The ELISA (enzyme-linked immunosorbent assay) is a plate-based method used to detect and quantify a variety of materials such as proteins, peptides, and small molecules. In general, the assay involves immobilization of the target (either directly or through a ‘capture reagent’), detection with a specific antibody or aptamer molecule, and quantification using a variety of fluorescent or colorimetric reagents. There are multiple approaches that can be used to immobilise, detect and quantify the amount of material depending on availability of specific antibodies or aptamer reagents which bind to the materials being measured.

Because of limited antibody availability and size constraints for small molecules, a common approach is to use a competitive assay where purified, labelled and unlabelled sample material are combined and compete for detection and measurement. This competitive assay format measures a loss of signal as the target in the sample competes for binding with the fixed amount of labelled target in the kit. This assay format is therefore an indirect measurement can be subject to variable background and matrix effects, leading to inaccuracy and reproducibility issues.

To overcome these limitations Aptamer Group has developed specific aptamers and a simple gain of signal assay format for small molecule detection, that shows a target concentration dependant response. This assay has been used to quantify small molecules in the presence of common sample matrices, including plasma, milk and urine.  An added advantage of using aptamers is that they are composed of nucleic acids, so they are readily amplified by polymerase chain reaction (PCR). Aptamer Group has therefore used qPCR to detect the aptamers and again shown the gain of signal ELISA, in that assay format.

In this experiment Aptamer Group used an aptamer developed to the small molecule antibiotic Moxifloxacin.  Fluorescently labelled aptamer was immobilized on a suitable ELISA plate and was then incubated with a concentration gradient of Moxifloxacin, prepared in 25% Plasma or Milk.  The fluorescence of the recovered material was measured and plotted against target concentration for each of the different matrices.

Figure 1. Fluorescently labelled aptamer shows concentration dependant response to its target in a gain of signal ELISA-like assay format, in complex matrices such as 25% diluted Plasma or Milk.

The displaced aptamers were also measured by quantitative PCR (qPCR) and normalized to an internal standard.

Figure 2. Aptamer quantification by qPCR shows concentration dependant response to its target in a gain of signal ELISA-like assay format, in complex matrices such as 25% diluted Plasma or Milk.

Conclusion:

Aptamer selected to Moxifloxacin demonstrates a concentration dependent gain of signal response in an ELISA like assay measured by both fluorescence and qPCR.

The responses in the two assay formats are similar but not identical as the assays are affected differently by the different matrices. An internal reference (calibration curve) would be included in the end assay to normalize each matrix, to compare and correlate the results. This aptamer and others will be available soon as part of our validated catalogue.

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