Direct Peptide Reactivity Assay (DPRA)

[MikeSimone]

Any consumer product testing people out there? Is anyone aware of the DPRA , DB-ALM Protocol no 154?

From my point of view as an analytical chemist, this protocol has some flaws. I am wondering if it is really in widespread use, has it been superseded by something which addresses the flaws?

Thanks, any insights are appreciated.

Mike

[bunnahabhain]

I am struggling with this test. The first thing I did was to replace the Agilent column with an ACE SuperC18 150x3 mm => much better peak shape. The positive control always falls out of range (too high reactivity and/or precipitation), the incubation conditions (especially times) are ridiculous, ...
What is your main point of criticism?

[MikeSimone]

Where do I start?

It really isn't quantitative, with degradation of the peptide standard material, the 0.5mM standard is roughly half that original concentration at the end of the incubation period before calibration. It's like a moving target calibration.

As written, it is difficult to verify the calibration against a second source material in the traditional sense.

We have issues with intermittent peak splitting on a Zorbax 4.6x50mm 1.8um column.

Certain lots of ACN seem to contribute to rapid degradation of the peptide, no one seems to understand what exactly causes this.

[bunnahabhain]

Where do I start?

It really isn't quantitative, with degradation of the peptide standard material, the 0.5mM standard is roughly half that original concentration at the end of the incubation period before calibration. It's like a moving target calibration.

Is this related to the acetonitrile batch you are using? (see also below)

As written, it is difficult to verify the calibration against a second source material in the traditional sense.

IMHO this is not necessary. As long as you have a purity certificate of the peptide, you compare the samples from a single stock solution, so the results should be consistent.

We have issues with intermittent peak splitting on a Zorbax 4.6x50mm 1.8um column.

I never liked the Zorbax columns. Go ahead and try the ACE column or Luna C18(2). Both are much better and ACE is the cheaper one. Easy enough to show comparative chromatograms of both columns to justify the selection.

Certain lots of ACN seem to contribute to rapid degradation of the peptide, no one seems to understand what exactly causes this.

This issue has already been addressed in the DB-ALM protocol No 154 (https://ecvam-dbalm.jrc.ec.europa.eu/me ... /key/t_41#)
I also don't know why this should happen, but at least it is a known issue and we know we are not alone...

The major problem I have with the principle of this test is, that the design is dependent on the molecular mass of the test substance. As for most other in vitro tests, also the DPRA should be based on a mass/volume concentration, the molar ratio is irrelevant as soon as you get in touch with the chemical in real life.