ELISA Assay for Bioanalysis of Oligonucleotides and Other Macromolecules

Oligonucleotide therapeutic bioanalysis is a continually evolving phenomenon that drives different aspects of the drug discovery and development process. Every method has been employed across other study purposes over time. To support both the clinical and pre-clinical studies, reliable data is generated by incorporating several critical parameters such as assay ruggedness, assay robustness, specificity, sensitivity, etc.

What is ELISA?

ELISA or Enzyme-linked immunosorbent assay is a plate-dependent technique explicitly designed to detect and quantify soluble substances such as proteins, peptides, hormones, and other forms of antibodies. The antigen is immobilized onto a solid surface in such techniques, followed by complexing it with an antibody linked to a reporter enzyme. The detection is accomplished through the activity measurement via incubating it and a substrate for generating a measurable product. One of the vital crucial elements for ELISA bioanalysis is its particular antigen-antibody reaction.

Although several variants of ELISA are available in the market, they all are based on some general elements –

Coating or Capture –

It involves direct or indirect antigenic immobilization onto the polystyrene microplate wall surface.

Plate blocking –

Addition of any irrelevant proteins or other related molecules to cover all the remaining unsaturated surface-binding sites on to the top surface of the microtiter wells.

Probing or detection –

This stage involves incubating the coated microtiter well with the antigen-specific antibodies bound with the antigens according to their antigenic specificity.

Signal detection or measurement –

The detection of the generated signal either through the direct or secondary tag on to the specific antibody involved.

Most appropriately involved enzyme labels are HRP/horse-radish peroxidases and AP/alkaline phosphatases. Other enzymes are inclusive of beta-galactosidases, catalases, and acetylcholinesterases. An extensive selection range is also available for performing ELISA either with an AP or HRP conjugate. Substrate selection depends on the sensitivity and instrumentation for detecting signals such as fluorometer, spectrophotometer, and luminometer. 

Hybridization ELISA, alternatively known as ELISA immunoassay, promises both the assay sensitivity and throughout in comparison to other similar methods. Such assays usually employ target oligonucleotide hybridization with a capture probe called immobilization and a detection probe called signalling. Thus, there is little to no sample clean-up requirement.

However, the assay offers a narrow range of calibration, usually lying between 20 to 50-fold, with its robustness heavily residing on the reagent quantification. Thus, the quantification of both the sensitivity and specificity of assay relies on selecting methods and reagents, along with the structure of the targeted oligonucleotide.

The LLOQ can lie as low as 100 pg per ml in plasma in case of a sandwiched oligonucleotide assay. However, the target oligonucleotide must be long enough to allow both the capture probe and the detection probe to bind stably to different molecular parts. Yet, this bioequivalence method usually falls short in distinguishing between the large metabolites and the target oligonucleotides.

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