P. aeruginosa mucoid exopolysaccharide specific binding peptides
a technology of aeruginosa and aeruginosa, which is applied in the field of prevention and treatment of pseudomonas aeruginosa (p. aeruginosa) infections, can solve problems such as serious infections, and achieve the effect of enhancing opsonization and phagocytosis
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[0188] Immunization of Patients with MEP
[0189] Culture of P. aeruginosa, purification of MEP, and immunization of subjects with MEP has been described before in U.S. Pat. No. 4,578,458, the entire contents of which are incorporated herein by reference. Briefly, subjects are preferably immunized with purified MEP harvested from P. aeruginosa 2192 isolates (ATCC No. 39324). Although P. aeruginosa strain 2192 is preferred, new strains that produce a polysaccharide having essentially the same antigenic determinants as present in P. aeruginosa 2192 may also be used. P. aeruginosa can be maintained in liquid basal medium containing essential nutrients and ingredients (e.g., Mian's Minimal Medium, Trypticase Soy Broth and deoxycholate citrate agar, preferably supplemented with divalent cations such as magnesium). Methods of bacterial culture are known in the art. MEP is purified from Mian's minimal medium, followed by precipitation of MEP with alcohol. The precipitat...
example 2
Materials and Methods
[0264] Binding Assays to P. aeruginosa and purified MEP
[0265] ELISA plates were coated with purified P. aeruginosa MEP at 2 μg / ml in 0.02 M phosphate buffer containing sodium azide overnight at 4° C. The plates were washed several times to remove unbound MEP in PBS with 0.05% Tween. The plates were then incubated overnight at 4° C. with PBS / BSA in order to block all other non-specific binding sites. Following several washes, antibodies were added to the plates at various concentrations ranging from 0.0625 to 2.0 μg / ml. The plates were incubated for 2 hours at room temperature. Bound antibody was detected with an alkaline phosphatase conjugated goat anti-human IgG antibody specific for the Fc domain. Assays could similarly be performed using intact bacteria, as described by Preston et al. Infection and Immunity 66:4137-4142, 1998.
[0266] Complement Deposition Assays
[0267] ELISA plates were coated with purified P. aeruginosa MEP at a concentration of 10 μg / ml i...
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