Tracking genetically engineered bacteria: monoclonal antibodies against surface determinants of the soil bacterium Pseudomonas putida 2440.
Assessment of potential risks involved in the release of genetically engineered microorganisms is facilitated by the availability of monoclonal antibodies (MAbs), a tool potentially able to monitor specific organisms. We raised a bank of MAbs against the soil bacterium Pseudomonas putida 2440, which is a host for modified TOL plasmids and other recombinant plasmids. Three MAbs, 7.3B, 7.4D, and 7.5D, were highly specific and recognized only P. putida bacteria. Furthermore, we developed a semiquantitative dot blot assay that allowed us to detect as few as 100 cells per spot. A 40-kDa cell surface protein was the target for MAbs 7.4D and 7.5D. Detection of the cell antigen depended on the bacterial growth phase and culture medium. The O antigen of lipopolysaccharide seems to be the target for MAb 7.3B, and its in vivo detection was independent of the bacterial growth phase and culture medium. MAb 7.3B was used successfully to track P. putida (pWW0) released in unsterile lake mesocosms.
Ramos-González
M I
MI
Consejo Superior de Investigaciones Científicas, Departamento de Bioquímica, Vegetal, Granada, Spain.
Ruiz-Cabello
F
F
Brettar
I
I
Garrido
F
F
Ramos
J L
JL
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D004783:Environmental Microbiology; D004797:Enzyme-Linked Immunosorbent Assay; D005818:Genetic Engineering / Q000379:methods*; D019081:O Antigens; D011135:Polysaccharides, Bacterial / Q000276:immunology / Q000302:isolation & purification; D016958:Pseudomonas putida / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D012988:Soil Microbiology; D014871:Water Microbiology
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D019081:O Antigens; D011135:Polysaccharides, Bacterial