I'm running some ELISA experiment plates coated with an extremely expensive protein. However, at the end of the experiment there was no signal.
The last step of this experiment was the incubation with TMB and stopped the reaction with 0,18M sulfuric acid.
I would like to know if it is possible to re-incubate again with the primary Ab and secondary or if the sulfuric acid would affect the binding properties of my protein to the Ab.
Grettings.
I have to say that only with the secondary Ab you can incubate your sample, 'cause the reaction with the primary antibody is irreversible (covalent, you need to denaturate to break the bound). Take it to pH 6 with citrate buffer and make the 2nd incubation .
You can, but you will loose a lot of quality.
Several prerequisites:
1. Don't overload your plates during coating (2 - 5 µg/mL are sufficient)
2. Don's stop your reaction with H2SO4, you can measure at a distinct time (e.i. 30 min) in the blue area of the spectrum.
3. Wash your plates 3 x with PBS, 0.1% Tween 20. Wash 3 times with PBS only. Break the antigen-antibody binding either be acidic conditions (0.05 mol/L Gylcin/HCl pH 2.0), wash 3 times 5 min. Neutralize with 3 PBS washings. Or incubate with 3 mol/L KSCN. 3 times 10 - 20 min. wash with PBS.
But, the better way instead of re-use the coated plates, use smaller volumes. 50 µl are sufficient for all reaction steps in an ELISA, except the substrate reaction. Here you should use 100 - 200 µL (Stop with 50 µl 2 mol/L H2SO4). When you are using instead of flat bottom plates round bottom - you can work also with 30 µl reaction volume.
I think the first experiment is a loss and you'll never get decent data even with rewashing and starting with the secondary antibody. I would start over. The reccomendations of limiting the coating concentration (2-5 ug/ml) and keeping the volume to 50 ul/well are good ways to minimize use of expensive coating protein.
I generally don't go below 50 ul/well even with flat bottom plates. My favorite plates are Nunc Maxisorp. I've gotten good results even with coating as low as 1 ug/ml.
Good luck.
It's not easy to advice when you don't know all steps of assay. Have you controls in the first experiment? Certanly you can rewashe plate and repeat ELISA with controls of all reaction steps, use not each cells of plate, enough 8-16 cells.
Do you have a protocol for this assay or are you developing one? You may want to run a checkerboard assay using you're protein and primary Ab.
You can re-use your antigen ( protein or antibody) just collected after first incubation and use again up to two times. But if you want you can also re-use your ELISA plates: Add 0.1-0.4M NaOH or an alkaline detergent at 60ºC, for 20 min, them live overnight, next day wash with tap water, dry it and re-use it!.... with your first antibody!
You can test if the plates are ok, just add second antibody in bloking buffer, wash then add substact and the OD value should be the same as (control plate) the new 96 well plate.
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