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TOPIC: T200 parameter setttings

T200 parameter setttings 05 Aug 2020 03:18 #1

dear all,

I have a question to ask for you. the Initial value of parameter settings about the Biacore T200 can be changed?

Under what conditions can I change this inintial value ?
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Last Edit: by Wang.

T200 parameter setttings 05 Aug 2020 08:39 #2

Hi,
You can always change the initial values because that is what they are. Initial values. The fitting algoritm uses them to start the fitting and will adjust them during the procedure. However, when the initial values are very different from the expected answer the fitting may go wrong and give a wrong answer. Generally you can see that by inspecting the fitting visualy.
You may set the RI to constant/zero for the first fittng to get an idea about the kinetic parameters and then fit again with bulk effect.
You cannot permanently change the initial values.
Kind regards
Arnoud

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T200 parameter setttings 05 Aug 2020 09:58 #3

thank you so much, Arnoud!

when the fit with seting the RI to constant/zero is necessary?

I have a kinetic data for a antigen and antibody, not exactly 1:1 binding( theoretically,it is 1:1 binding), I tried to fit the curve by seting the RI to 0, as follows: If the reult of 1:1 binding is reliable?

kindly regards.
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T200 parameter setttings 05 Aug 2020 19:42 #4

The fitting shows that the curves are not 1:1. In the RI.jpg the bulk (RI) is adjusted to have the 'best' fit and as you can see it does not fit the first part of the dissociation. In RI constant the fit does not match the association very well.
These are not reliable fits.
Is the antibody the ligand or analyte? Antibody is ligand --> 1:1; ab is analyte --> bivalent model.
What to do to get this better? Please visit www.sprpages.nl/experiments/troubleshooting and www.sprpages.nl/best-results/artifacts . Maybe these pages give some clues to optimize the conditions.
Arnoud

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Last Edit: by Arnoud.

T200 parameter setttings 06 Aug 2020 04:25 #5

hi,arnoud

thank you for your patient reply.

I have one more question: bivalent model have ka1, kd1, ka2,kd2 . Which one should I report as the equilibrium dissociation constant for the antibody?

kindly regards,
lily

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T200 parameter setttings 06 Aug 2020 19:39 #6

The bivalent analyte model results in to two sets of rate constants, one for each binding step. The meaning of the two sets of rate constants and particular the second set is difficult to interpret. The second association constant (ka2) is dependent on the first binding. In case of an antibody, both binding sites are equal, and consequently also the rate constants. However, since the second binding is dependent on the first interaction (it is to some extent directed) the second association rate constant is given in RU-1s-1. In addition, the first and second dissociation rate constant (kd) are interchangeable.

I don't have an accepted solution to this question. The best answer you get probably with the equilibrium analysis since this will give the overall affinity of the two antibody arms binding to the ligand. If this is not possible, and you really want to have the kinetic constants then you should immobilize the antibody as the ligand and flow the target over it.

Arnoud
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