Bioanalytical Community

Hi all,

I am reaching out to gather insights from those of you who have experience working with plate based neutralizing antibody (NAb) assays. Specifically, I am interested in understanding what you consider to be the most effective positive control for these assays (mAb vs pAb).

Could you please share your preferences and the reasoning behind your choices? 

Thank you in advance for your valuable input!

Best Regards,

Soumya

Hi Soumya,

I don't think there is a clear preference for mAbs over polyclonals or vice versa. Monoclonal antibodies used in the PK assay are typically neutralizing and could be repurposed for use in the nAb assay. Polyclonal antibody (if purified well on affinity column) is also likely to have strong neutralizing activity. Monoclonals are more prone to hook effect, but I don't expect to see a hook in a nAb assay. If you use a high affinity mAb, there may be a concern that it may not be representative of the human immune response. However, if Kd is > 10 pM, you should be fine (see Joyce et al, 2022 Determination of Anti-drug Antibody Affinity in Clinical Study Samples Provides a Tool for Evaluation of Immune Response Maturation - PubMed).

I would pick a control that is best behaved in your assay and with sufficient amount to support the study. At the end of the day, whether you select a polyclonal or a monoclonal antibody, it's just a positive control, and not the actual analyte.

Best regards,

Robert

I agree with John on the rationale to prefer mAb. That said, sometimes it is hard to get a good neutralizing PC from monoclonals. In that case, I tend to report sensitivity of ≤LLOD from polyclonal.

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Joleen White Ph.D.
AAPS 2024 Global Health Community Chair
Bioanalytical 101 Course Development
Senior Bioassay Development Lead
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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Hi Soumya,

Both pAb and mAb are suitable for NAb assays if they have adequate neutralizing capabilities (assay sensitivity). For long-term studies, maintaining consistency for a pAb may be challenging, so a combination of pAb and mAb may be considered in validation, with mAb for ongoing assay monitoring during sample analysis.

Best,

Sanjay