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About single concentration kinetic
- Wang
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6 years 2 months ago #1
by Wang
About single concentration kinetic was created by Wang
Dear SPR users,
Recently, a strange result appeared in my experiment, as described below:
I have detected the single concentration kinetic of 20 antibodies. All the antibody's capture level is about 90ru,
The analyte is antigen and the analyte concentration is 5nM.
The results show that binding response values are very different.
In theory, the antibody with low binding response value is due to insufficient purity or low activity.
However, for antibodies with low binding response value, the kinetic curve has no saturation trend。On the contrary, the kinetic curve showed a saturation trend for antibodies with high binding response value.
What causes this? I haven't thought of a reasonable explanation.
So, I hope the experienced SPR can give me some Suggestions.
Thank you very much
best wishes
Recently, a strange result appeared in my experiment, as described below:
I have detected the single concentration kinetic of 20 antibodies. All the antibody's capture level is about 90ru,
The analyte is antigen and the analyte concentration is 5nM.
The results show that binding response values are very different.
In theory, the antibody with low binding response value is due to insufficient purity or low activity.
However, for antibodies with low binding response value, the kinetic curve has no saturation trend。On the contrary, the kinetic curve showed a saturation trend for antibodies with high binding response value.
What causes this? I haven't thought of a reasonable explanation.
So, I hope the experienced SPR can give me some Suggestions.
Thank you very much
best wishes
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- Arnoud
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6 years 2 months ago - 6 years 2 months ago #2
by Arnoud
Replied by Arnoud on topic About single concentration kinetic
Hi,
I had to think about this one. I think that your results are explained by the differences in kinetic constants between the different antibodies. Analytes with a fast dissociation rate constant (kd) reach steady state faster (takes less contact time) than analytes with a slow kd. (You write about saturation but how can you determine saturation with one injection; do you mean steady state?)
Equally, a slow association rate constant (ka) takes more contact time to reach steady state.
Your remark about the purity I don't understand. When you capture 90 RU of ligand (by means of an anti Fc I presume) then all of this should be antibody. Do you mean that the captured antibody is not one species? In general you have to be cautious with comparing response values when using an non-pure sample or when the responses are on steady state. In addition, response values are also dependent on the analyte concentration.
In the SPRpages book ( www.sprpages.nl/contact-spr-pages/sprpages-book ) is an example of an affinity plot where you can see how different rate constants work out on the curve shape.
You can also try the SPRSimulation software ( www.sprpages.nl/spr-simulation ) to model the curves your self to experience which parameter is important for the response level and curve shape.
Kind regards
Arnoud
I had to think about this one. I think that your results are explained by the differences in kinetic constants between the different antibodies. Analytes with a fast dissociation rate constant (kd) reach steady state faster (takes less contact time) than analytes with a slow kd. (You write about saturation but how can you determine saturation with one injection; do you mean steady state?)
Equally, a slow association rate constant (ka) takes more contact time to reach steady state.
Your remark about the purity I don't understand. When you capture 90 RU of ligand (by means of an anti Fc I presume) then all of this should be antibody. Do you mean that the captured antibody is not one species? In general you have to be cautious with comparing response values when using an non-pure sample or when the responses are on steady state. In addition, response values are also dependent on the analyte concentration.
In the SPRpages book ( www.sprpages.nl/contact-spr-pages/sprpages-book ) is an example of an affinity plot where you can see how different rate constants work out on the curve shape.
You can also try the SPRSimulation software ( www.sprpages.nl/spr-simulation ) to model the curves your self to experience which parameter is important for the response level and curve shape.
Kind regards
Arnoud
Last edit: 6 years 2 months ago by Arnoud.
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