I want to coat bacterial cells at a cell number of 10 (^8). I need help in regards to how to design the strategy for coating of my cells, assuming that each well of the 96 well plate will have equal cell number.
Coating the plates with bacterial culture directly works. but if you coat O/N then there is high chance that the cells will grow. this would lead to the discrepancy in your results later on.
Coating of plates with culture for 1-3 hours usually works with low noise levels in your readings.
However, if you want to try to coat the plate O/N , then you can try coating and incubating at 4 degrees O/N. many labs also do it.
Yes, and the dilutions of cultures to coat can be made in 1X-PBS
thanks,
saby
hello Sabyasachi
Let me know the detail of your work, so are you wanna use the protein membrane of bacteria as antigen? As I know, that the antigen it must be in protein extract. so maybe first step you must extract your bacteria to get pure protein membrane. Second, you must to know the concentration of your protein (antigen), and than you can use pattern M1.V1=M2.V2
That's my experience to prepare the antigen, but my sample is from salivary gland. Sorry if my answer is ot specific for you, I just Try to share :D
If your antibody will recognize the surface protein, just coat the bacteria cell, then block with unrelated protein, then run the indirect ELISA. In this case you need to use anti species antibody (related to your antibody , poly or mono) that conjugated to enzyme to indicate the level of binding of your antibody the surface protein.
Yes, you can use whole cells to coat ELISA plates. Of course, the target antigen must be on cell surface. I have worked in whole cell ELISA of V. cholera and N. meningitidis. You can prepare a cell suspension with an optical density between 01 -0.2 (measured at 600 -620nm). Add 100 microliter per wells and after 16h at 37o C (wells must be fully dry) perform washing, blocking and others steps as a conventional ELISA.
I would recommend to treat the bacteria with formaldehyde previously to coat them to the plate, and then run a regular ELISA.
The use of formaldehyde treatment could modify structure on protein, so you be careful. If you need to have 108 cells per wells you can prepare a cell suspension at 109 cells/ml and then add 0.1 ml/well. So, each well shall have 108 cells.
Formaldehyde estabilize the proteins and the membrane. The changes are small and the antibodies will recognize the surface proteins anyway.
Yes, whole cell ELISA is possible. We've used whole cell ELISA for Salmonella and Bacillus family. In our procedure, we would centrifuge the liquid culture twice to wash out the growth media and then prepare cells to higher 1x et 8 cells in buffer before adding to ELISA plate.
yes it is possible to doing whole cell ELISA as per CHEN and OSCAR, but time and temperate have to standardize for coating and blocking. Further make sure about chemical nature of bacterial cell wall i.e. protein /lipid containing. coating and antibody binding may also depends on the nature of cell wall.
The rationale behind use of whole cell bacterium is not clear. ELISA is a very sensitive system. Moreover immune response in vivo occurs to processed bacterial cell, in other words to various components of the bacterium. Hence it is advised to use a group of specific antigens (of the bacterium) of interest to study an immune response. In this case surface proteins may be extracted and assessed.
HI
Can anybody advise on how exactly coat bacteria onto plates? Do you keep it in PBS e.g 100ul per well? In RT or in 37C? O/N or shorter time? Do you use ELISA plates (high absorbance)?. Thanks. Aga
Coating the plates with bacterial culture directly works. but if you coat O/N then there is high chance that the cells will grow. this would lead to the discrepancy in your results later on.
Coating of plates with culture for 1-3 hours usually works with low noise levels in your readings.
However, if you want to try to coat the plate O/N , then you can try coating and incubating at 4 degrees O/N. many labs also do it.
Yes, and the dilutions of cultures to coat can be made in 1X-PBS
thanks,
saby
Hey Saby
Thanks a lot for your answer. I was actually thinking of coating the plates with heat inactivated bacteria to reduce the variability in cell numbers. Is that done in your lab?
Would you use MaxiSorb type plates to adhere bacteria or regular tissue cultures plated would also work?
thanks a lot
aga
Currently I am in the U.S. and my current projects are different. However, when I was involved in those experiments some time back, I have tried both maxisorp and normal 96 well tissue culture plates, which also have polystyrene coatings. it doesnot affect your adherence much.
but, if you have nunc96 well plates or maxisorp ones, prefer them first.
Regarding the variability, when u process your plate after coating, it will lead to detach or wash-off of the cells in a proportionate manner. I guess, that loss of cells will be uniform enough across all the wells.
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