Below is a link to the protocol from Sigma that may address some of your questions. There are many protocols on this so it depends on what kit you use. You may be able to find out using an internet search of what you want.

https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/296/961/rab0039bul.pdf

Dear Prof Murakami,

Thank you very much for your kind answer. I am using exactly this kit. However, dilution factor is still my concern. I have limit (5-20 µl) sample for each. They state in the protocol that 100 µl prepared samples should be use for each well. Can we use half or one fourth of this amount with corresponding reduced volume of substrate and buffer? Thank you so much!

A long incubation time for the primary antibody should be no problem, I'd just be gentle to the HRP: 1hr in the fridge, if possible with gentle agitation, should be sufficient. Otherwise, if an instruction sheet came with your kit, just follow it and talk to the manufacturer's tech service. You have paid for it.

The datasheet states a range of 3 orders of magnitude, so it's very likely that your samples will yield signals within the standard curve, when you are using less sample volume than stated in the IFU. I'd dare to check how far one would get with 5ul.

If you're in doubt about the dilution of your samples and about the incubation times (or you need to dilute your samples), sacrifice a few wells and run a few samples together with a high and a low standard at the suggested conditions and see how far you get, before changing the protocol without thorough validation. Extending incubation time usually does not increase the sensitivity, you'd need to use more antibodies instead, which is ~impossible with an off-the-shelve- kit. Running a "blind shot" very likely will yield unusable results and you'll lose your samples. Test any modified conditions with non-valuable samples first and compare with the standard protocol.

I'd advise against reducing the amounts of buffers and reagents involved. The assay has been optimized for them. As you have less sample available than required, dilute them or try with a smaller sample volume, when no diluent is necessary.

If you expect to analyze huge amounts of samples, consider to have custom antibodies made and create your own assay.