Antibody measurements in serum

Hi,

I am still pretty new in SPR experiments and want to use it to quantify antibody titers in human serum. I have a couple questions regarding the optimization of the assay (mainly non-specific binding (NSB)) and study design.

I use the Reichert 8500 SPR and use the dextran chip from Reichert. I have immobilized neutravidin to the chip to which I have immobilized a biotinylated polymer.

q1) I understood that for concentration determination it is best to saturate the chip with your ligand. Why should you saturate your chip when for kinetic studies you immobilize less ligand as this enables more precise results?

Because I inject serum (25x diluted in PBS) I have the problem of non-specific binding. Besides non-specific binding I also believe that both IgG and IgM bind specifically to my polymer. So clearly by incubating the serum I am not able to quantify the amount of either IgG or IgM. Therefore I will inject an excess of secondary anti human IgG or IgM antibody to determine the amount bound. I do have monoclonal antibodies from which I will make a standard curve by injecting these monoclonal antibodies. As I will detect the antibodies in serum with a secondary antibody I will also inject secondary antibodies in the standards as follows: inject monoclonal antibody and wait till steady state is reached, then incubate an excess of anti-human IgG or IgM and wait till steady state is reached. The response of the secondary antibody will be used for the standard curve.

q2) Is it possible to quantify the amount of bound IgG or IgM with this method? I am wondering if the non-specific binding will have an effect on the response of the secondary binding antibody to the specifically bound IgG or IgM. Due to non-specific binding the values of response will be higher in the serum sample, but as the response in SPR is linear (within its limits) I expect that the response of the secondary antibody is the same in both serum and standards (as long as an excess of secondary antibody is used).

q3) I have been looking for articles about protein detection in serum using SPR. However, I haven't been able to find a nice article describing their methods. If someone has a nice article, please let me know.

Thank you very much,

Jan-Jaap

Hi Jan-Jaap

q1) If you want to determine concentration you want to have mass transport limitation. This will make the binding rate (and reponse) dependent of the analyte concentration and independent of the kinetics. In the case of kinetics you want to immobilize a little as possible ligand to have the interaction totally kinetic.

q2) I am not sure I understand, but if the IgG or IgM bind specifically to your polymer a reference channel should do the trick. By subtracting the reference channel you have the bound IgG to the polymer. By detecting the first interaction (antibody with the polymer) with a second the response can be more selective. You have to make the standard curve the same way.

When the interaction reaches steady state you are measuring kinetics. Although the steady state is dependent on the analyte concentration I am not sure this is a good thing to do.

q3) I have been looking for articles about protein detection in serum using SPR. However, I haven't been able to find a nice article describing their methods. If someone has a nice article, please let me know.

q3) Sorry have no articles at hand.

Things to try:

-Add some extra salt to you flow buffer and sample (u to 500 mM NaCl). A high affinity binding should tolerate it.

-Add cm-dextran up to 1 mg/ml

Arnoud Marquart

SPR-pages

Good morning,
I have seen that you work with serum. I also tried to find a good article about it but I couldn´t. I´m not familiar with serum. I see that you use a dilution 1:25. What do you think is the useful range of serum dilution in this kind of experiments? Do you think a 1:200 or 1:500 could be a right dilutions?
Thank you very much.
Papillon