Webinar Q&A follow up: Immunoassay signal amplification: bold new solutions for existing ELISAs

Increased sensitivity also means data may be misinterpreted as you might detect levels of biomarker that reflect physiological/baseline levels. If you increase the sensitivity using BOLD, does this also shift the upper limit of quantification (ULOQ) and, therefore, our ability to quantify real clinical values?

You are correct that increased sensitivity can lead to detection of concentrations that reflect physiological or baseline levels, which requires careful interpretation. When increasing sensitivity using BOLD, the ULOQ will most likely be lower, since a lower analyte concentration will generate a higher signal in the amplified assay. The exact shift in the ULOQ depends on the dynamic range and saturation characteristics of the instrument and detector; correspondingly, the enhanced sensitivity also results in a lower LLOQ.

The figure below shows an IL‑4 example (comparison of Standard vs Exazym® Human IL-4 ELISA) in which a standard assay and a BOLD‑amplified (Exazym®) assay are presented side by side, each using calibrators selected to match the sensitivity of the respective assay. As illustrated, the BOLD/Exazym‑amplified assay exhibits earlier signal saturation, resulting in a lower apparent ULOQ compared with the standard format.

Importantly, the increased sensitivity of the BOLD approach enables improved characterization of normal baseline levels and biological variability that may not be accessible using a standard immunoassay. If true clinical concentrations exceed the ULOQ of the BOLD‑amplified assay, the samples can be diluted to fall within the usable dynamic range of the calibration curve, allowing accurate quantification of clinically relevant concentrations.

Is there a reason why we should conjugate through click chemistry and not with an N-hydroxysuccinimide (NHS)-ester activated oligo?

We do use NHS chemistry initially to introduce azide groups on the antibody, but we avoid direct conjugation using NHS‑ester–activated oligos. The additional effort required for the ClickChemN approach is minimal, and we have found that dibenzocyclooctyl (DBCO)‑modified oligos are stable stock reagents, likely more stable than the hydrolysis‑sensitive NHS‑ester variant of the oligo‑dT primer. Based on our experience, these DBCO‑modified oligos exhibit long‑term stability of more than two years.

The use of click‑chemistry for conjugation gives good control over conjugation stoichiometry and results in high reproducibility and robustness. This strategy is well established and is also widely used in the development of ab-oligo conjugates, where controlled and reliable bioconjugation is critical.

What are the advantages of this technology compared to immuno-PCR (where the oligo on the detection antibody is amplified by PCR)?

Theoretically, immuno-PCR offers highly improved sensitivity, but in practice it’s often limited by contamination risk, and the need for repeated high temperature cycling. The structure of both the antibody and the antigen, if it is a peptide or protein, may be affected, resulting in a weaker antibody–antigen binding. The gain in sensitivity from the signal amplification can thus be counteracted by the decreased strength of the antibody–antigen binding.

BOLD avoids some of the shortcomings of iPCR by using low temperature isothermal, target‑dependent signal amplification, resulting in better signal‑to‑noise and more robust quantification. Most importantly, BOLD can be added to virtually any existing immunoassay or ELISA with only minimal modifications, making it a practical and flexible way to boost sensitivity without redesigning the entire assay workflow.

Can we apply this technique for PK determination? If so, what are the challenges associated with doing this?

Yes, BOLD can be applied to immunoassays for PK determination, and it is particularly useful when very low concentrations need to be quantified. Because BOLD can be added to an existing ELISA with only minor modifications, it fits well into established PK workflows. The main challenges are the same as for any highly sensitive PK assay: controlling background, ensuring specificity in complex matrices like serum or plasma. These challenges are typically addressed through assay optimization, appropriate calibration ranges, and sample dilution when/if needed, rather than being specific limitations of the BOLD technology itself.

Could you share if there are any challenges associated with preparing good critical reagents for the assay and maintaining them?

Answer: Overall, we have not experienced major challenges in preparing or maintaining critical reagents. All key components in the Exazym® kit system, including reagents used for antibody conjugation, polymerization, and signal detection are performance‑tested, and qualified by us. Their functionality is continuously monitored over time through stability and performance studies to ensure that customers receive reliable, well‑functioning products.

With respect to the Exazym® critical oligo‑dT detector conjugate, which is prepared by the user to link an existing ELISA to BOLD signal amplification, we recommend, depending on the stability of the detector antibody, adding an appropriate preservative and storing the conjugate in DNase/RNase‑free vials. When handled according to standard best practices, these reagents show good stability and are well suited for routine use.

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Disclaimer
The opinions expressed in this interview are those of the interviewee and do not necessarily reflect the views of BioTechniques or Taylor & Francis Group.

In association with Cavidi.