The sensorgram changes as the analysis proceeds.

Hi, I am Biacore T200 user and I have one question.

I developed an analytical method using His capture kit.
To verify repeatability, repeatability test was performed to analyze repeatedly 30 times about same sample diultion series.

My prediction was that the RU value would show a similar sensorgram even if it fell a bit. However, it was found that not only does the RU value go up as the analysis progresses, but also the sensorgram, which was well saturated in the beginning, is analyzed in a shape that does not saturate as the analysis proceeds.

(Similar to the shape marked 'not saturation' in the sensorgram section on this homepage ( www.sprpages.nl/sensorgram-tutorial/a-sensorgram )
In my experiment result, there is no mass transport but not saturated.

I cannot understand this result, so I have difficulties about troubleshooting of this experiment.
Any help with knowledge would be greatly appreciated.

Thanks for reading.

Hi,

You say that during the experiment the sensorgrams shift from 'normal' to a curve that keeps on rising during analyte injection.
Did you monitor the regeneration between the cycles? Is the regeneration sufficient in that all the analyte is removed and baseline is restored to the start of the experiment? Did you do the experiment in one run? I could that the storage of the diluted analyte in the flow buffer in the vial is detrimental to the analyte conformation. Try adjusting the sample compartment temperature to 10 degrees. If you try fresh diluted analyte do you still have the problem?

Kind regards
Arnoud

Dear Arnoud.

Thanks for your reply.

I am using 'His capture kit' and I used regeneration condition & buffer in His capture kit. I used that condition & buffer to other analysis, so I think that regeneration is sufficient in my condition.

I tried many method and design to figure out the abnormality so I tried fresh diluted analyte, capture ligand and even fresh sensor chip, but there is no effective result.

The strangest thing is that after the above-mentioned phenomenon occurs in the first analysis, the next day, when a fresh sample is used for reanalysis, the sensorgram is analyzed in an abnormal state at first time (I think this means that the state of sensorgram abnormality on the previous day does not recover even with a fresh sample and capture ligand the next day)

You said flow buffer in the vial can affect analyte conformation, so I will do sample compartment temp to 10 degrees.

I would really appreciate it if you could provide any additional advice I can try more.

Thanks for your advice.

Best regards,