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high affinity interaction analysis
- Priyanka
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6 years 4 months ago #1
by Priyanka
high affinity interaction analysis was created by Priyanka
I am using ProteOn XPR36 instrument (BIO-RAD). Currently we are working on molecules that has high affinity.
Very less dissociation is seen and this affects the kd values.
To overcome the dissociation issue, we started using saturating concentration of analyte.
Now in my sensorgram, upper two points are in saturation,( rest all points show proper curve) we are getting proper kd and chi2 values. But the problem is we cannot move ahead with such sensorgrams due to regulatory issues ( we have to submit the data in regulatory agencies).
Please suggest how can we improve the dissociation.
Kindly help!
Very less dissociation is seen and this affects the kd values.
To overcome the dissociation issue, we started using saturating concentration of analyte.
Now in my sensorgram, upper two points are in saturation,( rest all points show proper curve) we are getting proper kd and chi2 values. But the problem is we cannot move ahead with such sensorgrams due to regulatory issues ( we have to submit the data in regulatory agencies).
Please suggest how can we improve the dissociation.
Kindly help!
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- Arnoud
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6 years 3 months ago #2
by Arnoud
Replied by Arnoud on topic high affinity interaction analysis
Hi,
As you encountered, high-affinity interactions (especially slow dissociation rates: < 10-5) are not easy to solve in the Biacore. Generally prolonging the dissociation time is the easiest option to have enough decay in the response level needed to calculate the dissociation rate constant. The complex half-life for a kd of 1.10-5 is ~20 hours and the time to 5% decay ~ 90 minutes (See BiaCalculations ( www.sprpages.nl/downloads/software ). In principle this is feasible with a well equilibrated system and enough blank runs to perform double referencing (1,3).
An alternative is to do the equilibrium analysis, but due to the slow dissociation rate you need a long association time to reach equilibrium. This is generally not feasible with the injection volume available. D. Myszka (2) showed that you can add the analyte to the flow buffer and recirculate this until equilibrium is reached.
A second alternative is to use a different technique to determine the KD or the kinetics. You can think of ITC (for KD) of BLI which can provide long association and dissociation times without using much sample.
1. Katsamba, P. S., Navratilova, I., Calderon-Cacia, M., et al.; Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users. Analytical Biochemistry (352) 2: 208-221; 2006.
2. Myszka, D. G., Jonsen, M. D. and Graves, B. J.; Equilibrium analysis of high affinity interactions using BIACORE. Analytical Biochemistry (265) 2: 326-330; 1998.
3. Yang, D., Singh, A., Wu, H., et al.; Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms. Journal of Visualized Experiments : JoVE 122: 55659; 2017.
Regards
Arnoud
As you encountered, high-affinity interactions (especially slow dissociation rates: < 10-5) are not easy to solve in the Biacore. Generally prolonging the dissociation time is the easiest option to have enough decay in the response level needed to calculate the dissociation rate constant. The complex half-life for a kd of 1.10-5 is ~20 hours and the time to 5% decay ~ 90 minutes (See BiaCalculations ( www.sprpages.nl/downloads/software ). In principle this is feasible with a well equilibrated system and enough blank runs to perform double referencing (1,3).
An alternative is to do the equilibrium analysis, but due to the slow dissociation rate you need a long association time to reach equilibrium. This is generally not feasible with the injection volume available. D. Myszka (2) showed that you can add the analyte to the flow buffer and recirculate this until equilibrium is reached.
A second alternative is to use a different technique to determine the KD or the kinetics. You can think of ITC (for KD) of BLI which can provide long association and dissociation times without using much sample.
1. Katsamba, P. S., Navratilova, I., Calderon-Cacia, M., et al.; Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users. Analytical Biochemistry (352) 2: 208-221; 2006.
2. Myszka, D. G., Jonsen, M. D. and Graves, B. J.; Equilibrium analysis of high affinity interactions using BIACORE. Analytical Biochemistry (265) 2: 326-330; 1998.
3. Yang, D., Singh, A., Wu, H., et al.; Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms. Journal of Visualized Experiments : JoVE 122: 55659; 2017.
Regards
Arnoud
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- Priyanka
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6 years 3 months ago - 6 years 3 months ago #3
by Priyanka
Replied by Priyanka on topic high affinity interaction analysis
Thanks for your prompt reply...!!
I tried dissociation time of 1 hour.. But this did not help much and also I faced the issue of channel instability so it gave me high chi2 values.
I tried dissociation time of 1 hour.. But this did not help much and also I faced the issue of channel instability so it gave me high chi2 values.
Last edit: 6 years 3 months ago by Priyanka.
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