How Does Ultra-sensitive Protein Detection Work via Elisa Assay?

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One of the problems with an early-stage cancer diagnosis is the fact that the current techniques of detection and screening are not quite sensitive or accurate enough to give proper results. This is why a new non-invasive but blood-based detection system is extremely necessary for continuously monitoring the growth of tumors.

Since the current blood tests and imaging techniques are proving to be inadequate, scientists are looking into ultrasensitive ELISA assays for the measurement of some proteins, such as the increasing levels of PSA (Prostate-Specific Antigen) over a specific amount of time in the murine system. This is the antigen that is held responsible for the development of tumors. Single-Molecule Arrays (SiMoA) are utilized in these ultrasensitive ELISA assays.

ELISA assays are quantitative plate-based assays used to detect substances like proteins, hormones, antibodies, peptides, and small molecular drugs. Since these assays are ultrasensitive, they are also used to quantify the amount of each of these substances. ELISA assays have been in use since 1971.

This ELISA method has a very high sensitivity level. You can measure 1pg/ml of particular proteins even when they are present in a complicated matrix like a serum. Antibodies determine the sensitivity and specificity of the assays. Sometimes, polyclonal antibodies are used to increase the sensitivity of the assay because they have a higher affinity.

How Does an ELISA Assay Work?

What an ELISA assay does is that it immobilizes the suspected antigen or an antibody upon a flat plate surface with a 96-well format. Proteins (like bovine serum albumin) are used to block the non-specific sites on the plate. Then, the antigen has to be taken through the incubation process to capture the antibody. The antigen is then captured by the already immobilized antibody.

At this point, a particular primary antibody is bound to the analyte. If the primary antibody is not linked to an enzyme, a secondary antibody linked to an enzyme is utilized to attach the primary antibody. The substrate that will be developed subsequently will create a measurable signal.

These are the choices that you have to make while doing an ELISA assay: a choice of binding reagents, an assay diluent that is used to reduce nonspecific effects of the matrix, a coating method, and a readout detection method. The last one is ideal for all kinds of tissue homogenates and body fluids.

Various Applications of Ultrasensitive ELISA Assays:

  • Sandwich Method
  • Competitive or Direct Measure
  • Detection of proteins bound by membranes
  • Detection of intracellular antigens presents within adherent cells.
  • ELISpot is used to detect a cytokine signal from only one cell.

Several labs have been working on producing ultrasensitive assays to detect various protein biomarkers. The most viable experiments have come up with options of electrochemical microfluidic arrays, novel fluorescence systems, which are laser-induced (these are used for the detection of cancer biomarkers), and also electrochemical immunosensors that make use of gold nanoparticles.

The gold nanoparticles are made functional using magnetic multi-walled carbon. The problem with these new techniques is that many of them require extremely complicated setups for the assay. For some, the preparation is too lengthy, or the experiments are prone to sensor fouling.

Single Molecular Arrays have previously been used to monitor the recurrence of cancer (especially prostate cancer) after radical prostatectomy. It is also used to study the efficacy of treatment in the case of Crohn’s disease by studying the presence and quantity of interleukin 6 (also called IL-6) and tumor necrosis factor-alpha (also called TNF-α).

SiMoA is a method that has already been commercialized. It is also extremely straightforward, so it can be performed in a few hours and requires a minimal level of preparation. Unlike the other experimental techniques mentioned above, you can perform SiMoA with a fully automated instrument. This is exactly what makes SiMoA the most suited option for clinical usage.

During one of the studies, the measured quantities of Prostate-Specific Antigen were much below the normal detectable limits of a PSA ELISA assay obtained from a clinical diagnostic center and a commercially available PSA assay.

As a result, SiMoA was used in this situation because it was the only way to detect the previously negligible amount of protein biomarkers in the serum. This helped in the early detection of cancer. This method also offered a viable alternative to the usual commercial models to monitor the early stages of cancer. Additionally, this method is non-invasive as well.

Various Kinds of ELISA Assays That Can Be Performed at Laboratories:

  • Protein phosphorylation
  • Multiplex Assays- in which you can measure multiple analytes present within the same well.
  • RadioImmune Assay
  • Competitive ELISA to detect small molecules
  • Cell-Based Assays

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