Generation of a recombinant antibody for sensitive detection of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen that causes nosocomial infections and often exhibits antibiotic resistance. Therefore, the development of an accurate method for detecting P. aeruginosa is required to control P. aeruginosa-related outbreaks. In this study, we established an enzyme-linked immunosorbent assay method for the sensitive detection of three P. aeruginosa strains, UCBPP PA14, ATCC 27853, and multidrug-resistant ATCC BAA-2108. We produced a recombinant antibody (rAb) against P. aeruginosa V‐antigen (PcrV), which is a needle tip protein of the type III secretion system of P. aeruginosa using mammalian cells with high yield and purity, and confirmed its P. aeruginosa binding efficiency. The rAb was paired with commercial anti-P. aeruginosa Ab for a sandwich ELISA, resulting in an antigen-concentration-dependent response with a limit of detection value of 230 CFU/mL. These results suggest that the rAb produced herein can be used for the sensitive detection of P. aeruginosa with a wide range of applications in clinical diagnosis and point-of-care testing.

Flow cytometry-based rapid detection of Staphylococcus aureus and Pseudomonas aeruginosa using fluorescent antibodies

“…1B). According to the method described in our previous paper, 12,13 we expressed 6DW2 and 6CYF and purified each protein by using protein A affinity beads. We used these proteins for fluorescent labelling.…”

“…In our previous study, we generated recombinant full-sized IgGs against S. aureus 12 and P. aeruginosa 13 using mammalian cells. To produce recombinant anti- S. aureus antibodies, we constructed three mammalian cell-expressing plasmids with the insertion of the variable domain of three antibodies against wall teichoic acid on the surface of S. aureus : 6DWI, 6DW2, and 6DWC, whose amino acid sequences and CDR regions differed.…”

“…Mammalian cell-based 6DW2 and 6CYF expressions and purifications were performed according to our previous study. 12,13 Briefly, HEK293F cells were cultivated in 200 mL of Freestyle 293 medium in a flask at 37 °C in 8.0% CO 2 . When the density cells reached to 1 × 10 6 cells per mL, 1.25 μg per mL heavy chain and light chain genes of the anti-6DW2 antibody were co-transfected to the cells using 7.5 μg per mL PEI and incubated for 7 days with 120 rpm at 37 °C in 8.0% CO 2 .…”

“…Two S. aureus strains, WKZ-1 (NR-28984) and WKZ-2 (NR-28985), three P. aeruginosa strains, UCBPP PA14, ATCC 27853, and ATCC BAA-2108, and one E. coli strain, BL21(DE3) were cultured according to our previous study as follows: 12,13 the stocks of cultures were inoculated into 3 mL of LB media and incubated overnight at 37 °C with 200 rpm shaking. The sample was centrifuged (3500 rpm, 10 min, 4 °C) and the supernatant was discarded.…”

“…Polymerase chain reaction (PCR)-based detection 7–11 is faster than conventional culture- and colony-counting-based methods, but still requires 4–5 h for repeated amplification cycles. Our group recently developed an enzyme-linked immunosorbent assay (ELISA) systems for detecting S. aureus 12 and P. aeruginosa 13 by using commercially available antibodies and recombinant antibodies. We produced recombinant antibodies via a mammalian cell culture system and revealed that these antibodies had high antigen-binding efficiency and a low detection limit (LOD), which enabled the detection of S. aureus or P. aeruginosa under 10 4 CFU.…”