response at end of injection and BiaEval software

Hi there can you please help me clarify

if i have example 2 analytes i pass over a receptor (ligand coated on chip)

both analytes are run at the SAME conc

say analyte 1 = 300RU just before the end of the injection
analyte 2= 600RU just before the end of the injection its HIGHER

would this mean for analyte 2 the ka (on rate) caclculated should be higher than that of analyte 1

would the dissociation of analyte 2 be expected to be lower?


my other question is that I find with the BiaEvaluation software if i choose for kinetic analysis (simultaneous) diffrent areas of the curvefor assoc and dissoc my constants can be very diffrent

If I choose dissoc too close to the injection stop the chi are very high, so if i avoid a section after the injection stops its alot better but the calculated dissoc value is drastically different i don't understand there is no nonspecific binding either


thanks so much for your help

Hi,

Can you give the molecular weights of the ligand and the two analytes?
Which type of instrument/software version do you use?

You cannot deduce the k_a or the k_d from the height of the curve.

Can you show us the two pictures of the fitting where you select different analysis regions?

kind regards
Arnoud

Hi I attached powerpoint image here to show you of a single analyte concentration.
No1 is analysed the same as 2 but the dissoc in 2 is close to the injection stop.


its a pretty fast dissociation correct but the Rmax doesn't make sense does it?

This is 1:1 fitting (binding is fc region)

I find the fitting is always a problem after the injection stop. If i take it too close to the injection stop the chi values are very high always. The buffer is matched. where does the real dissociation start?


analyte is 150kDa (IgG antibody)

ligand is a receptor (25kda)

in the third image are 2 different IgG's purified from 2 diffrent persons and run at the same concentration over the ligand (we are trying to see if any diffrences in disease state of the individuals affecting the kinetics of binding to a receptor)

should the ka for the one that binds higher over the ligand be higher? I am confused as to whether this should be refelcted as a greater on rate. I thought you can tell by looking at the curves if one has reached say 500RU at the end of the injection and another identical in MW and concentration has reached say 250RU, surely the one that reached 500RU point just as the injection stops should have a higher on-rate i.e. after fitting the ka should be higher.

Thanks so much for your help

Biacore3000
BiaEvaluation software 4.1.1

we are doing highthouput screening of IgG to various receptors or antigens

attachment

sorry i dont think my files were addded before