I am developing a sandwich ELISA, for that I need to coat antibodies on plate. I need your suggestions about coating buffer, dilution factor, and blocking buffer. Someone told me you need a competitive protein to block. Can I use FCS for blocking?? Currently I am thinking to dilute and coat in PBS.
Coating should be done at pH 9.6 +/- 0.1. Only for some monoclonals an other pH is requried due to the sometimes uncommen isoelectric point.
Forget the blocking, Blocking is a kind of modern voodoo. There will be no free binding site anymore after coating with 1 ... 10 µg/mL. The binding capacity of polystyrene is much lower.
Beware: No detergents into your blocking buffer. The hydrophobic end of this molecules will bind much better than any protein. Thatswhy a detergent containing buffer should be used for the washing and incubation steps.
The coated plates can be stored frozen at -15 .... -80 °C until use (even several years) after most of the coating solution was removed.
Hi there. We coat our ELISA plates with carbonate/bicarbonate buffer pH 9,6, doesn't matter if it's an indirect or sandwich assay. For blocking we normally use a solution of PBS-milk 5% + BSA 1%. The dilution factor will depend on how concentrated your antibody is. If you don't have a clue, you should do a dilution scale (1/100; 1/200, 1/400, 1/800...1/3200, etc). But it's just a suggestion.
Here is the recipe for the coating buffer that we use.
500 ml ddH20
1.6 g Na2CO3
2.9 g NaHCO3
pH 9.5-9.6
For cytokine ELISA I coat plates with 1 ug/ml of capture antibody in coating buffer and for Ig isotype ELISA I coat them with 10 ug/ml in PBS only. The blocking buffer I use is 1% BSA in PBS. Washes are done with PBS-T. Standards and samples are diluted in blocking buffer (1% BSA/PBS) and make sure you do 2-3 different dilutions of the samples so that they fall in linear range on the standard curve. I typically start with 1:100 for Ig isotypes and do 5-fold dilution of that, twice. But the starting dilution is highly variable upon the isotype or cytokine being tested and the samples you are working with. So you might have to do some optimization. Hope this helps and good luck!
Should I add Tween in blocking solution?? I tried to make standard curve with 2 fold dilution, first 3-4 dilutions (high conc. to low conc.) are ok, but lower concentration are giving very strange results, I coudn't find any difference. I am using blocking solution (1% PBS + 0.05% tw20 + 5% FCS); is it OK?
Coating should be done at pH 9.6 +/- 0.1. Only for some monoclonals an other pH is requried due to the sometimes uncommen isoelectric point.
Forget the blocking, Blocking is a kind of modern voodoo. There will be no free binding site anymore after coating with 1 ... 10 µg/mL. The binding capacity of polystyrene is much lower.
Beware: No detergents into your blocking buffer. The hydrophobic end of this molecules will bind much better than any protein. Thatswhy a detergent containing buffer should be used for the washing and incubation steps.
The coated plates can be stored frozen at -15 .... -80 °C until use (even several years) after most of the coating solution was removed.
Hi Christopher,
be careful with NaN3. It's a highly potent inhibitor of the peroxidase. If the plates are stored frozen, no anti-microbiological agent is required.
In the case of Sandwich ELISA we dilute anti-MPO antibody at 1:2000 in coating buffer (PH 9.6). We prepare the coating buffer by addition of sodium carbonate and sodium bicarbonate to DW and adjust the PH by adding a weak acid/alkali as required.
In the case of blocking buffer we add PBS+20% sodium azide +BSA and wait 10 minutes after mixing.
We usually get good result.
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