% Activity Calculation and Importance

Hello,

I am new to the SPR forum, as well as Biacore analysis (Chromatography background). I have recently taken over analysis in my lab and am trying to craft reports and other compilations of data for customer delivery.

Most of the deliverable data is kd on, kd off, and KD values, however some of the legacy reports have a % activity reported. I vaguely understand this value to be a measurement of the % of the analyte that showed affinity during the association phase to the ligand. I am currently using the equation below:

Rmax from fit/ (RL*(MWA/MWL)*Sm)

where: RL is ligand density on sensor chip, MWA is analyte MWL is ligand, Sm is # of binding sites

Overall, I want to make sure that I am calculating this, perhaps a brief description if my understanding of %activity is correct/overall importance of the value, and if that are any additional resources readily available.


Thanks,
Sam

Hi Sam and welcome to the SPR.
First of all we speak about the association rate constant k_a (k_on) in M^-1s^-1; dissociation rate constant k_d (k_off) in s^-1 and equilibrium dissociation constant K_D in M.

When I understand you correctly this calculation gives an estimate of ligand molecules that is biological active after immobilization. The Rmax can be calculated during the fitting procedure or determined by saturating the ligand. Theoretical this value is between 0 and 100%. In general we find some lower value depending on the integrity and purity of the ligand and how the immobilization is 'destroying' the ligand. For the calculation of the kinetic constants the Rmax is not important in the sense that a low activity surface (or low density surface) should give the same kinetic values as a high activity surface (or high density). But you can use the % activity as a proxy for the suitability of the sensor surface.

Kind regards
Arnoud