comparison of ELISA protocols - (Jan/03/2008 )
Hi,
I've tested two different ELISA protocols. The differences are the following:
1. protocol: coating in PBS, washing in Millipore water, blocking buffer contains 1 mM EDTA, 0.05% Tween®20 in PBS, sample dilution in PBS
2. protocol: coating in methyl alcohol, washing with PBS, blocking buffer: 3% BSA in 0.05M carbonate bicarbonate buffer, pH 9.6, sample diluent contains 3% BSA, 2% normal goat serum in PBS containing 0.05% Tween20
Although I used the same concentrations for the coating antigens, the first antibody and the secondary antibody (goat anti-human IgG conjugated with HRP) the OD values are totally different. Whereas ELISAs according to the first protocol resulted in OD values about 3, the values resulting from the ELISAs following the 2nd protocol where lower (OD<1). The background is always the same. I'm not sure why there is such a big difference. Is the BSA in the sample diluent and blocking buffer responsible for that?
I hope someone can help me!
I've tested two different ELISA protocols. The differences are the following:
1. protocol: coating in PBS, washing in Millipore water, blocking buffer contains 1 mM EDTA, 0.05% Tween®20 in PBS, sample dilution in PBS
2. protocol: coating in methyl alcohol, washing with PBS, blocking buffer: 3% BSA in 0.05M carbonate bicarbonate buffer, pH 9.6, sample diluent contains 3% BSA, 2% normal goat serum in PBS containing 0.05% Tween20
Although I used the same concentrations for the coating antigens, the first antibody and the secondary antibody (goat anti-human IgG conjugated with HRP) the OD values are totally different. Whereas ELISAs according to the first protocol resulted in OD values about 3, the values resulting from the ELISAs following the 2nd protocol where lower (OD<1). The background is always the same. I'm not sure why there is such a big difference. Is the BSA in the sample diluent and blocking buffer responsible for that?
I hope someone can help me!
I preferred the 1st protocol.
Coating buffer: steril PBS;
Blocking buffer: 1%BSA in PBST;
Dilution buffer: 1% BSA in PBST.
Methyl alcohol may destroy coating antibody.
hey,
i wud agree with glcui..., check for the coating efficiencies (alcohol can precipitate the proteins)..
gud luk
I've tested two different ELISA protocols. The differences are the following:
1. protocol: coating in PBS, washing in Millipore water, blocking buffer contains 1 mM EDTA, 0.05% Tween®20 in PBS, sample dilution in PBS
2. protocol: coating in methyl alcohol, washing with PBS, blocking buffer: 3% BSA in 0.05M carbonate bicarbonate buffer, pH 9.6, sample diluent contains 3% BSA, 2% normal goat serum in PBS containing 0.05% Tween20
Although I used the same concentrations for the coating antigens, the first antibody and the secondary antibody (goat anti-human IgG conjugated with HRP) the OD values are totally different. Whereas ELISAs according to the first protocol resulted in OD values about 3, the values resulting from the ELISAs following the 2nd protocol where lower (OD<1). The background is always the same. I'm not sure why there is such a big difference. Is the BSA in the sample diluent and blocking buffer responsible for that?
I hope someone can help me!
I preferred the 1st protocol.
Coating buffer: steril PBS;
Blocking buffer: 1%BSA in PBST;
Dilution buffer: 1% BSA in PBST.
Methyl alcohol may destroy coating antibody.
I coudnt agree more with this......u should not use alcohol in coating, PBS or carbonate buffer may help in binding.
Washing rather than water u can do with PBST
I agree with respect to the alcohol issue. Coat with aqueous buffer.
But you mentioned your OD was quite high. You may discover you have a problem in that your 1st protocol has no blocking agent remove the surfactant and block with buffer and protein such as BSA, Casein, etc. Surfantant in the wash solution.
I've tested two different ELISA protocols. The differences are the following:
1. protocol: coating in PBS, washing in Millipore water, blocking buffer contains 1 mM EDTA, 0.05% Tween®20 in PBS, sample dilution in PBS
2. protocol: coating in methyl alcohol, washing with PBS, blocking buffer: 3% BSA in 0.05M carbonate bicarbonate buffer, pH 9.6, sample diluent contains 3% BSA, 2% normal goat serum in PBS containing 0.05% Tween20
Although I used the same concentrations for the coating antigens, the first antibody and the secondary antibody (goat anti-human IgG conjugated with HRP) the OD values are totally different. Whereas ELISAs according to the first protocol resulted in OD values about 3, the values resulting from the ELISAs following the 2nd protocol where lower (OD<1). The background is always the same. I'm not sure why there is such a big difference. Is the BSA in the sample diluent and blocking buffer responsible for that?
I hope someone can help me!
Contrary to what some others say, there are some times when coating in alcohol works, but they are very much the exception to the rule! What are you coating?
Next, try washing off the antigen with PBS, rather than water, and PBS-T during the assay. Also, your second assay might suffer because you are washing off antigen with the bicarbonate; try BSA in PBS.
I presume the ODs you mentioned are for the top standards. Also, what is the background?
I agree you need protein in the blocking solution.
I think carbonate/bicarbonate buffer is for coating. Well I could be wrong.
you don't have any BSA or FBS in your blocking buffer?
why don't you use blocking with protein to dilute your sample?
coating: carbonate buffer nahc03/na2co3
wash: PBST
block/dilute: BD Opteia blocking media