Human monoclonal antibody specific for lipopolysaccharides (LPS) of the pseudomonas aeruginosa IATS O11 serotype
A LPSIATSO11, human monoclonal antibody technology, applied in the direction of antibodies, DNA/RNA fragments, specific peptides, etc., can solve the problems of a large number of antibodies, antibody methods have no advantages, antibodies lack effector functions, etc., and achieve high protection effect
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Embodiment 1
[0113] Example 1: DNA and Amino Acid Sequence of 1BO11
[0114] Antibody specificity was determined by DNA- and amino acid sequence, respectively. The DNA sequences of the variable segments of the heavy and light chains were determined. Briefly, total RNA from hybridoma cells was isolated and reverse transcribed into complete cDNA using SMART technology (Becton Dickinson). By this method, a universal primer is added to the 5' end of cDNA. IgM and kappa variable regions and part of the constant regions were amplified by PCR using this primer and the CK and Cμ-specific primers described in Table III. PCR fragments were then obtained by excising from the agarose gel and used as templates for sequencing with the primers described in Table III.
[0115] Table III
[0116] Primers for PCR amplification and sequencing of variable regions of 1BO11 IgM heavy and kappa light chains
[0117] Primer
sequence
purpose
Conμ
Conκ
μ sequence
kappa...
Embodiment 2
[0122] Example 2: Recognition of monoclonal antibody 1BO11 to clinical isolates of Pseudomonas aeruginosa IATS O11 serotype
[0123]1BO11 was produced by immunizing healthy volunteers with octavalent OPS-Toxin A vaccine. The vaccine contained the IATS O11 reference strain FT-2. In order to investigate whether 1BO11 produced against this strain LPS would also recognize other isolates of IATS O11 serotype, a large number of clinical isolates were collected from different hospitals (see Table 2). The serotypes of all isolates were determined using a commercially available serotype agglutination kit. Serotypes were confirmed by PCR. 1BO11 binding was then tested on different clinical isolates of IATS O11 serotype (Figure 3a) as well as other serotypes (Figure 3b) by whole cell ELISA.
[0124] 1BO11 reacted strongly with all tested IATS O11 isolates except for two weak responses due to low expression of LPS on the surface of these isolates. Furthermore, binding was only observe...
Embodiment 3
[0125] Example 3: In vitro activity of 1BO11: opsonophagocytic activity
[0126] The in vitro biological activity of 1BO11 was evaluated using a flow cytometry-based opsonophagocytosis assay. FITC-conjugated Pseudomonas aeruginosa IATS O11 serotypes were incubated with serial dilutions of 1BO11 with normal rabbit serum as the source of complement. The opsonized cells were incubated with differentiated HL-60 cells (promyeloid cell line, ATCC: CCL-240; addition of 0.1M dimethylformamide, 3 days to complete differentiation to monocytes). Opsonophagocytosis was analyzed by FACS. Positive opsonophagocytic activity was determined by analyzing the green fluorescence of HL-60 cells compared to background staining (staining of HL-60 cells and FITC-bound cells in the absence of serum but in the presence of complement). The results are shown in Table 4.
[0127] 1BO11-mediated phagocytosis of Pseudomonas aeruginosa IATS O11 serotype in a dose-dependent manner (closed circles). Phagoc...
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