Method for detecting a gram-negative bacterial autoinducer molecule
The present invention relates to an immunogenic conjugate comprising a carrier molecule coupled to an autoinducer of a Gram negative bacteria. The immunogenic conjugate, when combined with a pharmaceutically acceptable carrier, forms a suitable vaccine for mammals to prevent infection by the Gram negative bacteria. The immunogenic conjugate is also used to raise and subsequently isolate antibodies or binding portions thereof which are capable of recognizing and binding to the autoinducer. The antibodies or binding portions thereof are utilized in a method of treating infections, a method of inhibiting autoinducer activity, and in diagnostic assays which detect the presence of autoinducers or autoinducer antagonists in fluid or tissue samples.
1. A method for detecting an autoinducer of a Gram negative bacterium in a sample comprising:
adding to the sample an antibody or a functionally active fragment thereof which binds immuno specifically to the autoinducer of the Gram negative bacterium having Formula (I):
wherein X is O, S, N—(C 1 -C 6 ) alkyl, NR 2 , N-phenyl; Y is C 1 -C 6 straight or branched alkyl, C 1 -C 6 straight or branched alkenyl, C 1 -C 6 straight or branched alkynyl; Z is C═O, C═S, CHOH, C═N—NR 1 , C═N—OH, C 1 -C 8 straight or branched alkyl, C 1 -C 8 straight or branched alkenyl, C 1 -C 8 straight or branched alkynyl; L is C 1 -C 18 straight or branched alkyl, C 1 -C 18 straight or branched alkenyl C 1 -C 18 straight or branched alkynyl, or —CO 2 H, —CO 2 R 1 , —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR 1 , —CH═CH—CH 2 Br, —CH═CH—CH 2 Cl, —SAc or SH, where R 1 is C 1 -C 6 straight or branched alkyl, m is 0 or 1; z is 0 or 1; R 2 is H, C 1 -C 6 straight or branched alkyl, C 1 -C 6 straight or branched alkenyl or C 1 -C 6 straight or branched alkynyl, or CO 2 H; and Q is CH or N; and n is 0-3 with the proviso that when n is 0, X is N—(C 1 -C 6 alkyl) or N-phenyl; and
detecting whether immonospecific binding occurs between the antibody, or the functionally active fragment thereof, and the autoinducer of the Gram negative bacterium, wherein said immunospecific binding indicates presence of the autoinducer in the sample.
2. The method according to claim 1 wherein the Gram negative bacterium is selected from the group consisting of Aerononas hydrophila, Agrobacterium tumefaciens, Burkholderia cepacia, Chromobacterium violaceum, Enterobacter agglomerans, Erwinia stewarti, Erwinia carotovora, Escherichia coli, Nitrosomas europea, Photobacterium fischeri, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobium leguminosarum, Serratia liquefaciens , and Vibrio harveyi.
3. The method according to claim 1 wherein the antibody or the functionally active fragment thereof comprises a label.
4. The method according to claim 3 wherein the label is an enzyme, a radioactive label, a fluorescent label, a chemiluminescent compound, or a bioluminescent compound.
5. The method according to claim 1 wherein said detecting is carried out by ELISA, radioimmunoassay, gel-diffusion precipitation reaction assay, immunodiffusion assay, agglutination assay, fluorescent immunoassay, protein A immunoassay, or immunoelectrophoresis assay.
6. The method according to claim 1 wherein the sample is a histological sample obtained from a patient.
7. The method according to claim 6 wherein said detecting is carried out in situ.
8. The method according to claim 1 wherein the sample is selected from the group consisting of biological fluids, tissue extracts, freshly harvested cells, and lysates of cultured cells.