A growing need to detect and quantify low abundance biomarkers is driving the development of novel immunoassay formats that promise improved sensitivity. To meet researchers’ demands to achieve this with only minimal sample usage, manufacturers of immunoassay kits, reagents, and instruments have had to be increasingly inventive. This article discusses established technologies to improve immunoassay sensitivity and introduces some more recent approaches.
Traditional colorimetric ELISAs that rely on substrates such as TMB have been a go-to immunoassay technology for decades. With good antibodies, these frequently achieve low pg/mL or even sub-pg/mL limits of detection (LOD), but some researchers choose to substitute a chemiluminescent substrate to further enhance sensitivity. “In our experience, compared to a colorimetric ELISA with the same antibodies, chemiluminescent detection provides 10 to 20-fold sensitivity enhancement in a best-case scenario,” reports Valerie Jones, Ph.D., director of sales and marketing at RayBiotech, Inc. “However, in some situations there may be no clear benefit, making it necessary to consider alternative approaches.”
One of these alternatives is electrochemiluminescent (ECL) detection. By converting electrical energy into light, ECL provides a broad dynamic range and low background, as exemplified by the well-known Meso Scale Discovery (MSD) technology. Based on electrochemical stimulation of a Sulfo-Tag label on the detection antibody, MSD ECL generates a measurable readout only when the tagged antibody is anchored to the microplate through antigen binding. This method has demonstrated a reduced sample requirement in comparison to traditional ELISA, making it popular among researchers working with only limited material. It is also extremely convenient, especially for those wishing to multiplex both high and low abundance targets within the same sample, yet a drawback of MSD ECL is that it requires a dedicated instrument and specialized consumables.
Immuno-PCR is a technology with no need for dedicated instrumentation, instead using a generic rtPCR thermal cycler to quantify an immobilized oligonucleotide. The immuno-PCR method is similar to an ELISA method wherein the enzyme-linked detection antibody is substituted with an antibody-oligonucleotide conjugate; this greatly reduces the LOD as a result of exponential amplification. The development of various enabling technologies has led to rapid uptake of immuno-PCR by researchers. These include user-friendly kits for conjugating antibodies to oligonucleotides, as well as products for straightforward conversion of a standard ELISA into an immuno-PCR format. RayBiotech’s IQELISA™ is a popular example of a ready-to-use immuno-PCR kit, requiring 10x less sample than a traditional sandwich ELISA and routinely providing 10-fold sensitivity improvement over the original assay. With high quality antibodies, the LOD can be reduced as much as 1,000-fold.
Like immuno-PCR, proximity ligation assay (PLA) is another immunoassay format that relies on antibody-oligonucleotide conjugates and an rtPCR readout to improve immunoassay sensitivity. In contrast to immuno-PCR, PLA uses two antibodies that have been raised against distinct epitopes on the same target molecule. When these are brought close together through target binding, the oligonucleotides ligate and undergo rolling circle amplification (RCA) to generate a quantifiable product. PLA is widely used to visualize individual proteins within cells or tissue samples on microscope slides, providing superior sensitivity to traditional ICC/IHC immunoassay formats. It also allows for processing of plated cells, making it suitable for high-throughput screening.
The sensitivities of protein-based detection methods have historically lagged behind those of nucleic acid-based technologies such as immuno-PCR and PLA. However, this issue is being addressed through the development of innovative single molecule assays such as Quanterix’ single-molecule arrays (Simoa®). Originally designed to detect sub-femtomolar concentration proteins in blood, Simoa traps antibody-coated beads in individual femtoliter-size wells to allow a precise, digital readout of each bead down to a single molecule level. Simoa is routinely 1,000x more sensitive than a conventional sandwich ELISA (often providing sub-fg/mL LOD), making it ideal for detection and quantification of biomarkers at concentrations that had previously been difficult or impossible to measure. To circumvent the need to purchase the costly analyzer instrument and consumables, RayBiotech offers a Simoa service for a wide range of target biomolecules.
Although the chosen immunoassay format will be largely dictated by research requirements, increased sensitivity is typically associated with increased cost. As such, tried and trusted immunoassay methods like ELISA, ICC, and IHC remain mainstream. But with more options available than ever before, researchers seeking enhanced sensitivity need not look far to deliver on their immunoassay objectives.
To learn more about RayBiotech’s immunoassay products and services, visit www.raybiotech.com.