Hey there, I do lot of ELISAs for HIV antigen/antibody detection.

First, in regards to your goal, 96 well plates are limited to a minimum of 25ul for the reaction because of the surface area of the well in a 96 well plate. 384 well plates (what we use) bring this down to as low as 5ul and the increased amount of wells also ups the throughput. Since ELISA are extremely sensitive, once you add in the dilution factor and your actually limited by the fact that you don't really want to pull less than 0.5 ul from a sample w/ a standard pipette. Washing/blocking is also dependent on the type of assay you’re trying to perform and your available detection reagents.

Here are the answers to your questions.

1. Depends on what you want to do. Direct ELISAs are usually highly optimized (need to be sure there is no interference from the primary labeling), rigid, and expensive. Sandwhich ELISAs are more flexible and cost effective but they take more time and you need to watch out for cross reactivity. Since you are looking for antgiens you are mostly likely going to coat w/ a capture antibody, block, add your sample, then add a detection antibody conjugated w/ a label or reagent for subsequent substrate reaction. You can research protocols by going to your library and accessing journals. Search for ELISAs based on the antigen you are trying to detect and read there materials and methods. Here is a basic protocol to give you an idea of the assay.
2. Coat your plate w/ 50ul per well capture antibody @ 2 ug/ml (excess is fine here because you want to saturate the bottom of the well). Incubate for 2 hours at room temp or over night at 4C.
3. Wash your plates 4x with 100 ul PBS. After flip them upside down and bang them on a paper towel to remove any remaining PBS.
4. Block your plates w/ 100 ul per well of either a blocking reagent or milk w/ tween & NGS (normal goat serum). Incubate for 2 hours at room temp or overnight at 4C. During this incubation prep your samples.
5. Wash your plates 4x with 100 ul PBS. After flip them upside down and bang them on a paper towel to remove any remaining PBS.
6. Add 50 ul of sample. Depending on the volume of sample you can start at net or dilute 1:2 or 1:10 then titrate from there. Incubate for 1-2 hours at room temp. Note: you will need to include controls (blank well, negative sample, positive sample of known titration that you can use as a standard to quantify the concentrations in your unknown samples).
   • Wash your plates 4x with 100 ul PBS. After flip them upside down and bang them on a paper towel to remove any remaining PBS.
7. Add 50 ul per well of your secondary antibody at 4 ug/ml, in this case lets say it’s monoclonal antibody specific to HIV gp140 conjugated to ALP (Alkaline phosphatase). Incubate for an hour at room temp.
8. Wash your plates 4x with 100 ul PBS. After flip them upside down and bang them on a paper towel to remove any remaining PBS.
9. Add 50 ul per well substrate (in this case p-nitrophenyl phosphate since you used ALP) and start a timer. Watch the OD of your positive control standards most concentrated well come up to 2 and then add 12.5 ul stop solution (4N NaOH for example). Read your plates.
10. Fit a 4PL curve to your positive control standard and use the slope to determine the concentrations of your unknowns.

A few problems you might run into

• Unless you have a good amount of money (at least 1k) you are not going to optimize and ELISA from scratch. This is due to reagents, buffers, plates, and other materials. You can buy a pre-optimized kit depending on what you are trying to detect but that's still going to cost you <$500 and you are going to be limited to a few assays.
• w/ out a spectrophotometer I doubt you could read your OD's to any dependable specificity to differentiate between the minute discrepancies in absorbency especially for immuno assays. How much of a problem this is depends on the seriousness of your project. With out software you are going to have to do all the maths to determine your concentrations manually in excel/prism/etc,

I think your best bet is to contact a lab that does ELISA’s in your area and see if you can get little funding from your school to cover the cost in materials to run an ELISA there. Or alternatively you can prep your samples and send them off and it will probably be around $100 per plate.

Good luck!