Coating antibody quantitation

[chrisg]

I am in the process of developing my own ELISA, and want to quantitate the amount of primary antibody that has bound to the microplate well after the initial coating step. Is anybody aware of an assay that can be used to measure this?

Thanks in advance!

[danfive]

Some rough ideas:

1. Manufacturers usually state the binding capacity of polypropylene as ng of protein/mm^2
2. You can bind anti-rabbit-HRP to detect the capture antibody, then add color reagent. Extrapolite Abs to binding efficiency. Should block first, of course.
3. Re-use the solution containing coating Ab to coat a 2nd, 3rd plate etc..until you see the re-use/dilution have a negative effect.
4. Before and after protein quantitation assays of coating Ab solutions.

[Zagami]

Method that used by me to evaluate efficiency coating, is micro determination of proteins bound to well surface through colorimetric BCA (bicinchoninic acid), which has a sensibility between 0.2 to 50 µg/500 µl (Zagami F.: On type-labda micromolecular mieloma case. Technical considerations. Bioch. Clin. Vol. 16, n° 3:237-241, 1992). Reagent is composed by 3 components : a) Reagent A ( dissolve in 80 ml dH2O, 8 g sodium carbonate monohydrate, 1.6 g sodium tartrate, adjuste pH to 11.25 with 10 M of NaOH and bring to 100 ml with dH2O. Stable 1 year at room temperature); b) Reagent B (dissolve in 80 ml dH2O, 4 g bicinchoninic acid sodium salt [BCA] and bring to 100 ml with dH2O. Stable 1 year at room temperature); c) Reagent C : dissolve 0.4 g cupric sulfate.5dH2O in 10 ml of dH2O. Stable 1 year at room temperature); d) Work solution (mix 1 volumes reagent C with 25 volumes reagent B, and then to add at mixture, 26 volumes of reagent A, that from a green color solution that it's stable for 1 week); e) Standards BSA or bovine gamma globulin stock solution (2 m mg/ml), before to use realize serial standards from 1, 3, 4, 6, 8, 12, 16 and 20 µg/ml in PBS pH 7.4. Procedure : After coating step, wash the wells with appropriate washing solution, presence of the detergents don't influence the BCA reaction. For to realize standard curve dispense in uncoated strips 50 µl of every standards, while in coated wells dispense 50 µl of PBS pH 7.4, and to shake, avoiding squirts, for 10 mins. Distribute in all wells 100 - 150 µl of work solution, cover sealing with adhesive plastics, and incube, in continuous shaking, at 37 °C for 2 hrs. Read, within 1 hr., Abs at 562 nm (540-570) nm. Employ for the calculation Excel software. Optimal coating has been considered when protein concentration result inclusive between 50 and 500 ng, depending from molecular characteristics of the proteins used. In every case, for values below this concentration the coating results less efficient.

[wendell]

Originally Posted by danfive

Some rough ideas:

1. Manufacturers usually state the binding capacity of polypropylene as ng of protein/mm^2
2. You can bind anti-rabbit-HRP to detect the capture antibody, then add color reagent. Extrapolite Abs to binding efficiency. Should block first, of course.
3. Re-use the solution containing coating Ab to coat a 2nd, 3rd plate etc..until you see the re-use/dilution have a negative effect.
4. Before and after protein quantitation assays of coating Ab solutions.

It's good!