WO2014063703A1

WO2014063703A1 - Monoclonal antibodies

Info

Publication number: WO2014063703A1
Application number: PCT/DK2013/000069
Authority: WO
Inventors: Anders Rhod LARSEN
Original assignee: Statens Serum Institut
Priority date: 2012-10-22
Filing date: 2013-10-16
Publication date: 2014-05-01

Abstract

The invention covers monoclonal antibodies that can be used for i) specific detection of the new PBP variant PBPLGA2SI found in some MRSA isolates and ii) detection of both PBP2a or PBP2'and PBP_LGA251 - This will make it possible to detect MRSA expressing PBP_LGA251 and integrate these antibodies into in vitro diagnostics for detection of MRSA. This will enhance the sensiti vity of existing MRSA detection tests and ultimately improve infection control.

Description

Monoclonal antibodies

Field of invention The present invention discloses monoclonal antibodies that bind to antigens in the PBP_LGA2S I protein expressed by a subset of MRSA strains harboring mecC and monoclonal antibodies that bind to both PBP2a and PBP_LGA2SI proteins and a kit for in vitro diagnostics detecting MRSA strains.

3 General background (State of the Art)

Staphylococcus aureus is a human commensal, colonizing 20-50 % of the population. As such S. aureus is harmless, but may cause infections when the skin or mucosal barrier is ruptured. S. aureus is typically involved in mild skin infections (i.e. impetigo, boils, furunculosis) but is also a predominant cause of severer hospital infections (postoperative and prosthesis related infections) and bacteraemia, which is fatal for 10-20% of patients (Lowy F. NEJM, 339, 520-532. 1998). Penicillin's are very effective against S. aureus however an increasing number of strains are resistant to most members of this class of antibiotics.

Methicillin resistant Staphylococcus aureus (MRSA) are essentially resistant to all β-lactam antibiotics, including penicillin, carbapenems, cephalosporins and may also be resistant to other classes of antimicrobials. This makes MRSA infections difficult to treat, which has a significantly negative impact on the morbidity and mortality of the patients compared to infections caused by methicillin susceptible S. aureus (MSSA) (Cosgrove SE et al.Clin Infect Dis. 2003 Jan

l ;36(l):53-9). Moreover, MRSA infections are more expensive to treat due to isolation procedures, longer time of hospitalisation and rehabilitation and use of typically more expensive antibiotics. For these reasons, MRSA are a great concern and infection control programmes are set up globally to diminish its spread. Precautions involve good hygiene standards, especially hand hygiene, screening and decolonization of patients and healthy persons at risk of carrying MRSA. As another cornerstone in diminishing spread is rapid and accurate diagnosis of MRSA.

The β-lactam antibiotics resistance in MRSA isolates is due to acquisition of a mobile 2.1 Kb mecA gene situated in the so-called Staphylococcal Cassette Chromosome (SCC) elements. The mecA gene encodes a 76 kDa Penicillin Binding Protein (PBP) 2a (sometimes referred to as 2') in addition to the four other PBPs found in both MSSA and MRSA. The binding of penicillin (and other β-lactams) to the PBPs interferes with cell wall synthesis by interrupting peptidoglycan cross- linking. Low affinity of PBP2a to β-lactams however allows continuous growth of MRSA in the presence of β-lactams antibiotics (Yukio Utsui and Takeshi Yokota, Antimicrobial Agents and Chemotherapy, Sept. 1985, p. 397-403; Berger Bachi and Rohrer, 2002, Arch. Microbiol, 178, 165-71 ; Deurenberg RH and

Stobberingh EE, The evolution of Staphylococcus aureus - review, Infection, Genetics and Evolution 8 (2008)747-63;).

Detection of the mecA gene has been the "gold standard" to diagnose MRSA since its discovery in the 1990s (Chambers, H. F. 1997. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin. Microbiol. Rev. 10:781-791 ; Brown DF, JAC2001, 2001 Jul; 48 Suppl 1 :65-70). MRSA confirmation and diagnosis typically involves in-house or commercially available PCR and real time PCR protocols directed against mecA. Another diagnostic method commonly used is a rapid (15 min) latex agglutination assay for the direct detection of PBP 2a from MRSA isolates after a rapid extraction procedure (Nakatomi Y, Sugiyama J. Microbiol Immunol. 1998;42(1 1):739-43). The MRSA-Screen latex agglutination assay is produced by Denka Seiken Co., Ltd. and distributed by various companies under different brand names (Eg. Penicillin Binding Protein

(PBP2') latex agglutination assay, (Oxoid, UK, Mastalex™ - MRSA (MAST, UK). Its accuracy has been evaluated similar to PCR detection of the mecA gene (A. van Griethuysen et al., JCM, Sept. 1999, p. 2789-2792; John H. Lee, J. Clin. Microbiol. 2004, 42(6):2780) and the test is widely used in routine laboratories for rapid diagnosis of MRSA.

WO8802028 describes monoclonal antibodies that are specific for the MRSA mecA gene product PBP2a and the use of this for diagnosis and WO20100104245 describes a monoclonal antibody that binds specifically to a fragment of the mecA gene product PBP2a. A proportion of phenotypic MRSA isolates has been denoted borderline oxacillin resistant S. aureus

(BORSA) or moderately resistant S. aureus (MODSA) as neither the mecA gene nor PBP2a is detectable. The apparent resistance in these strains has been attributed to hyper-production of beta- lactamases (BORSA) or altered PBPs (MODSA) (Chambers, H. F. 1997. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin. Microbiol. Rev. 10:781-791 ).

In 201 1 , a new gene was found in some MRSA in both human and bovine samples. The first isolate (LGA251 ) originated from bulk milk in the UK. The LGA251 genome was DNA sequenced (Deposited at EMBL accession numbers FR821779) whereby a new mecA homologue inserted in a new type of transmittable genetic element (SCCmec XI) was found (Garcia-Alvarez L et al. Lancet Infect Dis. 201 1 Aug; l l(8):595-603). The new mecA homologue was initially named mecAiGA25i after the LGA251 strain in which it was found, but has now been renamed mecC according to the criteria set by the international working group on SCC nomenclature (Ito T., et al. Antimicrob Agents Chemother. 2012 Oct; 56( 10), 4997-4999) . The sequences and annotations of the S. aureus strain LGA251 genome have been entered in the EMBL database (accession numbers FR821779).

No decision has been made about the nomenclature of the protein encoded by mecC. Garcia- Alvarez et al. used MecA_LG_A25! and Choonkeun et al. recently described the mecC encoded protein as PBP2A_LG_A25I _> however herein the term PBP_LG_A2SI will be used (Garcia-Alvarez et a/.Lancet Infect Dis. 201 1 Aug; l l (8):595-603; Choonkeun et al JBC Papers in Press. September 12, 2012.

Ml 12.395962).

Epidemiological data on the new type of MRS As are sparse, but our data suggest that these isolates may contribute with 2-5% of the total number of MRSA (Stegger M, et. al. Rapid Detection,

Differentiation and Typing of Methicillin-Resistant Staphylococcus aureus Harbouring either mecA or the new mecA Homologue mecA_LGA25 i . Clin Microbiol Infect. 2012 Apr; 18(4):395-400 and Petersen,

Andreas et al. Epidemiology of methicillin-resistant Staphylococcus aureus carrying the novel mecC gene in Denmark corroborates a zoonotic reservoir with transmission to humans", Clin Microbiol Infect Sept. 15, 2012: doi: 10.1 1 1 1/1469-0691.12036)

This estimation is based on a search we performed in the Staphylococcus Laboratory at

Statens Serum Institut. We searched our collections of S. aureus isolates dating back to 1957 for the presence of isolates harboring mecC and found a considerable number of such isolates (n=l 12). The first was from 1975 but the majority was found since 2003 and with the highest annual numbers of cases found in 2010 and 201 1 (n=21 and n=36, respectively). None of these isolates had previously been regarded as MRSA due to their lack of the mecA gene but had been annotated as BORSA or MODSA strains. With the detection of mecC these strains are now regarded as "true" MRSA replacing the BORSA/MODSA terms.

The change in annotations is however less important than the fact that these MRSA patients have not been detected and precautions according to the MRSA guidelines have not been followed causing a potentially risk for further transmissions of these new MRSAs.

A common characteristic of the isolates in addition to be carrying mecC is that they so far belong to only few genetic lineages that are not normally observed to cause infections in human. These lineages have by typing of the staphylococcal Protein A and by multi locus sequencing (MLST) been grouped into so called clonal complexes (CC) 130, CC2361 as well as ST425, which previously have mostly been detected in bovine samples (Hasman H et al.. Vet Microbiol. 2010 Mar 24;141(3-4):326-31., Shore A. et al., Antimicrob Agents

Chemother. 201 1 Aug;55(8):3765-73 ).

A likely connection between a bovine and a human reservoir of these MRSA has been substantiated by interviewing patients infected by MRSA due to mecC, as several patients have had direct contact with cattle (butchers, farmers). We have furthermore in two occasions isolated the same type of MRSA from dairy cattle as well as from sheep in vicinity to infected persons. These isolates have been whole genome sequenced and the high similarity of the isolates corroborates that animal reservoir exists with transmission to humans (Harrison EM et al, EMBO Mol Med. 2013 Apr;5(4):509-15). Furthermore, mecC MRSA isolates have been detected in a variety of different animal species including both domestics and wildlife:

Domestic dog, brown rats, a rabbit, a common seal, European otter and a chaffinch, which may also contribute to its spread (Paterson GK et al,. J Antimicrob Chemother. 2012

Dec;67(12):2809-13, Loncaric et al, J Antimicrob Chemother. 2013 Oct;68(10):2222-5).

Alignment of mecA and mecC nucleotide sequences showed only 70% similarity and only 63% when the nucleotides were translated into aminoacids. These dissimilarities make accurate detection of the new MRSA variants impossible when using conventional genetic tests detecting either mecA or the latex agglutination tests using antibodies directed against the PBP2a (Garcia-Alvarez et α/.Lancet Infect Dis. 201 1 Aug;l l(8):595-603). A commercial kit for detection of the mecC gene has recently been launched (ELITech Molecular

Diagnostics, MRSA/SA ELITe MGB® Kit), but at present there are no commercial available rapid antibody based MRSA detection systems to identify the new mecA PBPLGA2₅ I/PBP variants. A rapid commercial available detection of the PBPLGA2₅I will improve the diagnosis of this new MRSA type and diminish its spread.

4. Summary of the invention

Monoclonal antibodies according to the present invention are to an antigen selected from penicillin binding proteins, especially the PBP_LGA251 protein. The monoclonal antibodies either detects PBP_LGA.5I specifically and not PBP2a or detect both PBP_LGA25 I and PBP2a equally well. This will allow development of a confirmatory in vitro diagnostics test that detects MRSA due to mecC or MRSA due to either mecA or mecC.

The invention also comprises use of the monoclonal antibodies bound to various particles or solid phases with or without conjugated labels (flourophores, chemi-or bio-luminiscent, radioactive, ferromagnetic atom, or any other label). Additionally the invention is also directed to any therapeutic composition comprising the antibody for the antigen, as well as kits containing at least one labelled monoclonal antibody to the antigen.

5. Detailed disclosure of the invention

The present invention discloses i) monoclonal antibodies that binds to antigens in the

PBPLGA25I protein expressed by a subset of MRSA strains harboring mecC and ii) monoclonal antibodies that bind to both PBP2a and PBPLGA25 I proteins, which will enable detection of MRSA strains harboring either mecA or mecC.

Preferably said monoclonal antibody binds to amino acid fragment

YISVLVLLLIMIIITWLFKDDDIEKTISSIEKGNYNE (SEQ ID NO 1) which is specific for MRSA strains expressing the PBP_LGA251 protein.

In another preferred embodiment said monoclonal antibody binds to amino acid fragment KKEPLLNKFQITTSPGSTQKILTSIIALKENKLDKNTNFDIYG (SEQ ID NO 2) which is partly shared by both MRSA strains expressing the PBPLGA25 _I protein and MRSA strains expressing the PBP2a protein. The first half of SEQ ID NO 2

(KKEPLLNKFQITTSPGSTQKILT; SEQ ID NO 3) is conserved between MRSA strains and can be used for preparing monoclonal antibodies against MRSA strains expressing both the PBP2a and the PBP_LGA2_S I protein. The second half of SEQ ID NO 2

(SIIALKENKLDKNTNFDIYG; SEQ ID NO 4) is specific for MRSA strains expressing the PBPLGA25 I protein and can be used for preparing monoclonal antibodies against MRSA strains expressing the PBPLGA251 protein. Another object of the present invention is to provide for a kit for in vitro diagnostics detecting MRS A strains expressing the PBPLGA251 protein and optimally also MRS A strains expressing the PBP2a protein. The kit comprises at least a monoclonal antibody that specifically binds to the PBPLGA2₅I protein expressed by a MRS A strain harboring mecC or a monoclonal antibody for detection of both types of MRS A expressing either PBP2a or the PBPLGA25 I■ Alternatively the kit comprises monoclonal antibodies that specifically bind to the PBPLGA2₅ I protein and additionally monoclonal antibodies that specifically bind to the PBP2a protein expressed by the mecA gene. The monoclonal antibodies of the present invention can also be used for preparing a pharmaceutical composition for therapeutic use.

Definitions S. aureus means Staphylococcus aureus

MRSA means methicillin resistant Staphylococcus aureus

MSSA means methicillin susceptible Staphylococcus aureus

mecA means the gene that encode PBP2a or PBP^'

PBP means penicillin binding protein

PBP 1 , PBP, PBP2 and PBP4 means PBPs that are common in both MSSA and MRSA

PBP2a or PBP' means the PBP encoded by the mecA gene that cause a MRSA phenotype mecAiGA₂5i is identical to mecC and encodes PBPLGA25 I

PBPLGA25 I means the PBP that is encoded by the mecC gene

PBPLGA2₅I may soon change names according to a new nomenclature

SCC means Staphylococcal Cassette Chromosome

SCCmec means SCC containing the mecA or mecC gene

MLST means multi locus sequence type

CC means clonal complex based on MLST

BORSA means borderline oxacillin resistant S. aureus

MODSA means moderately resistant S. aureus

In order to obtain monoclonal antibodies either specific for PBPLGA ₅ I or specific for both

PBP2a and PBPLGA25I amino acid sequences of the two proteins derived from the nucleotide sequences of mecA and mecC were analysed: Initially, deduced protein sequences of the mecC and mecA genes from SCC elements of the different known types (I-X, Accession no.: CP000046, D86034, AB037671, JCSC6943, AB121219, AF41 1935, AB462393, FJ390057, JCSC 6943(AB5056329) and JCSC6945 (AB505630), respectively) were aligned to visualize the variable positions in the PBP2a compared to PBPLGA25 I using CLC DNA workbench® software (Figure 1).

We identified aminoacid (aa) stretches with i) low similarity, which could serve as target for the antibodies specific for PBPLGA2₅ I and ii) with high similarity that could serve as target for the monoclonal antibodies directed against both PBP2a and PBPLGA25I -

A fragment of 37 aa length (YISVLVLLLIMIIITWLFKDDDIEKTISSIEKGNYNE; SEQ ID NO 1) with specificity towards PBPLG_A2₅ I was found in the beginning of the PBPLGA251 sequence (position 5-41 in Figure 1). Another fragment of 42 aa (position 387-429 in Figure 1)

KKEPLLNKFQITTSPGSTQKILTSIIALKENKLDKNTNFDIYG (SEQ ID NO 2) was chosen due to its high conservation between PBP2a and PBPLGA2₅ I in the beginning (aa387- 409) and the specificity to PBP_LGA25 I in the end (aa410-429) meaning that PBP_LGA25 i specific antibodies and PBP2a / PBP_LGA25 i specific antibodies could be obtained from the same immunization.

The oligo amino acids described above were purchased with a purity >95% as determined by HPLC and used for immunization of mice for further generation of specific antibodies. In addition two oligoaminoacids representing the first KKEPLLNKFQITTSPGSTQKILT (aa387-409; SEQ ID NO 3) and second half SIIALKENKLDKNTNFDIYG (aa410-429) of the oligo amino acid (aa387-429: SEQ ID NO 4) were purchased to be used for screening of PBPLG_A25I and PBP2a / PBP_LGA251 specific antibodies obtained by immunization with the full length oligo amino acid.

The monoclonal antibodies of this invention are prepared by fusing spleen cells from mice immunized with the particular antigen, with the appropriate myoloma cell line. The resultant product is then cultured in HAT (hypoxanthine, aminopterin and thymidine) medium. Screening test for the specific monoclonal antibodies are employed using immunoassays as described in more details below.

The immunized spleen cells may well be derived from other mammal species but in this work murine antibodies were obtained. The mice are first immunized with the antigen in question by intravenously injection. Inoculation can be repeated over a suitable period to obtain high titters of antibodies. When a blood sample from the mouse shows a high yield of antibodies, the mouse will be euthanized and the spleen removed. The fusion can then be carried out by utilising the immunized spleen cells and an appropriate myeloma cell line. Fused hybridoma cell producing monoclonal antibodies with specificity to a given antigen will be found by screening for binding of the hybridoma to the antigen of interest.

Fused cells that gave a positive specific binding to the antigen were removed and cloned utilising any of the standard methods. The monoclonal antibodies from these clones were tested for their specificity towards the antigens of interest. Amounts of sufficient antibodies were produced by conventional batch cultures.

The monoclonal antibodies may be labelled with a multitude of different labels, such as flourophores, enzymes, or bound to ferromagnetic beads or sensitized latex particles for in vitro diagnostic use.

The antibodies are used for in vitro diagnostics to detect MRS A expressing PBPLGA2₅ I or PBP2a. Detection of MRSA-PBP_LGA25 I or both types of MRSA will improve existing diagnostics and thereby infection control, which is of importance for public health. The diagnostics could also be implemented in veterinary practices as the genetic lineages of MRSA isolates harbouring mecC has shown to be related to cattle/cows and sheep.

The antibodies can be incorporated in various detection systems that make use of antibodies. This could be diagnostic kits that resemble existing latex agglutination assays, where antibodies coupled to latex particles can mediate an agglutination reaction whenever MRS A- PBPLGA25 I is present. Other diagnostic tests in which the invented antibodies could be used are for example when the antibodies are coupled to particles or beads which could be e.g. silica or carboxyl beads (Bead array technology) or magnetic particles, beads used in purification/separation steps to isolate and visualize the bacteria that express PBPLGA2_SI, conventional sandwich ELISA tests, or other antibody based tests (e.g. dipstick tests) all well known in the art of immunodiagnosis.

A preferred commercial available diagnostic method for detecting MRSA is the Penicillin- Binding Protein (PBP2') latex agglutination test. The (PBP2') latex agglutination test kit comprises the antibodies coupled to latex particles for the detection of the target protein: penicillin binding protein 2'. The test is performed in 10-15 minutes from one loop full (3-5 μΐ) of bacterial cells taken from an overnight growth on agar plate. The bacteria are lysed in two steps including 3 minutes of boiling and a 5 minute centrifugation step with enzymatic lysis. The supernatants of the lysed bacterial culture are then tested by mixing 2 drops with reagents containing either test or control latex on a scorecard on which agglutination is inspected visually. The test reagent contains sensitized latex particles coupled to PBP2' specific antibodies whereas the control reagent contains particles coupled to some S. aureus specific antibodies. This test is rapid and easy to perform and was chosen as test system for the antibodies developed and described in this application.

Therapeutic use can also be a possibility with a composition comprising one or more antibodies to the particular antigen. E.g. antibodies directed against MRSA specifically would enhance the elimination of MRSA by the immune system without affecting the commensal MSSA population of the patient. The antibodies could also be used as vectors to deliver relevant antibiotics in the proximity of the MRSA, which could make the therapy more effective.

Figur legends

Figure 1. Alignment of DNA sequences of mecA genes derived from the main ten types of Staphylococcal Cassette Chromosome elements (I-X). Figure 2. Studies of the DNA sequences of mecC genes from isolates originating from different genetic lineages (CC130, ST425 and CC2361) of S. aureus isolated from cows and human in the United Kingdom and Denmark have shown that it is a highly conserved gene. Figure 3. The same is also true for mecA, and alignment of the aminoacid sequences of PBP2a and PBP_LGA25 i using Clustal W (CLC) shows that they overall have a homology of 63%.

Example 1 : Selection of sequences for targets of the monoclonal antibodies

Studies of the DNA sequences of mecA and mecC genes showed that both genes are highly conserved. Thus alignment of mecA originating from each of the described SCCmec elements type I-X showed a 100 % similarity (Figure 1) and the same was true for mecC by alignment of a 631 bp fragment in 79 isolates (Figure 2). The mecC sequences were obtained from isolates originating from different genetic lineages (CC130, ST425 and CC1946) of S. aureus isolated from cows and human in the United Kingdom and Denmark, thus maximum variability was included in the study.

The dissimilarities between mecA and mecC may therefore be expected to be conserved and alignment of the two translated genes was used to find areas of approx. 40aa, the preferred length for immunization, of low and high similarity, respectively (Figure 3).

Two amino acid sequences were chosen for preparation of monoclonal antibodies.

i) A sequence of low similarity was chosen for preparation of monoclonal antibodies with specificity for the PBPLG_A2₅I protein alone (Position aa5-aa41)

YISVLVLLLIMIIITWLFKDDDIEKTISSIEKGNYNE specific for PBP_LGA25i

ii) A sequence of high similarity was chosen for preparation of monoclonal antibodies with specificity to both PBP2a and the PBPLGA2₅I proteins (Position aa387-429) KKEPLLNKFQITTSPGSTQKILTSIIALKENKLDKNTNFDIYG containing an aminoacid stretch that is shared between PBPLG_A251 and PBP2a (aa387-aa 409) and another PBPLGA251 specific one (aa 410-aa 429) r

Example 2: Production of specific monoclonal antibodies

Prior to immunization the immunogen preparation was prepared by coupling the peptid (KKEPLLNKFQITTSPGSTQKILTSIIALKENKLDKNTNFDIYG) to BCG S3 (purified protein residues from degradation of Bacillus Calmette-Guerin bacterias) in a peptid: carrier ratio of 2: 1 by using glutaraldehyd as coupling reagent. Three NMRI mice were

subcutaneously immunized with 0,5 ml 0,5 μg/ml immunogen and Al(OH)₃ as adjuvant.

Immunization was repeated 4 times during a period of 2½ months. After the second immunization the antibody titer was tested in ELISA (see below) 10 days after immunization. When the antibody titer was at least 1 : 1600 the mice were ready for fusion. However, in order to boost the production of antibody producing B-cells, mice were injected intraperitoneally with immunogen without adjuvant 3-4 days prior to fusion. Post-mortem blood was collected from the heart and serum was saved and used as a positive control when testing for antibodies in culture supernatants from hybridomas. Spleen cells were harvested and mixed with the Balb/c myeloma cell line X63.AG8.653 in a ratio of 5: 1 in serum-free SSI-modified DMEM. The cell mixture was centrifuged at 400 x g for 10 min. Cell pellet was gently resuspended in 1 ml 37 °C warm Fusogen (PEG 47% v/v, DMSO 7,5% v/v) during stirring for 2 min. Then 2 ml 37 °C warm serum-free SSI-modified DMEM was added while stiring for 3 min. Finally, 7 ml 37 °C warm HAT (hypoxanthine-aminopterin-thymidine) medium with 1% HybER (growth supplement) was added. The fused cells were seeded in a volumne of app. 225 μΐ per well in 96 well microculture plates. Only fused immortal hybridomas survive in the HAT medium, since the spleen cells are mortal and the immortal X63.AG8.653 cell line lack the enzyme hypoxanthine:guanosine phosphoribosyl transferase that are required for survival in the HAT medium. The HAT medium with HybER was replaced after 7 days. After another 3 days, the culture supernatant was harvested from wells with cell clones.

Culture supernatants were tested by ELISA for antibodies against the PBP unspecific peptid (aa 387-409: KKEPLLNKFQITTSPGSTQKILT) and the PBP_LGA2₅i specific peptid (aa410- 429: SIIALKENKLDKNTNFDIYG).

Cells with positive antibody reactions were transferred to cell culture flasks and cultured for another 10-14 days in order to eliminate unstable clones. Culture supernatants were tested again and clones were selected for further cloning to monoclonality by seeding approximately 1 cell per well. When all wells with clones in the microculture plate were positive and had the same absorbance the clone was considered monoclonal.

Example 3: Selection of clones

ELISA was used for antibody measurement of antibody titer during immunization and for selection of clones after fusion. Microtiter plates were coated over night at 4 °C with either 100 μΐ 2 μg/ml PBP unspecific peptid (aa 387-409) coupled to ovalbumin in a ratio of 5: 1 or 100 μΐ ^g/ml PBPLGA25 I specific peptid (aa410-429) coupled to ovalbumin in a ratio of 5: 1. Phosphate buffered saline (PBS) was used as coating buffer. The antigen coated microtiter wells were incubated with 100 μΐ serum from immunized mouse diluted 1 : 100-1 :3200 (when testing during immunization period) or culture supernatant diluted 1 : 10 (when testing after fusion/ during cloning) in ELISA buffer (0.5 M NaCl, 2,7 mM KC1, l,2mM KH₂P0₄, 6,5 mM Na₂HP0₄, 0,1 % Triton x-100) for 1 h at room temperature. All wells were washed 3 times with 200 μΐ ELISA buffer and incubated for 1 h with 100 μΐ peroxidase conjugated rabbit anti-mouse immunoglobulin diluted 1 : 1000 in ELISA buffer. Wells were washed 3 times with 200 μΐ ELISA buffer. Wells were shortly washed with 100 μΐ substrate buffer (67 mM

Na₂HP0₄, 35 mM citric acid) followed by addition of 100 μΐ/well freshly prepared substrate (2.5 mg/ml OPD and 0.01 % H₂0₂ in substrate buffer). After incubation for 30 min at room temperature in the dark the colour reaction was stopped by addition of 150 μΐ 1 M H₂S0₄. The absorbance was immediately read at 490 nm with background subtraction at 650 nm. The clones that gave rise to the highest absorbance with PBP unspecific peptid (aa 384-406) or PBPLGA251 specific peptid (aa407-426) or both antigens were selected for further cloning.

Normal mouse serum, normal human serum, irrelevant antibody and SSI-modified DMEM were used as negative controls. When testing culture supernatants, serum from the heart of the fusion mouse was used as positive control. Example 4: Development of a slide latex agglutination test

A slide latex agglutination test was developed using a commercial available MRSA detection kit (Denka Seiken MRSA Screen). Prior to the testing the contained mecA specific latex particles in the test were removed by filtering (22μιη) the "test solution". These were replaced by the developed specific monoclonal antibodies coupled to latex particles for the detection of PBPLGA2₅ I- The test was performed in 10-15 minutes from one loop full of bacterial cells taken from a 5% horse blood agar plate. The bacteria were lysed in two steps including 3 minutes boiling and a second 5 minutes step of enzymatic lysis. After adding sensitized and control latex particles to the lysates visual inspection of agglutination reaction within 3 minutes was performed. Positive (LGA251-MRSA mecAi_ga25 i) and (ATCC33591 - MRSA mecA) and a negative (ATCC29213 -MSSA) control strains were tested.

Eaxample 5: Diagnostic Clinical samples obtained as part of the routine at SSI was included in the evaluation of the PBPLG_A2₅I detection. Test strains were chosen to represent 25 mecC positive, 20 mecA positive MRSA and 20 MSSA as confirmed by PCR (Stegger et al). The mecC positive strains were chosen to represent the maximum genetic diversity (by spa types), mecA MRSA strains were chosen to include the globally most prevalent types found including ST8-IV: USA300, ST1-IV:USA400, ST59-V, ST72-V, ST30-IV, ST80-IV, ST22-IV and ST5-IV. MSSA isolates were chosen to represent the most abundant types found in Denmark since 2007.

Sequence lists

Claims

1. A monoclonal antibody for detection of MRSA variants expressing the PBP_LGA25_I protein

2. A monoclonal antibody according to claim 1 which binds specifically to SEQ ID

NO 1 or SEQ ID NO 4 and does not bind to SEQ ID NO 3.

3. A monoclonal antibody according to claim 1 which also detects MRSA expressing the PBP2a protein

4. A monoclonal antibody according to claim 3 which binds specifically to SEQ ID

NO 3.

5. A kit for detecting MRSA comprising a monoclonal antibody according to claim 1 to 4.

6. Use of a monoclonal antibody according to a previous claim for preparing a

pharmaceutical composition for therapeutic use.