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Monoclonal antibodies against the pbp2-a protein and homologous sequences for the treatment of infections by and immunodiagnostics of bacteria of the firmicutes phylum

A monoclonal antibody and sequence technology, applied in the direction of antibodies, anti-enzyme immunoglobulins, anti-bacterial immunoglobulins, etc., can solve the problem that vaccines cannot produce protective antibodies in time for bacterial infection.

Active Publication Date: 2012-10-17
FUNDACAO OSWALDO CRUZ FIOCRUZ
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0011] However, according to the foregoing, the vaccine cannot produce protective antibodies in time to control bacterial infection

Method used

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  • Monoclonal antibodies against the pbp2-a protein and homologous sequences for the treatment of infections by and immunodiagnostics of bacteria of the firmicutes phylum
  • Monoclonal antibodies against the pbp2-a protein and homologous sequences for the treatment of infections by and immunodiagnostics of bacteria of the firmicutes phylum
  • Monoclonal antibodies against the pbp2-a protein and homologous sequences for the treatment of infections by and immunodiagnostics of bacteria of the firmicutes phylum

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0067] 4. Preparation of Monoclonal Antibody

[0068] Lymphocytes taken from the spleen were fused with SP2 / 0-Ag14 myeloma cells (ATCC 1581) according to the record of monoclonal antibody production in Current Protocols in Immunology (22), using polyethylene glycol fusion, at 37°C, 10 %CO 2 cultured in hypoxanthine-aminopterin-thymus medium. Synthetic hybridomas were evaluated after 14 days by ELISA assay using purified recombinant protein as antigen as described below. The best hybridoma cells were used for cloning, the best clones were selected by ELISA, and these clones were then stored in liquid nitrogen.

[0069] 5. Enzyme immunoassay—ELISA

[0070] Maxisorb 96-well plastic plates were coated with recombinant protein (PBP2a fragment) in carbonate / bicarbonate buffer at 500 ng / well and incubated overnight at 4°C. On the following day, the plate was washed three times with PBS containing 0.05% Tween 20 and blocked for two hours at 37°C in PBS and 5% skim milk. Samples f...

Embodiment 2

[0122] 1 Acquisition of mouse anti-PBP2a monoclonal antibody

[0123] A group of animals was immunized according to the previously described immunization protocol. The results evaluated by ELISA are described in figure 1 middle.

[0124] After the degree of fusion (fusion of 90-LATAM), suspensions from 96-well plates (hybridomas) were analyzed via ELISA detection. From this total, the five best samples are selected to expand the cells (clones). Then, again, the resulting suspension was analyzed by the ELISA method. Results obtained by ELISA were confirmed by immunoblotting against purified recombinant protein (PBP2a) and confirming positive samples. The final results are in Table I.

[0125] figure 1 Results obtained by enzyme immunoassay (ELISA) for the production of antibodies against PBP2a in the sera of vaccinated animals are shown. Each jumping wave corresponds to a 1:100 dilution of sera from vaccinated animals. Positive control sera are angled columns [ ]. ...

Embodiment 3

[0241] A second study using the CEB MRSA strain, using the same method as described in Example 1, section 7.2, was assisted by two pulses of vortex agitation, 15 seconds each, after each wash to help grapevine aureus Dissociation of cocci clumps and increased exposure of PBP2a to antibodies.

[0242] In control samples (i pure bacteria, not exposed to mAb; and ii pure bacteria plus FITC or PE marker) and mAb-treated samples, after labeling with PE or FITC for analysis, Detection markers FITC (fluorescein isothiocyanate) and PE (phycoerythrin). Read in linear mode in a FACsalibur instrument.

[0243] Figure 10 is a graph of flow cytometry analysis of MRSA samples in the presence of FITC-labeled anti-PBP2a antibody. Curve (x) corresponds to unlabeled samples, curve (y) corresponds to labeled samples.

[0244] The results obtained showed that approximately 22% of the labeled population was instrumentally detected, confirming the recognition of the anti-PBP2a antibody for the...

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Abstract

The present invention relates to monoclonal antibodies capable of recognising and binding to the PBP2-a protein and to other proteins having sequences homologous to PBP2-a, including pathogenic species such as the methyciline-resistant Staphylococcus Aureus (MRSA), coagulase-negative Staphilococcus, Staphylococcus sciuri and Enterococcus, and any other bacteria containing the PBP2-a protein or homologous sequences. The invention also relates to the use of the monoclonal antibodies capable of recognising and binding to the PBP2-a protein and to other proteins having sequenceshomologous to PBP2-a in a complementary immunodiagnostic test for detecting resistance to beta-lactam antibiotics.

Description

technical field [0001] The present invention relates to monoclonal antibodies capable of recognizing and binding to other proteins of the PBP2a protein and other proteins homologous to PBP2a, including methicillin-resistant Staphylococcus aureus-MRSA, coagulase-negative Staphylococcus and Staphylococcus squirrel ( Staphylococcus sciuri ), Enterococcus ( Enterococcus spp .) and any other bacterial pathogen that has PBP2a or a sequence homologous to this protein. The present invention also relates to the use of monoclonal antibodies capable of recognizing and binding PBP2a protein and other proteins expressed in homologous sequences with PBP2a in complementary immunodiagnosis for detecting β-lactam resistance. Background technique [0002] Infections caused by methicillin-resistant Staphylococcus aureus-MRSA are a major concern for clinicians, exhibiting higher mortality and morbidity than infections caused by methicillin-susceptible Staphylococci (1). Furthermore, these in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K16/40A61K39/40
Inventor 若泽·普罗科皮奥·莫雷诺·塞纳乔奥·路易斯·桑帕约·凯罗斯纳迪娅·玛丽亚·巴特罗玛丽亚·达·格劳瑞娅·马丁斯·特谢拉
Owner FUNDACAO OSWALDO CRUZ FIOCRUZ
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