Immunological tests for the presence of bacteria which make use of antibodies obtained using a specific method
View Patent ↗The use of a bacterial antigen obtained from bacterial cells in the production of antibodies for immunological assays for detecting bacterial cells.
1. A method of detecting a specific bacteria in a sample, comprising the steps:
i) suspending the sample in a buffered solution;
ii) contacting the sample with an antibody specific against a surface antigen of the bacteria to be detected, wherein the antibody was raised against a bacterial cell which has been denuded of bacterial mucus via washing the bacterial cell with water, then with a buffered solution of glycine-HCl , then with a buffered solution of glycine-Na OH, and then with water;
iii) filtering the solution through a membrane: and
iv) detecting a presence of bacteria tagged via the antibody on the membrane, wherein the presence of bacteria tagged via the antibody on the membrane is evidence of the bacteria in the sample.
2. The method according to claim 1 , wherein the antibody is tagged with colloidal gold.
3. The method according to claim 1 , wherein the membrane of step iii) is low porosity and impermeable to bacteria.
4. The method according to claim 1 , wherein the membrane of step iii) is high porosity and permeable to bacteria.
5. The method according to claim 1 , wherein the bacteria is subspecies Clavibacter michiganensis subsp. sepedonicus.
6. The method according to claim 1 , wherein the bacteria is other than subspecies Clavibacter michiganensis subsp. sepedonicus.
7. The method according to claim 1 , wherein the presence of bacteria tagged via the antibody on the membrane is detected by using antibodies in conjugation with an enzymatic contrasting agent.
8. The method according to claim 2 , wherein the presence of bacteria tagged via the antibody on the membrane is detected by using the reduction of silver ions on the colloidal gold.
9. The method according to claim 1 , wherein the membrane of step iii) is an activated polycarbonate membrane modified with glutaryl aldehyde and containing immobilized antibodies.
10. The method according to claim 9 , wherein the immobilized antibodies are specific against a surface antigen of bacteria of the subspecies Clavibacter michiganensis subsp. sepedonicus.
11. The method according to claim 9 , wherein the presence of bacteria tagged via the antibody on the membrane is determined using antibodies in conjugation with an enzymatic contrasting agent.
12. The method according to claim 9 , wherein the presence of bacteria tagged via the antibody on the membrane is determined using antibodies tagged with colloidal gold.
13. The method according to claim 12 , wherein the antibodies tagged with colloidal gold are detected using the reduction of silver ions on the colloidal geld.
14. The method according to claim 9 , wherein the presence of bacteria tagged via the antibody on the membrane is determined using antibodies tagged with a fluorochrome.
15. The method according to claim 14 , wherein the fluorochrome is carbocyanine Cy3.
16. The method according to claim 1 , further comprising step v) after step iv), said step v) comprising culturing bacterial cells on the membrane by placing the membrane on the surface of a medium containing nutrients for bacterial cells.
17. The method according to claim 9 , wherein the activated polycarbonate membrane is prepared by coating a membrane with a polymer of aniline, chemical modification of the polymer of aniline with glutaryl aldehyde, coating with antibodies, blocking and rinsing with a buffer.
18. The method according to claim 1 , wherein the antibody is tagged with colloidal gold, the presence of bacteria tagged via the antibody on the membrane is detected by using the reduction of silver ions on the colloidal gold, and the bacteria is subspecies Clavibacter michiganensis subsp. sepedonicus.