PBS/TBS buffer in ELISA - Using TBS increases the signal, why? (Mar/21/2011 )

Hello,

I’m developing an indirect ELISA, and I have a question about the use of PBS and TBS buffers.

The assay starts with a blocking step (37 degrees Celsius, one hour) followed by plate washing and an incubation step with human serum. HRP conjugated goat anti-human IgE is used as secondary antibody.
Initially I used PBS (pH 7,4) with 3% BSA as blocking buffer and PBS with 0,05%Tween20 and 0,1%BSA as dilution buffer. But by using TBS (pH 7,5) instead of PBS, the OD value became twice as high. The value of blank on the other hand stayed the same.
This is nice, but I don’t understand why the signal increases. Has someone an explanation for this change in OD value? Can the small difference in pH cause this effect?

Thanks in advance,
Naomi

Naomiw on Mon Mar 21 15:53:47 2011 said:

The whole curve OD shifted or just the high points (with the 0 remaining constant)? Was the M of the buffers the same? If the curve shifted did the control values remain constant?

Thanks voor your answers.
PAO_ahac, as reaction on your questions: I don’t use controls. Summarized, at this point I’m just looking at different dilutions of human plasma on a plate (coated with protein). But the OD of every dilution became higher.
The TBS buffer contains 20 mM Tris and 137 mM NaCl. PBS contains also 137 mM NaCl, but next to that 2,68 mM KCl, 8,1 mM sodium phosphate dibasic and 1,47 mM potassium dihydrogen phosphate. So there are differences. Meanwhile I also discovered that increasing the NaCl concentration from 136 mM to 500mM, makes the OD decrease from 0,75 to 0,35.

Kind regards,
Naomi

I generally use 2% BSA in PBS (.01% Tween-20). Never really had any problems.

from the reading of your post I think that the matter related to the different Abs between TBS and PBS it is probably in the ionic strength among the two buffers from you used. The ionic strength of the reaction environment, as it is known, can also have a profound effect on the rate of the antigen-antibody reaction. Presumably, the ionic strength influences the interaction between antibodies and both hydrophobic and hydrophilic target molecules. For proteins of course, these observations have to be expect, because of the well-known influence of ionic strength on the electrophilic behaviour of charged particles. As the ionic strength increases, the depth of the electrical double layer that forms around a charged molecule is compressed, reducing the distance over which repulsive forces that keep molecules apart can act. This, in effect, leads to the promotion of aggregation. The reduction in charge will also influence the electrostatic attraction between oppositely charged species, which may then reduce specific binding. I remember to myself, that electrostatic interactions are influenced by the protonation state of the reacting molecules. Buffers with low ionic strength may contribute to the non-specific bindings of the conjugate to the capture antibody, while increase concentration of ionic strength can be used to minimize non-specific interactions, but further increases in ionic strength could cause decrease in binding affinity between antibodies and coated antigens, up to hindrance of antibody-antigen binding. If the interaction is dominated by ionic interactions, such as well as immunoglobulin versus immunoglobulin (e.g.: IgE/anti-IgE), high salt concentration lowers the affinity. In addition, when the specific binding between antigen/antibody is achieved in a reaction solution with low ionic strength, subsequent washing with a low-ionic-strength buffer solution lead to complete dissociation of the complexes (weak binding), while, when the specific binding reaction is performed in a high ionic-strength solution, the bond that produces him is to high affinity and most of the binding into complex are maintained even after the washing with low ionic strength solution. Insofar, it is fundamental in the immunometric diagnostic systems realization, to calculate and subsequently appraise the ionic strength of the used buffers. For Tris-HCl buffer, which weak acid (WA) TrisHCl dissociates in TrisH+ and Cl-, and the conjugated base (CB) Tris-base is electrostatically neutral (Z=0), her ionic strength (0.1 M at pH 7.4) calculated is 0.084, while phosphate buffer at identical pH and concentration show an ionic strength of 0.270. In both cases ionic strength can be increased adding NaCl up to that non specific bonds are reduced, while those specific kept.

Thank you very much!
Kind regards,
Naomiw