That depends on the plate, whether it is binding by hydrophobic interactions only or has been activated to bind the antibody covalently. Best follow manufacturers recommendations and optimise things from there.

However, if you are developing a new assay, consider using dot blots instead of ELISA. The protein of interest is bound to a PVDF membrane by filtration in a 96-well vacuum manifold. This binds the protein near quantitatively, and you can detect it with your detection antibody directly (no capture antibody needed). Since you use nearly 100% of the protein in the sample (ELISA usually less than 5%), dot blots are much more sensitive than ELISAs, in addition they save a lot of time and chemicals.

That depends on the plate, whether it is binding by hydrophobic interactions only or has been activated to bind the antibody covalently. Best follow manufacturers recommendations and optimise things from there.

However, if you are developing a new assay, consider using dot blots instead of ELISA. The protein of interest is bound to a PVDF membrane by filtration in a 96-well vacuum manifold. This binds the protein near quantitatively, and you can detect it with your detection antibody directly (no capture antibody needed). Since you use nearly 100% of the protein in the sample (ELISA usually less than 5%), dot blots are much more sensitive than ELISAs, in addition they save a lot of time and chemicals.

PBS works perfectly fine for most antibodies and most plates, I always use it. Coating buffer (pH 9.6) is widely used, as you mention. Some unique antibodies need more careful optimization, but this is not common. For instance I had an antibody that could only be immobilized on certain plystyrene o plates at pH 4-5.

The best answer to your question will depend on your ultimate goal for your project. Some of those have already been presented in the above replies to your question. Aside from choosing the type of pale you want to use, test the binding affinity of your mAb to the plates you have chosen and see what makes it stick best meaning is the binding through hydrophobic interaction, charge-charge complexation or do you need to perform covalent conjugation. Also, test the non-specific adsorption of your mAb to the plates and see if this is sufficient for your goal. All of these different ways of attaching your mAb on your plate would require specific pH conditions, specific buffers for optimum performance, and specific time frame to allow optimum exposure for binding or adsorption.

Hi,

You already got very important answers to your question. Anyway, It seems to me you're new to the subject and, If I am right, take a nice reading about your question in these easy manuals below:

1 - go to corning website and use the codes below:

ELISA Workflow Guide - CLS-DD-076 A4

Five ELISA Application Notes are available at www.corning.com/lifesciences.

Immobilization Principles – Selecting the Surface for ELISA Assays (CLS-DD-AN-454)

Optimizing the Immobilization of Protein and Other Biomolecules for ELISA Assays (CLS-DD-AN-455)

Effective Blocking Procedures in ELISA Assays (CLS-DD-AN-456)

Optimizing the Separation Step on 96-well Microplates for ELISA Assays (CLS-DD-AN-457)

Selecting the Detection System – Colorimetric, Fluorescent, Luminescent Methods for ELISA Assays

(CLS-DD-AN-458)

It may be a nice starting point for you, If you're new to it!

Hope it helps.

Thank you all for the very helpful replies! I have plenty of ideas and resources to play with now!

You are most welcome. There are plenty of materials you can access to help you figure out what's best for your needs and for your applications.