Bioanalytical Community

Hello everyone,

I would like to start a discussion about analyte stability with high bias problem. In ligand binding assay, when an analyte is unstable most commonly we see a percentage of decrease when compared with sample at T0 or the theoretical concentration. I had two biomarker studies, when we evaluated the F/T short term stability, the stressed samples had high bias, which means that the samples that had gone throw multiple F/T cycles had higher concentrations or activity when compared with the unstressed samples. In this case how to properly address the stability in your validation report? If it's a stability issue then how come your stressed sample is more active?

Any input is appreciated!

For this, it is helpful to remember that for ligand binding assays, stability encompasses the three dimensional structure of the protein-protein interactions. If the F/T cycles disrupt the secondary, tertiary, or quaternary structure of the binding surface, you will see changes in the assay even if the molecule primary structure remains intact.

In this case, it may be that the binding surface of a disrupted structure has higher affinity than the binding surface of the unstressed sample.

Another potential cause is that what is unstable is some sort of interference in the assay so that removing that component increases the effective concentration.

Either way, my recommendation would be to strictly control the F/T and match cycles for your calibrators, QCs, and standards. Annoying, but scientifically appropriate.

Hello everyone,

I would like to start a discussion about analyte stability with high bias problem. In ligand binding assay, when an analyte is unstable most commonly we see a percentage of decrease when compared with sample at T0 or the theoretical concentration. I had two biomarker studies, when we evaluated the F/T short term stability, the stressed samples had high bias, which means that the samples that had gone throw multiple F/T cycles had higher concentrations or activity when compared with the unstressed samples. In this case how to properly address the stability in your validation report? If it's a stability issue then how come your stressed sample is more active?

Any input is appreciated!

For this, it is helpful to remember that for ligand binding assays, stability encompasses the three dimensional structure of the protein-protein interactions. If the F/T cycles disrupt the secondary, tertiary, or quaternary structure of the binding surface, you will see changes in the assay even if the molecule primary structure remains intact.

In this case, it may be that the binding surface of a disrupted structure has higher affinity than the binding surface of the unstressed sample.

Another potential cause is that what is unstable is some sort of interference in the assay so that removing that component increases the effective concentration.

Either way, my recommendation would be to strictly control the F/T and match cycles for your calibrators, QCs, and standards. Annoying, but scientifically appropriate.

Hello everyone,

I would like to start a discussion about analyte stability with high bias problem. In ligand binding assay, when an analyte is unstable most commonly we see a percentage of decrease when compared with sample at T0 or the theoretical concentration. I had two biomarker studies, when we evaluated the F/T short term stability, the stressed samples had high bias, which means that the samples that had gone throw multiple F/T cycles had higher concentrations or activity when compared with the unstressed samples. In this case how to properly address the stability in your validation report? If it's a stability issue then how come your stressed sample is more active?

Any input is appreciated!

For this, it is helpful to remember that for ligand binding assays, stability encompasses the three dimensional structure of the protein-protein interactions. If the F/T cycles disrupt the secondary, tertiary, or quaternary structure of the binding surface, you will see changes in the assay even if the molecule primary structure remains intact.

In this case, it may be that the binding surface of a disrupted structure has higher affinity than the binding surface of the unstressed sample.

Another potential cause is that what is unstable is some sort of interference in the assay so that removing that component increases the effective concentration.

Either way, my recommendation would be to strictly control the F/T and match cycles for your calibrators, QCs, and standards. Annoying, but scientifically appropriate.

Hello everyone,

I would like to start a discussion about analyte stability with high bias problem. In ligand binding assay, when an analyte is unstable most commonly we see a percentage of decrease when compared with sample at T0 or the theoretical concentration. I had two biomarker studies, when we evaluated the F/T short term stability, the stressed samples had high bias, which means that the samples that had gone throw multiple F/T cycles had higher concentrations or activity when compared with the unstressed samples. In this case how to properly address the stability in your validation report? If it's a stability issue then how come your stressed sample is more active?

Any input is appreciated!

Thanks so much Joleen! If it's a 3D structure or matrix interference caused problem, would it be preferable to develop an assay to get rid of these matrix/structure interference or is it generally acceptable as long as these issues being properly discussed and controlled?

For this, it is helpful to remember that for ligand binding assays, stability encompasses the three dimensional structure of the protein-protein interactions. If the F/T cycles disrupt the secondary, tertiary, or quaternary structure of the binding surface, you will see changes in the assay even if the molecule primary structure remains intact.

In this case, it may be that the binding surface of a disrupted structure has higher affinity than the binding surface of the unstressed sample.

Another potential cause is that what is unstable is some sort of interference in the assay so that removing that component increases the effective concentration.

Either way, my recommendation would be to strictly control the F/T and match cycles for your calibrators, QCs, and standards. Annoying, but scientifically appropriate.

Hello everyone,

I would like to start a discussion about analyte stability with high bias problem. In ligand binding assay, when an analyte is unstable most commonly we see a percentage of decrease when compared with sample at T0 or the theoretical concentration. I had two biomarker studies, when we evaluated the F/T short term stability, the stressed samples had high bias, which means that the samples that had gone throw multiple F/T cycles had higher concentrations or activity when compared with the unstressed samples. In this case how to properly address the stability in your validation report? If it's a stability issue then how come your stressed sample is more active?

Any input is appreciated!

Hello everyone,

I would like to start a discussion about analyte stability with high bias problem. In ligand binding assay, when an analyte is unstable most commonly we see a percentage of decrease when compared with sample at T0 or the theoretical concentration. I had two biomarker studies, when we evaluated the F/T short term stability, the stressed samples had high bias, which means that the samples that had gone throw multiple F/T cycles had higher concentrations or activity when compared with the unstressed samples. In this case how to properly address the stability in your validation report? If it's a stability issue then how come your stressed sample is more active?

Any input is appreciated!

Hello everyone,

I would like to start a discussion about analyte stability with high bias problem. In ligand binding assay, when an analyte is unstable most commonly we see a percentage of decrease when compared with sample at T0 or the theoretical concentration. I had two biomarker studies, when we evaluated the F/T short term stability, the stressed samples had high bias, which means that the samples that had gone throw multiple F/T cycles had higher concentrations or activity when compared with the unstressed samples. In this case how to properly address the stability in your validation report? If it's a stability issue then how come your stressed sample is more active?

Any input is appreciated!

You say it is a biomarker study, and I therefore assume this is related to stability of the biomarker. If you use spiked recombinant protein instead of endogenous biomarker you can sometimes get really odd results. It relates to as @Joleen White write to the disruption of the secondary, tertiary, or quaternary structure. 
For biomarkers it is therefore essential that you assess the stability of the endogenous biomarker level instead of the spiked recombinant protein. This will give you the true stability of the biomarker. If you are measuring the endogenous biomarker with no additional spike of recombinant protein, and you then see this increase , then your samples are only stabile till you reach this threshold. For many biomarker assay the biomarker is only stable after one F/T cycle, and you only have one shot to analyse the aliquot. Therefore it is also crucial to have several aliquots prepared at the clinical site.  

All the best

Marianne

------------------------------
Marianne Fjording
CEO
Biolyzr

[email protected]

Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.

Original Message:
Sent: 03-02-2023 14:17
From: Anonymous Member
Subject: Analyte stability high bias

This message was posted by a user wishing to remain anonymous

Hello everyone,

I would like to start a discussion about analyte stability with high bias problem. In ligand binding assay, when an analyte is unstable most commonly we see a percentage of decrease when compared with sample at T0 or the theoretical concentration. I had two biomarker studies, when we evaluated the F/T short term stability, the stressed samples had high bias, which means that the samples that had gone throw multiple F/T cycles had higher concentrations or activity when compared with the unstressed samples. In this case how to properly address the stability in your validation report? If it's a stability issue then how come your stressed sample is more active?

Any input is appreciated!

You say it is a biomarker study, and I therefore assume this is related to stability of the biomarker. If you use spiked recombinant protein instead of endogenous biomarker you can sometimes get really odd results. It relates to as @Joleen White write to the disruption of the secondary, tertiary, or quaternary structure. 
For biomarkers it is therefore essential that you assess the stability of the endogenous biomarker level instead of the spiked recombinant protein. This will give you the true stability of the biomarker. If you are measuring the endogenous biomarker with no additional spike of recombinant protein, and you then see this increase , then your samples are only stabile till you reach this threshold. For many biomarker assay the biomarker is only stable after one F/T cycle, and you only have one shot to analyse the aliquot. Therefore it is also crucial to have several aliquots prepared at the clinical site.  

All the best

Marianne

Hello everyone,

I would like to start a discussion about analyte stability with high bias problem. In ligand binding assay, when an analyte is unstable most commonly we see a percentage of decrease when compared with sample at T0 or the theoretical concentration. I had two biomarker studies, when we evaluated the F/T short term stability, the stressed samples had high bias, which means that the samples that had gone throw multiple F/T cycles had higher concentrations or activity when compared with the unstressed samples. In this case how to properly address the stability in your validation report? If it's a stability issue then how come your stressed sample is more active?

Any input is appreciated!

Hello everyone,

I would like to start a discussion about analyte stability with high bias problem. In ligand binding assay, when an analyte is unstable most commonly we see a percentage of decrease when compared with sample at T0 or the theoretical concentration. I had two biomarker studies, when we evaluated the F/T short term stability, the stressed samples had high bias, which means that the samples that had gone throw multiple F/T cycles had higher concentrations or activity when compared with the unstressed samples. In this case how to properly address the stability in your validation report? If it's a stability issue then how come your stressed sample is more active?

Any input is appreciated!