Monoclonal antibody affinity (Kd constant) determination using ELISA - (Apr/16/2008 )

Hello,

I have been trying to determine dissociation constant for a monoclonal antibody in solution using an ELISA-based protocol first described by Friguet et al.. The same protocol is also described in the "Antibody Engineering, A practical approach" handbook and I believe it is quite often used as a simple technique of obtaining antibody dissociation constants.

Basically, indirect ELISA is used to determine concentration of the free antibody in a series of solutions, pre-incubated with varying concentrations of the antigen. An initial experiment has to be performed beforehand to ascertain that: (a) the antibody concentration used is in the linear range of the ELISA response (so that the absorbance is proportional to the Ab concentration); (b) only a small fraction of the total free antibody in solution is retained on the plate (so that the measurement does not significantly affect the Ab-Ag equillibrium in solution).

For (b), two plates are coated with Ag and a series of Ab solutions with different concentrations is added to the wells. After incubation, antibody solutions are transferred to the second plate. Special care is taken that all the incubation and detection steps are the same for both plates. By comparing the signals from both plates, you can determine the fraction of the free antibody that is retained on the first plate (by comparing the slopes of absorbance vs. concentration graphs). No more than 10% of the antibody should be retained by the plate, if it is more, you should coat less antigen and repeat the experiment.

I am having problems with this initial experiment, as the slopes differ by more than 10%. I lowered the concentration of the antigen twice (now down to 15 nM), but I see no improvement. In the last experiment I even got a bigger difference in slopes than when using double concentration of antigen and now I am losing my confidence in the reliability of the assay. It seems to me that the method is too sensitive to even very slight variations in the treatment of the two plates and that the differences can not be reliably attributed to the amount of the retained primary Ab.

Is there anybody out there with experience with this method? Did it work for you? Do you have any suggestions or do you recommend any other approach of determining antibody Kd (ELISA-based would be favored; I will be doing Biacore analysis also, but that can be fishy, too). Any help will be gladly appreciated.

Thank you in advance.

Simon

Hi,
I had done affinity determination of monoclonal antibodies (directly from culture supernatants) according to Rath et. al (1988). Firstly, an indirect elisa is performed with varying dilutions of culture supernatants to determine the half titration point. The antibody is incubated with varying amount of antigen in tube separately at the dilution point of the culture supernatant, and then added the solution after incubation to antigen coated plates. the concentration of antigen required for 50% competition is considered the Kd value
all the best
Leelaram

Leelaram,

thank you very much for pointing me to the paper by Rath. I will try this approach. Looks like the method only gives relative estimate of the affinity compared to another antibody, but that might in fact suffice for me, too.

Thanks again,

Simon