obtained in step 2) above Rabbit serum in a ratio of 1: 30, 1: 100, 1: 300, 1: 1000, 1: 3000 and 1: 10,000 with PBS containing 5% cattle rumalbumin and 5% bovine serum globulin were each 0.05 ml of the diluted serum obtained on each well distributed. This was followed by a 1 hour incubation 37 ° C (non-immunized for the negative control  Rabbit serum diluted in the same way, ver turns.

In the next step, the wells were washed, then a solution of anti-rabbit IgG goat serum labeled with horseradish peroxidase (product from Cappel Co.) was added in an amount of 0.05 ml each and added for 1 hour Let stand at 37 ° C. After washing the wells, 0.1 ml of OPD solution was added, followed by a 15-minute reaction at room temperature. The individual wells were then mixed with 0.1 ml of 1N sulfuric acid to stop the reaction with horseradish peroxidase. The absorbance at 492 nm for the individual wells of the microtiter plate was then measured using a photometer. It was found that the antibody obtained using the synthetic peptide as an antigen showed an antigen-antibody reaction with MRSA (see Fig. 3).

Example 4

This example illustrates a sandwich RIA process for Detection of MRSA using the one in Example 3-2) kept immunized rabbit serum.

1) Purification of MRSA-specific antibody from immune serum

The synthetic peptide A) obtained in Example 3 was coupled with CNBr-Sepharose B® gels (product of Pharmacia Co.) according to the conventional procedure. After washing the gel with 0.05 M phosphate buffer, pH 7.4, the gel was transferred to a column. The serum obtained in step 2) of Example 3 was then circulated through the column using a peristaltic pump at 4 ° C. overnight in order to react MRSA-specific antibodies in the immune serum with the synthetic peptide coupled to the gel. After removing uncoupled serum from the gel by washing, 1 M acetic acid was added to elute MRSA-specific antibody A). After pH elution of the eluate with K ₂ HPO ₄ solution, it was confirmed that the activity of the antibody was not impaired when the method mentioned in Example 2-2) was used.

A corresponding treatment was carried out with that in Example 3 holding synthetic peptide B) with the formation of MRSA-spe specific antibody B) performed.

2) Preparation of antibody immobilized on beads for RIA

Polystyrene beads were used as a carrier for the reaction a diameter of 6.25 mm (commercial product). Per Beads were cleaned of 5 µg of the above Antibody B) with 0.16 ml of 0.05 m phosphate buffer solution given a content of 0.15 M NaCl. The mixed one obtained Solution was added to the beads to cover them with the anti conjugate body. After washing with buffer, one obtained Beads of immobilized antibody used for the RIA Test were used.

3) Preparation of an RIA antibody tracer ( ¹²⁵ I-labeled antibody)

Radioactive iodine can be introduced into the side chain of the aromatic ring of tyrosine residues of proteins to form a stable compound which acts as a tracer. The iodination of the above-mentioned purified antibody A) was carried out in accordance with the known chloramine T method. Antibody A) was mixed with a solution of ( ¹²⁵ I) sodium iodide and chloramine T. Ten or more seconds later, the mixture obtained was treated with sodium disulfite as a reducing agent, whereby the reaction was terminated. The resulting mixture was subjected to gel filtration chromatography using a column packed with Sephadex G® (PD10 column, product of Pharmacia Co.) to remove free iodine from the iodinated antibody A). After the ¹²⁵ I-labeled antibody A) had been diluted with PBS containing 3% bovine serum albumin and 1% bovine globulin, the tracer product obtained was used for the RIA test.

4) Test procedure

A test was carried out using the sandwich RIA method the immobilized beads obtained above Antibody and tracer performed.

After obtaining bacterial cells from MRSA cultures (cultivation overnight at 37 ° C in a bacterial culture medium The bacterial cells were shaken by centrifugation with Lysostaphin (product from Sigma Co.) a pro teolytic enzyme, lysed, taking 2 mg of enzyme from 100 ml of culture medium were used. An MRSA lysate was obtained.

The MRSA lysate was diluted with PBS containing 1% bovine serum albumin and 1% bovine serum globulin to lysate solutions containing 300, 100, 30, 10, 3, 1 and 0.3 µg / ml (methicillin-sensitive Staphylococcus aureus lysates (MSSA lysates were diluted as controls as appropriate). The lysate (0.1 ml) and 0.2 ml of buffer were each placed in a well of the reaction plate with 60 wells (commercial product). A bead of immobilized antibody was then added to each well. After overnight incubation at room temperature and sufficient promotion of the reaction of MRSA antigens with the immunoglobulin on the bead, the beads were washed 3 times with physiological saline. In the next step, each bead was added with 0.2 ml of tracer, followed by a 4 hour incubation at room temperature to react ¹²⁵ I-labeled immunoglobulin with MRSA antigens that had been captured by the bead. The beads were then washed 3 times with physiological saline and then transferred to a test tube for measurement. The measurement was carried out using the Aloka V radiation detector (product from Aloka) (cf. FIG. 4).

Example 5

This example illustrates a competitive RIA test for Detection of MRSA using the synthetic peptide as a labeled antigen.

1) Preparation of RIA tracer ( ¹²⁵ I) -labeled synthetic peptide

Ten or more seconds after mixing the synthetic peptide A) obtained in Example 3 with a solution mixture containing ( ¹²⁵ I) sodium iodide and chloramine T, the mixture obtained was mixed with sodium disulfite as a reducing agent, whereby the reaction was terminated. The resulting mixture was subjected to gel filtration chromatography using a column packed with Sephadex-25® (PD10 column, product of Pharmacia Co.) to remove free iodine from the iodinated antibody A). After the antibody A) had been diluted with PBS containing 3% bovine serum albumin and 1% bovine globulin, a tracer product was obtained which was used for a RIA test.

2) Test procedure

A competitive RIA test was made using the previous one tracers produced.

After obtaining bacterial cells from MRSA cultures (cultivation overnight at 37 ° C in a bacterial culture medium The bacterial cells were shaken by centrifugation with Lysostaphin (product of Sigma Co.), a proteo lytic enzyme, lysed. 2 mg of enzyme were added to the 100 ml of culture medium used amount of bacteria used. Man he held an MRSA lysate.

The MRSA lysate was mixed with 0.1 m borate buffer containing 0.1% bovine serum albumin and 0.1% sodium azide to lysate solutions containing 1000, 300, 100, 30, 10, 3 and 1 µg / mg diluted (methicillin sensitive Staphylococcus aureus lysates (MSSA lysates) were diluted accordingly and used as controls). The lysate (1.0 ml each), the tracer, 10% bovine serum globulin, borate buffer and the borate buffer solution of the purified antibody A) obtained according to Example 4-1) were combined in a test tube. The mixture was stirred and left to stand at room temperature overnight. The mixture was then mixed with 1.0 ml of 25% polyethylene glycol 6000 solution and then stirred. The mixture was then centrifuged to precipitate antigen-antibody complexes, globulin and purified antibody. After removing the supernatant liquid formed in the centrifugation treatment and containing free tracer, the precipitate was measured using the radiation detector Aloka V (product from Aloka Co.). It was found that the reactive bacterial lysates were occupied by MRSA lysates. In addition, the amount of antibody-antigen complexes with the purified antibody increased with an increasing amount of MRSA. In contrast, the amount of antibody-antigen complexes between the tracer and the purified antibody decreased. Thus the amount of radioactivity measured in the sediment decreased with increasing amount of MRSA. From these data it follows that the synthetic peptide is suitable as a labeled antigen and that this method can be used in an effective manner for the detection of MRSA (cf. FIG. 5).

Example 6

This example illustrates the preparation of peptides with 7 or 12 amino acid residues according to the procedure of Example 1 and an EIA test for the detection of anti-MRSA-Ab. This EIA Test shows that these peptides are MRSA-spe specific antigens.

1) Production of peptides with 7 or 12 amino acid residues

c) Tyr-Gly-Ser-Thr-Gln-Lys-Ile

d) Gln-Ile-Thr-Thr-Ser-Pro-Gly-Ser-Thr-Gln-Lys-Ile

The aforementioned peptides c) and d) were according to the procedure synthesized from Example 1. The peptide c) has 7 amino acids  rests on, with Tyr on the N-terminal side of the peptide with 6 amino acid residues, which are part of the peptide a) from Form example 1 is coupled. The peptide d) has 12 ami nosic acid residues on which part of the peptide a) from example 1 form.

2) EIA test

Following the procedure of Example 2-1), EIA were microtiter plates for immobilizing the synthetic peptides c) and d) manufactured. The EIA test was carried out according to Example 2-2) guided.

Rabbit serum that had been immunized with purified MRSA antigen was diluted with PBS containing 5% bovine serum albumin as an additive in a ratio of 1: 30, 1: 100, 1: 300 and 1: 1000. 0.05 ml each of the dilutions thus prepared were added to a well, followed by a 1 hour incubation at 37 ° C (non-immunized rabbit serum was used in the same manner as a negative control). After washing the wells, 0.05 ml of a 1: 1000 diluted anti-rabbit IgG goat serum (product from Cappel Co.) labeled with horseradish peroxidase (product from Cappel Co.) was contained in PBS containing 5% bovine serum albumin Enzyme-labeled secondary antibody added. The reaction was promoted by incubation at 37 ° C for 1 hour. After washing, a solution of o-phenylenediamine (OPD) containing hydrogen peroxide (20 mg OPD and 10 µg 35% hydrogen peroxide was dissolved in 50 ml 0.1 M citric acid, pH 5.0) as a substrate in the wells given. After standing at room temperature for 15 minutes, color development began by decomposing the substrate due to the activity of the horseradish peroxides. The individual wells were then mixed with 0.05 ml of 1N sulfuric acid in order to stop the enzymatic reaction and to interrupt the color development. This color development was measured as an absorption at 492 nm. The synthetic peptide was found to react with rabbit serum immunized with MRSA (see Figures 6 and 7). It is therefore clear that the peptides with 7 or 12 amino acid residues behave as MRSA-specific antigens.

Example 7

This example illustrates the preparation of MRSA liquid sity samples using urea instead of Ly sostaphin and the MRSA measurement of these samples.

After obtaining bacterial cells from MRSA cultures (cultivation overnight at 37 ° C. in a bacterial culture medium with shaking) by centrifugation, the bacterial cells were diluted with a bacterial culture medium and then additionally diluted with 8 m urea solution, a 1: 10- Solution received. Bacterial solutions with 1 × 10 ⁸ , 1 × 10 ⁷ , 1 × 10 ⁶ , 1 × 10 ⁵ and 1 × 10 ⁴ pcs cells / ml were obtained. As a control, a bacterial cell solution was prepared by dilution with 0.1 M phosphate buffer solution (pH solution 7.4) without using a urea solution.

The bacterial solution obtained in this way was measured according to the RIA method of Example 4 (cf. FIG. 8).

Example 8

This example explains a procedure for the detection of MRSA by hemagglutination using MRSA-specific Antibodies sensitized according to Example 4-1) Sheep erythrocytes.

Fixed sheep erythrocytes (product of Sigma Co.) were three times by centrifugation with 0.05 M acetate buffer solution solution (pH 4.0). Then the erythrocytes again in the same buffer solution to an erythrocyte suspension with a solids content of 0.5% dispersed. The erythrocyte suspension was treated with a solution of the anti body against the synthetic peptide A), wherein one a mixture with a final concentration of 10 µg antibody per / ml. Then 1 hour at room temperature  temperature stirred to the antibody with the sheep erythrocytes to couple. After removing uncoupled antibodies by centrifugation at 1500 rpm for 5 minutes, the Ge mix again in a 0.1 M glycine buffer (pH 8.2) di sperged, whereby one sensitized with antibody Erythrocyte suspension received.

After growing MRSA overnight at 37 ° C in a culture an MRSA culture solution was obtained medium. As a control solution MSSA was grown in the same way. A hemaggluti nation was used according to the microtiter method the bacterial solution obtained. Here were ever because 25 µl phosphate buffer solution with a pH of 7.2 in a ver well of the microtiter plate. Then the MRSA culture solution and the MSSA culture solution using of a diluter is gradually diluted to a 1: 2 solution. Each because 25 ul of the sensitized erythrocyte suspension into a solution containing the above dilution ben. The mixture was vibrated on a mixer mixes. After standing for 1 hour at room temperature a visual reading was performed (see Table III).  

Table III

Example 9

This example explains a procedure for cleaning PBP2 ', an MRSA-specific protein, from bacterial cells using the purified obtained in Example 4-1) Antibody.

3 mg of MRSA-specific antibodies were coupled with 0.6 g of CNBr-Se pharose 4 B® gel (product from Pharmacia) according to a conventional method. After equilibrating the gel with 0.05 M phosphate buffer (pH 7.4), the gel was mixed with 50 mg (dry weight) lysostaphin lysates from MRSA. The mixture was reacted at 4 ° C overnight. After washing the gel sufficiently with 100 ml of 50 millimolar Tris hydrochloric acid buffer (pH 7.4) to remove uncoupled bacterial cells, the gel was washed with a 2.5 m magnesium chloride solution (pH 6.0) transferred. The PBP2'-eluate obtained was collected in amounts of 0.5 ml each. The high absorption fractions at 280 nm were pooled. The combined eluate was dialyzed overnight to remove the magnesium chloride against phosphate buffer solution (pH 7.4) and then brought to dryness. 0.8 mg of purified PBP2 'are obtained (cf. FIG. 9).

Example 10

This example illustrates a process for making anti-MRSA-PBP2′-monoclonal antibody using the synthetic peptides of Example 1.

1) Preparation of a hybridoma

25 µg peptide (synthetic peptide A) -BSA from Example 3-1)) were dissolved in 0.1 ml of physiological saline. The Solution was sent to Freund’s Kom with the same volume mixed and emulsified. The emulsion was in Intervals of 2 weeks in total 7 times subcutaneously over 7 weeks old BALB / c mice (female) administered for immunization. After the last immunization, one was solved by dissolving 25 µg of the synthetic peptide a) from Example 1 in 0.5 ml phy saline solution of the mouse intra injected peritoneally. 3 days later the spleen cells were removed Mouse isolated. The isolated cells were diagnosed with mouse myeloma cells (P3-X63-Ag8-U1: P3U1) using polyethylene Glycol (PEG) fused. After selecting the fused Cells in a HAT growth medium underwent activity anti-PBP2'-antibody in the supernatant of the hybridoma medium below Use of PBP2′-HRPO (the one obtained from the bacterium PBP2'-antigen from Example 9 was made with horseradish peroxidase (HRPO) marked, checked. Then a cloning of the monoclonal anti-PBP2′-antibody-producing hybridomas (DM2- 12, DM2-13, DM2-39, DM2-40 and DM2-45) using one limiting dilution analysis performed.

In a similar manner, monoclonal antibodies from anti-MRSA directed PBP2′-antibody-forming hybridomas (DM4-11, DM4-17, DM4-20, DM4-26 and DM4-32) as well as the monoclonal anti Synthetic peptide b) antibody-forming hybridomas (DM3-1, DM3- 19 and DM3-26).  

The hybridoma cell lines were used in fermentation research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Japan (FRI) deposited.

DM2-12: Filing number FERM P-13240;
DM4-17: Filing number FERM P-13242;
DM3-1: Filing number FERM BP-4065; and
DM3-19: Filing number FERM P-13241.

2) Production of monoclonal antibodies

The anti-PBP2'-monoclonal antibody-forming hybridomas obtained in this way were incubated on a large scale in DME culture medium (product from Flow) with a content of 15% FCS (fetal calf serum). The cells were administered intra-peritoneally in an amount of 5.0 × 10 ⁶ to BALB / c mice (male, which had previously been treated with pristane (2,6,10,14-tetramethylpentadecane). Two weeks later Ascites fluid containing Mab was obtained and the ascites fluid was salted out by 45% saturation with ammonium sulfate to give monoclonal antibody (Mab) products.

Example 11

In this example, the EIA procedure confirms that the Mab of Example 10 is an MRSA specific Antigen acts that no cross-reaction with MSSA un is subject.

1) Production of EIA plates with immobilized antigen

Made with Lysostaphin (product of Sigma Co.) Lysates from MRSA were dissolved in 0.1 M carbonate buffer solution (pH 9.6) dissolved in a concentration of 10 µg / ml. The lysate was added to the wells in a volume of 0.1 ml each a 96-well microtiter plate (product of the company Flow Co.) poured. After standing overnight at room temperature temperature for coupling the bacteria in the individual Wells of the plate became the next step  Bacterial lysate removed and the wells washed. A Blocking solution (PBS containing 5% bovine serum albu min) was added to the wells, after which a 1 followed by incubation at 37 ° C for hours. After incubation the plate was washed. EIA plates were included immobilized antigen.

In the same way, plates with immobilized An made from MSSA lysates.

2) Test procedure

The Mab obtained in Example 10 was in a volume of 0.05 ml each in the wells of the EIA plate with immo bilized antigen, after which a 1 hour In incubation at 37 ° C. After the incubation, the Wells washed 3 times. Each 0.05 ml one with sea radish peroxidase labeled anti-mouse IgG goat serum (Product from Cappel Co.) was placed in a well poured, followed by a 1 hour reaction. On finally, the wells were washed 4 times and then was 0.1 ml of a solution of o-phenylenediamine (ODD) in the Poured well, after which 15 minutes at room temperature was set. Then the wells were each with because 0.05 ml of 1N sulfuric acid is added to the enzyme reaction to end. The absorption of the individual wells of the Mi Criteria plate at 492 nm was measured.

The monoclonal anti Example 10 body around an MRSA-specific antibody acts immunochemically with MRSA in the sense of an antigen Antibody response reacts while using MSSA at all not responding (see Tables IV to VI).  

Table IV

EIA test for monoclonal anti-MRSA-PBP2′-synthetic peptide a) antibodies

Table V

EIA test for monoclonal anti-MRSA derived PBP2′-antibodies

Table VI

EIA test for monoclonal anti-MRSA-PBP2′-synthetic peptide b) antibodies

Example 12

This example illustrates a sandwich RIA process for Detection of MRSA using the Mab from Example 10.

1) Preparation of RIA reaction beads

Polystyrene beads with a Diameter of 6.25 mm (commercial product) used. Mab, DM3-1 salted out at 45% ammonium sulfate saturation, was in 0.05 M phosphate buffer solution containing of 0.15 M NaCl dissolved in such an amount that an antibody amount of 5 µg per bead resulted. The bead was conjugated to the antibody of the solution formed. The solid phase serving as a carrier was washed with buffer solution washed. RIA beads were obtained.  

2) Production of RIA antibody tracer ( ¹²⁵ I-labeled antibody)

Tracers were made from the following 4 types of monoclonal anti body made:

DM2-12 (anti-synthetic peptide a) antibody, DM3-1 and DM3-19 (anti-synthetic peptide b) antibody) and DM4-17 (from anti-MRSA derived PBP2′-antibody).

The iodine labeling was carried out according to the known chloramine T method. The antibody was mixed with [¹²⁵I] sodium iodide and chloramine T solution for labeling purposes. 10 seconds later, sodium disulfite was added as a reducing agent to stop the reaction. Free iodine was removed from the iodinated monoclonal antibodies by gel filtration chromatography using Sephadex G-25 ^R (PD10 column, product of Pharmacia Co.). The eluate was diluted with PBS containing 3% serum albumin and 1% bovine serum globulin. Tracer products were obtained.

4) Ria sandwich test

Using the antibody beads and the above a RIA sandwich test was made leads.

Bacteria were grown from MRSA cultures under overnight Shake at 37 ° C in a bacterial culture medium had been obtained by centrifugation. Lysostaphin (Product of Sigma Co.) became the bacterial cells in an amount of 2 mg to a 100 ml culture medium given amount of bacteria. MRSA bacteria were obtained. lysates.

The MRSA lysate was diluted 1: 300, 1: 100, 1:30, 1: 3 with PBS containing 1% bovine serum albumin and 1% bovine serum globulin as diluent; Diluted 1: 1 and 1: 0.3. MSSA lysates were appropriately diluted as control samples. After adding 0.1 ml of bacterial lysate solution and 0.2 ml of buffer solution into the individual wells of a reaction plate with 16 wells (commercial product), 1 bead was added to the diluted sample. The mixture was then incubated at room temperature overnight. After washing three times with physiological saline, the beads were mixed with 0.2 ml of tracer and incubated for 4 hours at room temperature. The beads were then washed three times with physiological saline. After transferring the beads to a test tube, the radiation dose was measured with the Aloka V radiation detector (product from Aloka Co.) (cf. FIG. 10).

The detection and measurement methods according to the invention stop simple and expedient procedures that are highly sensitive and MRSA-specific proof as well as the easy opening find MRSA infection in humans and animals possible chen.

The synthetic peptide according to the invention is suitable as Im munogen since there are no other types of protein contaminants are included. That with such an immunogen Antiserum is valuable because it is specific to MRSA responds.

In addition, the synthetic peptide according to the invention can be used as Immunogen for immunoassays such as EIA and RIA are used when a measurement of extremely small amounts of MRSA Antibodies should be carried out.

Since the antibody according to the invention is also a pronounced one Antigen specificity, it can be used as an antibody for Immu noassays such as EIA and RIA for the detection of MRSA and for measurement of extremely small amounts of MRSA partial antigen be applied.  

Brief description of the drawings

Fig. 1 is an RP-HPLC chart showing the synthetic peptide a) of Example 1.

<06162 00070 552 001000280000000200012000285910605100040 0002004238806 00004 06043BOL <Fig. Figure 2 shows an RP-HPLC diagram of the synthetic peptide b) from Example 1.

Fig. 3 illustrates the reactivity of the synthetic peptides obtained in Example 3.

Figure 4 illustrates the results of a sandwich RIA test using immunized rabbit serum.

Fig. 5 illustrates the results of a competitive RIA test using the synthetic peptide as a labeled gene.

Fig. 6 illustrates the results of EIA assays Ver application of the immobilized synthetic peptide c) when the game 6.

Fig. 7 illustrates the results of EIA assays Ver application of the immobilized synthetic peptide d) of In Game 6.

Figure 8 illustrates the results of a RIA test when applied to the urea bacterial dilution of Example 7.

Fig. 9 shows an elution diagram of PBP2 'in the gel coupled with the MRSA-specific antibody of Example 9.

Figure 10 shows the results of a sandwich RIA test using various types of monoclonal antibodies from Example 12.

Sequence lists

Information for SEQ ID No. 1:

• (i) Sequence properties:
  • (A) Length: 32 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 1:

Information for SEQ ID No. 2:

• (i) Sequence properties:
  • (A) Length: 31 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 2:

Information for SEQ ID No. 3:

• (i) Sequence properties:
  • (A) Length: 7 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 3:

Information for SEQ ID No. 4:

• (i) Sequence properties:
  • (A) Length: 12 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 4:

Information for SEQ ID No. 5:

• (i) Sequence properties:
  • (A) Length: 16 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 5:

Information for SEQ ID No. 6:

• (i) Sequence properties:
  • (A) Length: 24 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 6:

Information for SEQ ID No. 7:

• (i) Sequence properties:
  • (A) Length: 20 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 7:

Information for SEQ ID No. 8:

• (i) Sequence properties:
  • (A) Length: 22 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 8:

Information for SEQ ID No. 9:

• (i) Sequence properties:
  • (A) Length: 21 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 9:

Information for SEQ ID No. 10:

• (i) Sequence properties:
  • (A) Length: 22 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 10:

Information for SEQ ID No. 11:

• (i) Sequence properties:
  • (A) Length: 27 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 11:

Information for SEQ ID No. 12:

• (i) Sequence properties:
  • (A) Length: 23 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 12:

Information for SEQ ID No. 13:

• (i) Sequence properties:
  • (A) Length: 20 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 13:

Information for SEQ ID No. 14:

• (i) Sequence properties:
  • (A) Length: 32 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 14:

Information for SEQ ID No. 15:

• (i) Sequence properties:
  • (A) Length: 31 amino acids
  • (B) Type: amino acid
  • (D) Topology: linear
• (ii) Molecule type: peptide
• (xi) Sequence designation: SEQ ID No. 15:

Claims (14)

1. A method for the detection of methicillin-resistant Staphylococcus aureus (MRSA) or fragments thereof in a sample, comprising the competitive reaction of at least one peptide from the following group:
Peptides of the formulas: Gly-Ser-Lys-Lys-Phe-Glu-Lys-Gly-Met-Lys-Lys-Leu-Gly-Val-Gly-Glu-Asp-Ile-Pro-Ser-Asp-Tyr-Pro Phe,
Pro-His-Leu-Leu-Lys-Asp-Thr-Lys-Asn-Lys-Val-Trp-Lys-Lys-Asn-Ile-Ile-Ser-Lys-Glu,
Met-Gln-Gln-Val-Val-Asn-Lys-Thr-His-Lys-Glu-Asp-Ile-Tyr-Arg-Ser-Tyr-Ala-Asn-Leu-Ile-Gly,
Ile-Gln-Asp-Arg-Lys-Ile-Lys-Lys-Val-Ser-Lys-Asn-Lys-Lys-Arg-Val-Asp-Ala-Gln-Tyr-Lys,
Met-Gln-Lys-Asp-Gln-Ser-Ile-His-Ile-Glu-Asn-Leu-Lys-Ser-Glu-Arg-Gly-Lys-Ile-Leu-Asp-Arg,
Met-Asp-Glu-Tyr-Leu-Ser-Asp-Phe-Ala-Lys-Lys-Phe-His-Leu- Thr-Thr-Asn-Glu-Thr-Glu-Ser-Arg-Asn-Tyr-Pro Leu-Gly,
Pro-Ile-Asn-Ser-Glu-Glu-Leu-Lys-Gln-Lys-Glu-Tyr-Lys-Gly-Tyr-Lys-Asp-Asp-Ala-Val-Ile-Gly-Lys or
His-Thr-Leu-Ile-Glu-Lys-Lys-Lys-Lys-Asp-Gly-Lys-Asp-Ile-Gln-Leu-Thr-Ile-Asp-Ala; derivatives thereof, which are obtained by adding at least one Ami nosacid residue or by substitution of at least one amino acid residue with another amino acid residue or fragment, and analogues thereof with less than 32 amino acid residues containing at least a part thereof and for immunological reaction with an antibody against MRSA-penicillin binding protein 2 ′ (MRSA- PBP2 ′) are able;
Peptides of the formula: X-Gly-Ser-Thr-Gln-Lys-Ile-Y, in which X means Tyr-Asn-Lys-Leu-Thr-Gl-Asp-Lys-Lys-Glu-Pro-Leu-Leu -Asn-Lys-Phe-Gln-Ile-Thr-Thr-Ser-Pro or a corresponding group in which one or more arbitrary amino acids have been removed from the N-terminal end of the peptide chain or by substitution of at least one amino acid residue has been obtained by another amino acid residue, -Y has the meaning -Leu-Thr-Ala-Met or denotes a group in which one or more arbitrary amino acid residues have been removed from the C-terminal end of the peptide chain, or by substitution of at least an amino acid residue thereof has been obtained by another amino acid residue,
Peptides of the formula: X'-Lys-Ile-Asp-Gly-Lys-Gly-Trp-Gln-Lys-Asp-Lys-Ser-Trp-Gly-Gly-Tyr, in which X 'is Met-Ile-Gly -Leu-Asn-Asn-Lys-Thr-Leu-Asp-Asp-Lys-Thr-Ser-Tyr- or a group in which any one or more amino acids have been removed from the N-terminal end of the peptide chain or the by substituting at least one amino acid residue thereof with another amino acid residue, derivatives thereof obtained by adding at least one amino acid residue or substituting at least one amino acid residue with another amino acid residue or a fragment, and analogs thereof with less than 32 amino acid residues, which contain at least part of it and are capable of immunological reaction with anti-MRSA-PBP2'-antibodies;
with an antibody against MRSA in the presence of the sample to form the immunological complexes of the peptide and MRSA or its fragment in the sample with the anti-MRSA-Ab and the detection of the immunological complex formed or the reaction of the anti-MRSA formed by the peptide -Ab with the sample to form an immunological complex of anti-MRSA-Ab with MRSA or its fragment in the sample and the detection of the immunological complex formed. 2. A method for the detection of an anti-MRSA antibody in a sample, comprising immobilizing at least one peptide from the following group:
Peptides of the formulas: Gly-Ser-Lys-Lys-Phe-Glu-Lys-Gly-Met-Lys-Lys-Leu-Gly-Val-Gly-Glu-Asp-Ile-Pro-Ser-Asp-Tyr-Pro Phe,
Pro-His-Leu-Leu-Lys-Asp-Thr-Lys-Asn-Lys-Val-Trp-Lys-Lys-Asn-Ile-Ile-Ser-Lys-Glu,
Met-Gln-Gln-Val-Val-Asn-Lys-Thr-His-Lys-Glu-Asp-Ile-Tyr-Arg-Ser-Tyr-Ala-Asn-Leu-Ile-Gly,
Ile-Gln-Asp-Arg-Lys-Ile-Lys-Lys-Val-Ser-Lys-Asn-Lys-Lys-Arg-Val-Asp-Ala-Gln-Tyr-Lys,
Met-Gln-Lys-Asp-Gln-Ser-Ile-His-Ile-Glu-Asn-Leu-Lys-Ser-Glu-Arg-Gly-Lys-Ile-Leu-Asp-Arg,
Met-Asp-Glu-Tyr-Leu-Ser-Asp-Phe-Ala-Lys-Lys-Phe-His-Leu- Thr-Thr-Asn-Glu-Thr-Glu-Ser-Arg-Asn-Tyr-Pro Leu-Gly,
Pro-Ile-Asn-Ser-Glu-Glu-Leu-Lys-Gln-Lys-Glu-Tyr-Lys-Gly-Tyr-Lys-Asp-Asp-Ala-Val-Ile-Gly-Lys or
His-Thr-Leu-Ile-Glu-Lys-Lys-Lys-Lys-Asp-Gly-Lys-Asp-Ile-Gln-Leu-Thr-Ile-Asp-Ala; derivatives thereof, which are obtained by adding at least one Ami nosacid residue or by substitution of at least one amino acid residue with another amino acid residue or fragment, and analogues thereof with less than 32 amino acid residues containing at least part thereof and for immunological reaction with an anti-MRSA-PBP2'-antibody in the Location;
Peptides of the formula: X-Gly-Ser-Thr-Gln-Lys-Ile-Y, in which X means Tyr-Asn-Lys-Leu-Thr-Gl-Asp-Lys-Lys-Glu-Pro-Leu-Leu -Asn-Lys-Phe-Gln-Ile-Thr-Thr-Ser-Pro or a corresponding group in which one or more arbitrary amino acids have been removed from the N-terminal end of the peptide chain or by substitution of at least one amino acid residue has been obtained by another amino acid residue, -Y has the meaning -Leu-Thr-Ala-Met or denotes a group in which one or more arbitrary amino acid residues have been removed from the C-terminal end of the peptide chain, or by substitution of at least an amino acid residue thereof has been obtained by another amino acid residue;
Peptides of the formula: X'-Lys-Ile-Asp-Gly-Lys-Gly-Trp-Gln-Lys-Asp-Lys-Ser-Trp-Gly-Gly-Tyr, in which X 'is Met-Ile-Gly -Leu-Asn-Asn-Lys-Thr-Leu-Asp-Asp-Lys-Thr-Ser-Tyr- or a group in which any one or more amino acids have been removed from the N-terminal end of the peptide chain or the by substituting at least one amino acid residue thereof with another amino acid residue, derivatives thereof obtained by adding at least one amino acid residue or substituting at least one amino acid residue with another amino acid residue or a fragment, and analogs thereof with less than 32 amino acid residues, which contain at least part of it and are capable of immunological reaction with anti-MRSA-PBP2'-antibodies;
on a solid support, reacting the immobilized peptide with the sample to form an immunological complex of the immobilized peptide with the anti-MRSA antibody and detecting the immunological complex formed, or com petitive reaction of an anti-MRSA antibody formed by this peptide with the Sample to form both immunological complexes of anti-MRSA antibodies and detection of the immunological complex formed. 3. The method according to claim 1 or 2, characterized records that the peptide is either by substitution of  at least one amino acid residue from cysteine or tyrosine or derived by adding cysteine or tyrosine. 4. Isolated, essentially pure MRSA-penicillin binding protein 2 ′ (MRSA-PBP2 ′) antigen, characterized by a purification by affinity chromatography, comprising contacting a gel which is coupled with an antibody against at least one peptide from the following group:
Peptides of the formulas: Gly-Ser-Lys-Lys-Phe-Glu-Lys-Gly-Met-Lys-Lys-Leu-Gly-Val-Gly-Glu-Asp-Ile-Pro-Ser-Asp-Tyr-Pro Phe,
Pro-His-Leu-Leu-Lys-Asp-Thr-Lys-Asn-Lys-Val-Trp-Lys-Lys-Asn-Ile-Ile-Ser-Lys-Glu,
Met-Gln-Gln-Val-Val-Asn-Lys-Thr-His-Lys-Glu-Asp-Ile-Tyr-Arg-Ser-Tyr-Ala-Asn-Leu-Ile-Gly,
Ile-Gln-Asp-Arg-Lys-Ile-Lys-Lys-Val-Ser-Lys-Asn-Lys-Lys-Arg-Val-Asp-Ala-Gln-Tyr-Lys,
Met-Gln-Lys-Asp-Gln-Ser-Ile-His-Ile-Glu-Asn-Leu-Lys-Ser-Glu-Arg-Gly-Lys-Ile-Leu-Asp-Arg,
Met-Asp-Glu-Tyr-Leu-Ser-Asp-Phe-Ala-Lys-Lys-Phe-His-Leu- Thr-Thr-Asn-Glu-Thr-Glu-Ser-Arg-Asn-Tyr-Pro Leu-Gly,
Pro-Ile-Asn-Ser-Glu-Glu-Leu-Lys-Gln-Lys-Glu-Tyr-Lys-Gly-Tyr-Lys-Asp-Asp-Ala-Val-Ile-Gly-Lys or
His-Thr-Leu-Ile-Glu-Lys-Lys-Lys-Lys-Asp-Gly-Lys-Asp-Ile-Gln-Leu-Thr-Ile-Asp-Ala; derivatives thereof, which are obtained by adding at least one Ami nosacid residue or by substitution of at least one amino acid residue with another amino acid residue or fragment, and analogues thereof with less than 32 amino acid residues containing at least a part thereof and for immunological reaction with an antibody against MRSA penicillin binding protein 2 '(MRSA PBP2 ') are able to;
Peptides of the formula: X-Gly-Ser-Thr-Gln-Lys-Ile-Y, in which X means Tyr-Asn-Lys-Leu-Thr-Gl-Asp-Lys-Lys-Glu-Pro-Leu-Leu -Asn-Lys-Phe-Gln-Ile-Thr-Thr-Ser-Pro has or a corresponding group in which one or more arbitrary amino acids have been removed from the N-terminal end of the peptide chain or by substitution of at least one Amino acid residue has been obtained by another amino acid residue, -Y has the meaning -Leu-Thr-Ala-Met or means a group in which one or more arbitrary amino acid residues have been removed from the C-terminal end of the peptide chain, or which have been substituted by at least one amino acid residue thereof has been obtained from another amino acid residue;
Peptides of the formula: X'-Lys-Ile-Asp-Gly-Lys-Gly-Trp-Gln-Lys-Asp-Lys-Ser-Trp-Gly-Gly-Tyr, in which X 'is Met-Ile-Gly -Leu-Asn-Asn-Lys-Thr-Leu-Asp-Asp-Lys-Thr-Ser-Tyr- or a group in which any one or more amino acids have been removed from the N-terminal end of the peptide chain or the by substituting at least one amino acid residue thereof with another amino acid residue, derivatives thereof obtained by adding at least one amino acid residue or substituting at least one amino acid residue with another amino acid residue or a fragment, and analogs thereof with less than 32 amino acid residues, which contain at least part of them and are capable of immunological reaction with anti-MRSA-PBP2′-antibodies;
with a culture medium or lysate of MRSA binding an MRSA-PBP2 'component to the immobilized anti-MRSA-PBP2' antibody and eluting MRSA-PBP2 'from the in the munological complex. 5. MRSA-PBP2'-antigen according to claim 4, characterized records that the peptide either by substitution of min  at least one amino acid residue from cysteine or tyrosine or is derived by adding cysteine or tyrosine. 6. Procedure for the detection of MRSA in a sample because characterized in that an antibody against MRSA PBP2 'according to claim 4 with the sample. 7. Method for the detection of anti-MRSA antibodies in a sample, characterized in that MRSA-PBP2 'according to Claim 4 implemented with the sample. 8. Peptide selected from the following group:
Peptides of the formulas: Gly-Ser-Lys-Lys-Phe-Glu-Lys-Gly-Met-Lys-Lys-Leu-Gly-Val-Gly-Glu-Asp-Ile-Pro-Ser-Asp-Tyr-Pro Phe,
Pro-His-Leu-Leu-Lys-Asp-Thr-Lys-Asn-Lys-Val-Trp-Lys-Lys-Asn-Ile-Ile-Ser-Lys-Glu,
Met-Gln-Gln-Val-Val-Asn-Lys-Thr-His-Lys-Glu-Asp-Ile-Tyr-Arg-Ser-Tyr-Ala-Asn-Leu-Ile-Gly,
Ile-Gln-Asp-Arg-Lys-Ile-Lys-Lys-Val-Ser-Lys-Asn-Lys-Lys-Arg-Val-Asp-Ala-Gln-Tyr-Lys,
Met-Gln-Lys-Asp-Gln-Ser-Ile-His-Ile-Glu-Asn-Leu-Lys-Ser-Glu-Arg-Gly-Lys-Ile-Leu-Asp-Arg,
Met-Asp-Glu-Tyr-Leu-Ser-Asp-Phe-Ala-Lys-Lys-Phe-His-Leu- Thr-Thr-Asn-Glu-Thr-Glu-Ser-Arg-Asn-Tyr-Pro Leu-Gly,
Pro-Ile-Asn-Ser-Glu-Glu-Leu-Lys-Gln-Lys-Glu-Tyr-Lys-Gly-Tyr-Lys-Asp-Asp-Ala-Val-Ile-Gly-Lys or
His-Thr-Leu-Ile-Glu-Lys-Lys-Lys-Lys-Asp-Gly-Lys-Asp-Ile-Gln-Leu-Thr-Ile-Asp-Ala; derivatives thereof, which are obtained by adding at least one Ami nosacid residue or by substitution of at least one amino acid residue with another amino acid residue or fragment, and analogues thereof with less than 32 amino acid residues containing at least a part thereof and for immunological reaction with an antibody against MRSA penicillin binding protein 2 '(MRSA PBP2 ') are able to;
Peptides of the formula: X-Gly-Ser-Thr-Gln-Lys-Ile-Y, in which X means Tyr-Asn-Lys-Leu-Thr-Gl-Asp-Lys-Lys-Glu-Pro-Leu-Leu -Asn-Lys-Phe-Gln-Ile-Thr-Thr-Ser-Pro has or a corresponding group in which one or more arbitrary amino acids have been removed from the N-terminal end of the peptide chain or by substitution of at least one Amino acid residue has been obtained by another amino acid residue, -Y has the meaning -Leu-Thr-Ala-Met or means a group in which one or more arbitrary amino acid residues have been removed from the C-terminal end of the peptide chain, or which have been substituted by at least one amino acid residue thereof has been obtained from another amino acid residue;
Peptides of the formula: X'-Lys-Ile-Asp-Gly-Lys-Gly-Trp-Gln-Lys-Asp-Lys-Ser-Trp-Gly-Gly-Tyr, in which X 'is Met-Ile-Gly -Leu-Asn-Asn-Lys-Thr-Leu-Asp-Asp-Lys-Thr-Ser-Tyr- or a group in which any one or more amino acids have been removed from the N-terminal end of the peptide chain or the by substituting at least one amino acid residue thereof with another amino acid residue, derivatives thereof obtained by adding at least one amino acid residue or substituting at least one amino acid residue with another amino acid residue or a fragment, and analogs thereof with less than 32 amino acid residues, which contain at least part of it and are capable of immunological reaction with anti-MRSA-PBP2'-antibodies. 9. Peptide according to claim 8, characterized in that the peptide by substitution of at least one Ami  Residual acid by cysteine or tyrosine or by addition derived from cysteine or tyrosine. 10. Polyclonal or monoclonal antibody which is used for specific reaction with MRSA-PBP2 'is capable. 11. Antibody according to claim 10, characterized in that he is using the peptide according to claim 8 or 9 as an immuno gene has been formed and isolated. 12. Antibody according to claim 10, characterized in that it was formed as an immunogen using MRSA-PBP2 ' and is capable of specific reaction with MRSA-PBP2 '. 13. Antibody according to claim 10, characterized in that that the monoclonal antibody by one of the following Hy bridome has been formed: DM2-12, DM2-13, DM2-39, DM2-40, DM2-45, DM4-11, DM4-17, DM4- 20, DM4-26, DM4-32, DM3-1, DM3-19 and DM3-26. 14. The method according to claim 1, characterized in that that the antibody is by one of the following hybridomas has been formed: DM2-12, DM2-13, DM2-39, DM2-40, DM2-45, DM4-11, DM4-17, DM4- 20, DM4-26, DM4-32, DM3-1, DM3-19 and DM 3-26.