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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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  • A61K39/104—Pseudomonadales, e.g. Pseudomonas
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
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  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2469/00—Immunoassays for the detection of microorganisms
  • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

• Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is capable of causing fatal acute lung infections in critically ill individuals (1) .
• the ability of the bacterium to damage the lung epithelium has been linked with the expression of toxins that are directly injected into eu aryotic cells via a type III- mediated secretion and translocation mechanism (2, 3) .
• the proteins encoded by the P. aeruginosa type III secretion and translocation apparatus demonstrate a high level of amino acid identity with members of the Yersinia Yop regulon (4-6) . Of all the type III systems discovered in Gram-negative bacteria, only P.
• aeruginosa possesses a homologue to the Yersinia V antigen, PcrV (see 6 for review of type III systems).
• Homologous proteins to the secretion and translocation apparatus are encoded by both plant and animal pathogenic bacteria. These organisms include human pathogens such as Salmonella typhimurium, Shigella flexneri , En teropa thogenic E . coli , Chlamydia spp . , and plant pathogens such as Xanthamonas campestris, Pseudomonas syringae, Erwinia amylovora and Ralstonia solanacearum . However, only P. aeruginosa and Yersinia encode the V antigen.
• Yahr, et al. , 1997 discloses the sequence of the operon encoding PcrV and compares the sequence to the LcrV protein.
• the amino acid sequence of PcrV is known and is available under accession number AF010149 of GenBank.
• the present invention involves methods and compositions developed from our observation that the Pseudomonas V antigen can be used to protect animals from a lethal lung infection.
• the present invention is a method of inhibiting Pseudomonas infection comprising inoculating a patient with an effective amount of PcrV antigen.
• DNA encoding PcrV is used as a gene vaccine.
• the antigen is expressed as a recombinant protein and used tc --.mru ⁇ -ie patients at risk.
• the patient is completely protected from infection.
• the DNA encoding PcrV (called pcrV) or a DNA fragment may be used diagnostically to detect P. aeruginosa infection.
• the recombinant protein is used diagnostically to detect antibodies from patients.
• Patient antibody response to PcrV may be associated with prognosis. Therefore, in this embodiment the recombinant protein is used as a prognostic indicator by measuring the patient's antibody titer.
• the present invention also provides a method for inhibiting a Pseudomonas infection in an individual by contacting the individual with an effective amount of a PcrV inhibitor, in particular with a PcrV antibody, antibody derivative or fragment, or antibody mimic.
• PcrV antibodies, antibody derivatives and antibody fragments are also provided.
• Figs. 1A and IB are a stained gel (Fig. 1A) and Western blot (Fig. IB) illustrating the phenotypic analysis of PA103 ⁇ pcrV.
• Figs. 2A and 2B are a graph (Fig. 2A) and set of bar graphs (Fig. 2B) illustrating the survival and ' lung injury of P. aeruginosa parental and mutant strains.
• Figs. 3A and 3B are a graph (Fig. 3A) and a set of bar graphs (Fig. 3B) illustrating the effect of immunization on survival, lung injury, and bacterial colonization.
• Fig. 4 is a graph of the number of animals surviving a challenge with 5 x 10 5 CFU/mouse of strain PA103 after passive administration of polyclonal IgG specific for PcrV, ExoU, PopD or control IgG from an unimmunized animal .
• Fig. 5 is a graph (Fig. 5A) and a set of bar graphs (Fig. 5B) illustrating survival and protection from lung injury by concomitant administration of IgG to different bacterial antigens and bacterial challenge.
• One-way ANOVA for lung injury, p 0.026, and lung edema, p ⁇ 0.C005.
• PcrV has a novel regulatory effect on expression of the type III secreted products, is involved in the translocation of type III toxins, and is the first antigen that protects against lung injury induced by P. aeruginosa infection.
• Vaccination against PcrV prior to the airspace instillation of anti-PcrV IgG not only ensured the survival of challenged animals but also decreased lung inflammation and injury caused by the bacteria.
• LcrV or the V antigen
• PcrV is a multifunctional protein that regulates secretion/translocation of the Yop effector proteins and plays an extracellular role in pathogenesis by altering the host cytokine response to Yersinia infection (7-11) .
• the only known homologue of this critical pathogenic factor is an extracellular protein encoded by P. aeruginosa , termed PcrV.
• PcrV P. aeruginosa
• One embodiment of the present invention is a method of moderating or inhibiting a Pseudomonas infection by immunizing a patient with an effective amount of the PcrV antigen.
• an amount of PcrV antigen effective to show some moderation or inhibition of Pseudomonas infection as compared to control subjects or animals who have not been treated with the antigen.
• modify we mean that infection is innibited by at least fifty percent compared to a non-immunized animal. Preferably, infection is completely prevented.
• a quantitative assessment of infection would preferably include the examination of the amount of bacteria in the bloodstream or pleural fluids and/or an examination of lung injury parameters. For example, the absence of bacteria in the blood stream or pleural fluids would indicate prevention of infection. A reduction in lung injury parameters would indicate that infection is moderated.
• Infection could be quantitatively assessed by several other clinical indicators, including the reduction of bacterial load in the sputum, blood or pleural fluids, reduction in the size of the infiltrate, oxygenation improvement, reduction in the length of time on mechanical ventilation, reduction m fever and reduction in white blood cell count.
• PcrV antigen we mean that portion or fragment of the PcrV protein that is necessary to invoke an immune response which prevents or moderates infection.
• one of skill in the art can map the protective epitope on the molecule. For example, we will be producing monoclonal antibodies and screening them for the antibodies that prevent cytotoxicity in tissue culture. Antibodies that prevent cytotoxicity will be tested in the acute lung infection model for passive protection against infection. Monoclonal antibodies generally recognize a small region of amino acids and when the amino acids are contiguous, the epitope can be defined to as few as 6-9 amino acids.
• recombinant PcrV or "rPcrV” we mean the protein produced from a PcrV gene that has been placed in a non-native host.
• chemically synthesize amino acid stretches to define the epitope.
• These chemically synthesized epitopes can be attached to carrier molecules and used for immunization to determine if protection is achieved.
• the PcrV antigen may be most easily obtained by the method we used, a commercially available bacterial expression plasmid called petl6b from Novagen.
• the pcrV gene was first cloned from the P. aeruginosa chromosome as part of an operon. The coding region was amplified and inserted into two different vectors.
• One vector is for expression from P. aeruginosa as shown in Fig. 1. This is a vector from Heroert Schweizer (reference 19) which we modified to contain an appropriate promoter sequence such that PcrV expression is coordmately regulated with the rest of the delivery and intoxication apparatus of the bacterium.
• the second plasmid, pETl ⁇ b is for expression and purification purposes from E . coll .
• the advantage of this system is that we do not have to worry about contaminating P. aeruginosa proteins, the protein is produced in great abundance, and there is a one-step purification process.
• the PcrV coding region is amplified to be cloned in frame with a histidine tag provided on the pET16b vector.
• the multiple histidine residues fused to the amino terminus of PcrV allow affinity chromatography using a nickel-NTA column. Therefore, a preferable PcrV antigen is a recombinant version of the natural PcrV protein.
• human or humanized monoclonal or polyclonal antibodies to PcrV are administered to prevent or treat infections with P. aeruginosa .
• antibodies could be administered for prevention of infection.
• antibodies may be administered after the onset of infection to treat the infection.
• antibodies can be administered alone or in combination with antibiotics. Administration of antibodies in conjunction with antibiotics may allow the administration of shorter courses or lower doses of antibiotics, thereoy decreasing the risk of emergence of antibiotic-resistant organisms.
• a healthy individual at risk of serious injury or burn would be immunized with the vaccine by a methodology (either injection or mtranasal) that would give long- lived protection.
• a booster would be given on admission (intramuscular injection) to the hospital after injury.
• a patient who is being subjected to mechanical ventilation would be given on admission (intramuscular injection) to the hospital after injury.
• Immunization may be done systemically or intranasally. Immunization of these individuals would preferably start during normal vaccination procedures for other childhood diseases. We would predict long-lived protection with booster doses probably around ages 5 and 10.
• the coding region for PcrV is at nucleotides 626-1510.
• a successful probe is one that will hybridize specifically to the PcrV DNA and not to other regions.
• nucleic acid diagnostic techniques would be suitable, for example, hybridization of the single-stranded 40 nucleotide probe to DNA or RNA extracted from a patient's sputum.
• patient's sputum would be used as a source for bacterial DNA or RNA to serve as a template for the PCR or RT-PCR reaction, respectively.
• the DNA encoding PcrV is used as a gene vaccine using standard molecular biological methods.
• Davis, H.L., et al. "DNA vaccine for hepatitis B: Evidence for lmmunogenicity in chimpanzees and comparison with other vaccines," Proc. Natl. Acad. Sci. 93:7213-7218, 1996; Barry, M.A., et al., "Protection against mycoplasma infection using expression-library immunization," Nature 377:632-635, 1995; Xiang, Z.Q., et al .
• an amount of vaccine effective to moderate or eliminate Pseudomonas infection or Pseudomonas infection symptoms.
• the protein or antigen could also be used diagnostically to detect antibodies in patients and, thus, predict the patient's infection status. One would preferably contact a sample obtained from an individual suspected of Pseudomonas infection with the PcrV protein or fragment thereof and detect protein/antibody binding. One would then correlate enhanced antibody binding (as compared with a control sample) with Pseudomonas aeruginosa infection in the individual.
• the antibody is preferably "humanized".
• the monoclonal antibody is obtained the heavy and light chain variable regions are cloned. These cloned fragments are then inserted into a human antibody backbone (constant regions).
• we can control the class of antibody IgG, IgA, etc. in addition to providing the binding specificity.
• the PcrV antibody may be a monoclonal antibody or polyclonal.
• the antibodies may be human or humanized, particularly for therapeutic applications.
• Antibody fragments or derivatives, such as an Fab, F(ab')_ or Fv, may also be used.
• Single-chain antibodies for example as described in Huston, et al. (Int. Rev. Immunol. 10:195-217, 1993) may also find use m the methods described herein.
• effective amount of the PcrV antibody or antibody fragment we mean an amount sufficient to moderate or eliminate Pseudomonas infection or infection symptoms.
• small molecule peptidomimetics or non-peptide mimetics can be designed to mimic the action of the PcrV antibodies in inhibiting or modulating Pseudomonas infection, presumably by interfering with the action of PcrV.
• Methods for designing such small molecule mimics are well known (see, for example, Ripka and Rich, Curr . Qpin. Chem. Biol. 2:441-452, 1998; Huang, et al . , Biopolvmers 43:367-382, 1997; al-Obeidi, et al. , Mol. Biotechnol. 9:205-223, 1998).
• Small molecule inhibitors that are designed based on the PcrV antibody may be screened for the ability to interfere with the PcrV-PcrV antibody binding interaction.
• Candidate small molecules exhibiting activity in such an assay may be optimized by methods that are well known in the art, including for example, m vi tro screening assays, and further refined in m vivo assays for inhibition or modulation of
• Pseudomonas infection by any of the methods described herein or as are well known in the art.
• Such small molecule inhibitors of PcrV action should be useful in the present method for inhibiting or modulating a Pseudomonas infection.
• PcrV protein may be used to identify a PcrV receptor which may be present in the host cells, particularly in human cells, more particularly in human epithelial cells or macrophages. Identification of a PcrV receptor allows for the screening of small molecule libraries, for example combinatorial libraries, for candidates that interfere with PcrV binding. Such molecules may also be useful in a method to inhibit or modulate a Pseudomonas infection.
• PcrV will be fused to glutathione S-transferase (GST) and attached to column matrix for affinity chromatography of solubilized cellular extracts. Proteins binding specifically to PcrV will be eluted and subjected to amino terminal sequencing for identification.
• GST glutathione S-transferase
• PcrV will be subjected to yeast two-hybrid analysis. In this case PcrV is fused in frame with the DNA binding domain of Gal4. Once the clone is obtained it will be transformed into a suitable yeast host strain.
• the yeast strain containing the Gal4PcrV construct will be transformed with a Hela cell cDNA bank cloned in frame with the Gal4 activation domain. Double transformants that complement the ability to utilize histidine and produce beta galactosidase (proteins that interact -with PcrV) will be analyzed genetically and at the nucleotide sequence level.
• the receptor is a cellular glycolipid we will utilize an overlay technique where glycolipids are separated by thin-layer chromatography and then probed with radiolabeled bacteria. The binding to specific components will be monitored by autoradiography.
• epithelial and macrophage proteins will be separated by SDS-PAGE, blotted onto nitrocellulose and overlaid with radiolabeled bacteria or labeled PcrV. Again, the protein components to which the bacteria bind are then identified by autoradiography.
• Pseudomonas species are known to infect a wide spectrum of hosts within the animal kingdom and even within the plant kingdom.
• the compositions and methods disclosed herein may have use across a wide range of organisms in inhibiting or modulating diseases or conditions resulting from infection by a Pseudomonas species.
• the compositions and methods of the present invention are described herein particularly for application to Pseudomonas aeruginosa but it is well within the competence of one of ordinary skill in the art to apply the methods taught herein to other species.
• PA103 ⁇ pcrV was characterized by the expression of several extracellular products that are secreted by the P. aeruginosa type III system which include the ExoU cytotoxin (3), PcrV (5), and a protein required for the translocation of toxins, PopD (14).
• SDS-polyacrylamide gel electrophoresis of concentrated culture supernatants indicated that the parental strain, PA103 is induced for production and secretion of the type III proteins by growth in medium containing a chelator of calcium, nitrilotriacetic acid (NTA) (Fig. 1) .
• NTA nitrilotriacetic acid
• PA103 ⁇ pcrV exhibits a calcium blind phenotype; extracellular protein production is strongly induced in both the presence and absence of NTA. These results suggest that the secretory system is fully functional but deregulated. This deregulated phenotype is in contrast to the calcium independent phenotype reported for an LcrV defective strain which fails to produce the extracellular Yops, grows at 37°C regardless of the presence or absence of calcium, nd shows only partial induction of the Yops (7). Complementing PA103 ⁇ pcr ⁇ with a clone expressing wild-type PcrV restored normal regulation of extracellular protein production in response to NTA induction . To test the contribution of PcrV to P. aeruginosa pathogenesis, two infection models were used. In an m vi tro model the parental and several mutant derivative strains were compared for their ability to cause cytotoxicity in a CHO cell infection assay (3) . The negative controls in this experiment included
• PA103popD : ⁇ , which has been previously shown to be defective in the translocation of type III virulence determinants (14) and PA103 ⁇ exoC7, which is non-cytotoxic due to the absence of ExoU production (3, 15) .
• CHO cells were unable to exclude trypan blue with the wild-type and ⁇ .pcrV strain complemented with a plasmid construct expressing PcrV. Staining did not occur when CHO cells were infected with the negative control strains or with PA103 ⁇ pcrV (data not shown) .
• PcrV expression is required for cytotoxicity.
• Purified recombinant PcrV was not cytotoxic when added exogenously to tissue culture cells. Since secretion of the type III proteins required for translocation was unaffected by the deletion of pcrV (Fig. 1A and B) , PA103 ⁇ pcr ⁇ appears to be defective in ExoU translocation.
• Figs. 1A and IB are a stained gel (Fig. 1A) and Western blot (Fig. IB) illustrating the phenotypic analysis of PAl03 ⁇ pcrV.
• the extracellular protein profile (Fig. 1A) was analyzed on a SDS-polyacrylamide gel (10%) stained with Coomassie blue. The migration of the P.
• aeruginosa-encoded type III proteins is indicated to the left and the migration of molecular weight markers is indicated on the right.
• Fig. IB is a Western blot of a duplicate gel using antibodies specific for ExoU, PcrV, and PopD and 125 I-Protein A to detect bound IgG.
• Wild-type and mutant P. aeruginosa strains were tested in an acute lung infection model using low and high challenge doses of bacteria. Survival measurements indicated that PcrV and PopD were required to induce a lethal infection (Fig. 2A) .
• the flux of labeled albumin from the airspaces of the lung to the bloodstream the flux of labeled albumin from the airspaces of the lung to the pleural fluids, and the wet/dry ratio, which measures lung edema
• PA103popD: were no different than the uninfected control animals (Fig. 2B) .
• Figs. 2A and 2B are a graph (Fig. 2A) and set of bar graphs (Fig. 2B) illustrating the survival and lung injury of P. aeruginosa parental and mutant strains.
• mice were challenged with 5 x 10 5 cfu of each of the indicated strains and survival was monitored for one week.
• lung injury was assessed by the flux of labeled albumin from the airspaces of the lung to the blood (lung epithelial injury) , to the pleural fluid (pleural fluid) or by measuring the wet/dry ratio (lung edema) .
• Two bacterial infectious doses were used as denoted by the solid and striped bars.
• Figs. 3A and 3B are a graph (Fig. 3A) and a set of bar graphs (Fig. 3B) illustrating the effect of immunization on survival, lung injury, and bacterial colonization.
• Fig. 3B lung injury assessment and bacterial colonization of vaccinated animals 4 hours after installation of PA103.
• the final number of bacteria in the lung is indicated as the number on the Y axis x 10" CFU.
• mice were passively immunized with preimmune rabbit IgG or rabbit IgG specific for rPcrV, rExoU, or rPopD one hour prior to airspace instillation of PA103 at a concentration of 5 xlO 5 CFU/mouse.
• Antibodies to rPcrV provided complete protection to a lethal infection (Fig. 4).
• Anti-rExoU IgG provided partial survival, which was significantly different from the administration of control IgG, although all the surviving animals appeared severely ill during the trial. Survival was not improved by the passive transfer of antibodies to another of the type III translocation proteins, PopD. From these results we conclude that antibodies to PcrV are highly protective in the acute lung infection model and that PcrV may be exposed on the bacterial surface or in a soluble form that is available for antibody-antigen interactions.
• Fig. 4 is a graph of the number of animals surviving a challenge with 5 x 10 s CFU/mouse of strain PA103.
• Animals were pretreated with 100 ⁇ g of immune IgG or control IgG from an unimmunized rabbit (rPcrV, preimmune serum).
• N 10 for each group; *p ⁇ 0.05 versus control group for treatment with anti-PcrV and anti-ExoU IgG preparations by Mantel-Cox log rank test. If PcrV is accessible for neutralization, then concomitant administration of the bacterial inoculum with anti-rPcrV IgG should completely protect against lung injury and lethality.
• IgG preparations were mixed with the inoculum (10-fold higher dose than the lethal inoculum) prior to instillation of the bacteria into the lung and survival was measured. Only anti-rPcrV IgG was protective against this extreme infection (Fig. 5A) . Lung injury was measured in animals infected with the normal lethal dose of 5 x 10 5 bacteria. The efflux of labeled albumin from the airspaces of the lung was only 3% more than uninfected controls (Fig. 5B) after co- administration of anti-rPcrV IgG. The decreased efflux of labeled protein from the lung to the pleural fluids was the same as the uninfected controls when anti-PcrV was included with the inoculum.
• Fig. 5 Curiously lung edema, as measured by the wet/dry ratio, was significantly reduced by the addition of either anti-rPcrV or anti-rPopD. (Fig. 5B) .
• the concomitant administration of anti- rPcrV IgG with the bacteria was even more effective in normalizing all the lung injury parameters than vaccination.
• These data support the accessibility of PcrV for antibody-mediated neutralization and document a clinically relevant decrease in lung injury; antibodies to PcrV may serve as therapeutic reagents in the treatment of severe nosocomial pneumonia caused by Pseudomonas aeruginosa .
• Fig. 5 is a graph (Fig. 5A) and a set of bar graphs (Fig.
• IgG 5B
• IgG 5 ⁇ g
• P. aeruginosa strain PA103 P. aeruginosa strain PA103. This mixture was instilled into the lungs and survival (Fig. 5A) or lung injury (Fig. 5B) was assessed.
• PcrV must be a component of the type III translocation complex in P.
• a gene encoding tetracycline resistance was cloned into the HindiII site of the vector (pNOT ⁇ pcrV) .
• the MOB cassette (17) was added as a WotI fragment.
• Selection of merodiploids, resolution of plasmid sequences, and confirmation of allelic replacement was performed as previously described (18).
• a shuttle plasmid (pUCP, 19) was used to construct a clone to complement the pcrV deletion.
• the coding sequence for PcrV was amplified and cloned behind the control of the ExoS promoter region (20) .
• ExoS The transcription of ExoS is coordinately regulated with the operons that control type III secretion and translocation in P. aeruginosa (2) .
• the nucleotide sequence was confirmed for each DNA construct involving s-.ce specific mutagenesis, PCR amplification, or in-frame deletion.
• P. aeruginosa were grown under inducing (+NTA) or non-inducing conditions (-NTA) for expression of the type III secreted products (18). Cultures were harvested based on optical density measurements at 540 nm and supernatant fractions were concentrated by the addition of a saturated solution of ammonium sulfate to a final concentration of 55%. Each lane of an SDS-polyacrylamide gel (11%) was loaded with 3 ⁇ l of a 20-fold concentrated supernatant and stained with Coomassie blue. An identical gel was subjected to Western blot analysis as previously described (3-5) using a cocktail of rabbit antisera, which specifically recognizes ExoU, PopD, and
• Protein A labeled with 125 I was used as a secondary reagent to identify bound IgG.
• CHO Chinese Hamster Ovary cells
• m vi tro infection model designed to measure cytotoxicity and type III translocation (21) . Briefly, a bacterial inoculum was prepared in tissue culture medium without serum. CHO cells, which were propagated in serum containing medium, were washed and infected with various P. aeruginosa strains at a multiplicity of infection of 5:1. Cultures were incubated under tissue culture conditions for 3 hours (37°C, 5% C0 2 ) , washed, and stained with trypan blue. Permeability to the dye was determined from phase contrast photographs.
• the lungs, pleural fluids, tracheas, oropharynxes, stomachs, and livers were harvested, and the radioactivity was measured.
• the percentage of radioactive albumin that left the instilled lungs and entered the circulation or the pleural fluid was calculated by multiplying the counts measured in the terminal blood samples (per ml) times the blood volume (body weight X 0.07).
• the wet-dry ratios of the lungs were determined by adding 1 ml of water to the lungs and homogenizing the mixture. Homogenates were placed in preweighed aluminum pans and dried to constant weight in an 80°C oven for three days. Lung homogenates were also sequentially diluted and plated on sheep blood agar for quantitative assessment of bacteria. Production of rabbit antiserum to PcrV, PopD, and
• ExoU. rPcrV, rPopD, and rExoU were produced as histidine tagged fusion proteins in pETl ⁇ b and purified by nickel chromatography as previously described (22) .
• Rabbits were injected intradermally (10 sites) with 300 ⁇ g of recombinant protein emulsified in Freund' s complete adjuvant, boosted with antigen in Freund' s incomplete adjuvant, and periodically bled at 7 day intervals.
• the IgG fraction was isolated using Protein A column chromatography (Pierce Chemicals, Rockford, IL) . Mice were injected with 100 ⁇ g IgG
• mice were immunized with 10 ⁇ g of purified, LPS- free, recombinant PcrV in Freund' s complete adjuvant and boosted two weeks later with the same dose of antigen emulsified in Freund' s incomplete adjuvant. Immunizations were performed subcutaneously. Spleens were harvested from mice one week after booster doses of PcrV in Freund' s incomplete adjuvant.
• a single spleen was placed in 5 ml of tissue culture medium without serum, cut into pieces and gently homogenized. Large pieces of tissue were allowed to settle from the homogenate and the supernatant, single- cell suspension was removed and subjected to centrifugation at 1200 rpm for 10 minutes. The pelleted cells were resuspended in 10 ml of a solution to lyse red blood cells for 5 minutes and subsequently underlaid with 10 ml of fetal bovine serum. The material was centrifuged at 1200 rpm for 8 minutes, the supernatant was discarded and the cells were suspended in 30 ml of medium.
• Spleenic cells and myeloma cells were harvested by centrifugation at 1200 rpm for 10 minutes, and each pellet was separately suspended in 10 ml of tissue culture medium.
• 10" spleen cells and 2 x 10 " myeloma cells were mixed and pelleted together by centrifugation at 1200 rpm for 6 minutes. The supernatant was removed by aspiration and 1 ml of 35% polyethylene glycol (PEG) was added. The cells were suspended in this solution gently and centrifuged at 1000 rpm for 3 minutes. In some experiments centrifugation was eliminated.
• PEG polyethylene glycol

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• Food Science & Technology (AREA)

Priority Applications (5)

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Publications (3)

Family

ID=26807543

Family Applications (1)

Country Status (10)

Cited By (5)

Families Citing this family (18)

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• 1999
  • 1999-11-23 CA CA002318536A patent/CA2318536C/en not_active Expired - Fee Related
  • 1999-11-23 EP EP06000111A patent/EP1666058B1/en not_active Expired - Lifetime
  • 1999-11-23 EP EP99971223A patent/EP1049488B1/en not_active Expired - Lifetime
  • 1999-11-23 AU AU35808/00A patent/AU3580800A/en not_active Abandoned
  • 1999-11-23 WO PCT/US1999/027796 patent/WO2000033872A2/en not_active Ceased
  • 1999-11-23 DE DE69930166T patent/DE69930166T2/de not_active Expired - Lifetime
  • 1999-11-23 JP JP2000586362A patent/JP4707234B2/ja not_active Expired - Fee Related
  • 1999-11-23 AT AT06000111T patent/ATE539349T1/de active
  • 1999-11-23 ES ES99971223T patent/ES2255329T3/es not_active Expired - Lifetime
  • 1999-11-23 ES ES06000111T patent/ES2379801T3/es not_active Expired - Lifetime
  • 1999-11-23 US US09/448,339 patent/US6309651B1/en not_active Expired - Lifetime
  • 1999-11-23 AT AT99971223T patent/ATE318613T1/de not_active IP Right Cessation
  • 1999-11-23 DK DK99971223T patent/DK1049488T3/da active
• 2001
  • 2001-01-26 US US09/770,916 patent/US6827935B2/en not_active Expired - Lifetime
• 2010
  • 2010-12-08 JP JP2010273433A patent/JP2011068672A/ja not_active Ceased

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