Bioanalytical Community

This message was posted by a user wishing to remain anonymous

Hi all. I am developing a human MSD assay to detect anti-human pAbs (vaccine-induced) against a target. Unfortunately, I cannot find any high concentration (titer) human antisera against the proteins of the disease I'm targeting. I do have low concentration human antisera from NIBSC but it's not high enough to assess parallelism. I also would like to use this high-titer sera as a reference standard. 

I have a couple of questions:

1. One thought was to SULFO-Tag the coating antigen to generate a sandwich assay. Can anyone suggest any other assay formats?
2. I do have a mAb that I could use as a reference standard; however, there is a risk that it may not demonstrate parallelism once we assay in-study clinical samples. Does anyone have any other suggestions to make this reference standard?
3. Lastly, if the assay is validated and we later confirm that the reference standard does not demonstrate parallelism with the endogenous analyte, what are the paths forward to address this?

Thank you all in advance!

I am tagging @Shara Dellatore from the vaccine working group as this falls well within their scope.

In the absence of a guidance document, I can share some high-level starting points:
1) Whether monoclonal or polyclonal, there is no guarantee of parallelism with antibodies generate to a vaccine by any individual.
2) My understanding is that the reference standard evolves over time for ​vaccine assays and that starting with a monoclonal is probably okay. Bridging between reference standards is part of that process.

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Joleen White Ph.D.
AAPS 2023 Global Health Community Past Chair
Bioanalytical 101 Course Development
Head of Bioassay Development
Gates Medical Research Institute
Cambridge MA
[email protected]

Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
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Original Message:
Sent: 01-13-2023 12:57
From: Anonymous Member
Subject: In need of a human pAb reference standard

This message was posted by a user wishing to remain anonymous

Hi all. I am developing a human MSD assay to detect anti-human pAbs (vaccine-induced) against a target. Unfortunately, I cannot find any high concentration (titer) human antisera against the proteins of the disease I'm targeting. I do have low concentration human antisera from NIBSC but it's not high enough to assess parallelism. I also would like to use this high-titer sera as a reference standard. 

I have a couple of questions:

1. One thought was to SULFO-Tag the coating antigen to generate a sandwich assay. Can anyone suggest any other assay formats?
2. I do have a mAb that I could use as a reference standard; however, there is a risk that it may not demonstrate parallelism once we assay in-study clinical samples. Does anyone have any other suggestions to make this reference standard?
3. Lastly, if the assay is validated and we later confirm that the reference standard does not demonstrate parallelism with the endogenous analyte, what are the paths forward to address this?

Thank you all in advance!

Parallelism can be conducted using instudy samples and included in your sample analysis plan tied to your validation / qualification protocol.
If you are using an end point titer method, you could identify the samples with the highest titers and conduct a dilution series to ascertain parallelism, assuming that the treatment generates sufficiently high titers.
Curious though: 
Is the final method output quantitative or qualitative? i.e is the output based on relative to a reference polyclonal control or in form of arbitrary titer?


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Ritankar Majumdar
Senior Lead Scientist
Covance Inc. - Chantilly, Va
Chantilly VA
[email protected]

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

Original Message:
Sent: 01-13-2023 12:57
From: Anonymous Member
Subject: In need of a human pAb reference standard

This message was posted by a user wishing to remain anonymous

Hi all. I am developing a human MSD assay to detect anti-human pAbs (vaccine-induced) against a target. Unfortunately, I cannot find any high concentration (titer) human antisera against the proteins of the disease I'm targeting. I do have low concentration human antisera from NIBSC but it's not high enough to assess parallelism. I also would like to use this high-titer sera as a reference standard. 

I have a couple of questions:

1. One thought was to SULFO-Tag the coating antigen to generate a sandwich assay. Can anyone suggest any other assay formats?
2. I do have a mAb that I could use as a reference standard; however, there is a risk that it may not demonstrate parallelism once we assay in-study clinical samples. Does anyone have any other suggestions to make this reference standard?
3. Lastly, if the assay is validated and we later confirm that the reference standard does not demonstrate parallelism with the endogenous analyte, what are the paths forward to address this?

Thank you all in advance!