Assay Development

Real Time Immunoassays

With Axela’s technology, novel multiplex assays can be easily created. New protein biomarkers can be analyzed using one of our panelPlus™ Labeling Kits, and numerous assay conditions can be quickly evaluated thanks to the automated capabilities of our flow-through microarray and diffractive optics platforms.

The development of a new immunoassay often requires adjusting a large number of parameters including the choice of reagents and buffer conditions. Normally, these steps would require testing each parameter individually, which can be very tedious and time consuming. Real time diffractive optics can aid in rapidly developing optimized immunoassay conditions by providing real time information on binding interactions and by its ability to sequentially probe an analyte or reagent. In the example below, a capture antibody was sequentially probed for cross reactivity against 6 different reagents and buffers during an assay that was less than 90 minutes. (see Figure 1).

Figure 1. Sequential probing of capture antibody for cross-reactivity.

In order to translate protein biomarkers for future clinical applications, a quick and easy method is needed to evaluate the performance of these biomarkers when combined into a single assay. For instance, cross-reactivity between various reagents and analytes often complicates multiplex assay development. Fortunately, with real time diffractive optics analysis, potential cross reactivity can now be clearly detected during the early stages of assay development, as demonstrated below with a set of ovarian cancer biomarkers: CA125, free bhCG and AFP (see Figure 2).

Figure 2. Triplex ovarian cancer assay.

One of the challenges facing multiplex immunoassays is the detection of analytes present over an extreme range of concentrations without the need for sample dilutions to cluster analytes into narrower concentration ranges. Ideally, the assay has a large enough dynamic range to encompass all analytes in a single sample dilution regardless of their concentrations. With the Ziplex® System we can achieve over 4 logs units of concentration range in one dilution using chemiluminescence detection. With diffractive optics, a large number of signal amplification techniques can be used to increase the dynamic range to over 6 log units (see Figure 3). These include techniques various oxidation/precipitation reactions catalyzed by HRP conjugated detector antibodies, or antibodies conjugated gold nanoparticles.

Figure 3. A duplex assay of two cardiac markers: NT-proBNP and C-reactive protein (CRP) at 25 pg/mL and 25μg/mL, respectively, spanning 6-orders of magnitude in concentration using TMB oxidation.

Literature on Immunoassay Assay Development by Axela

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Flow Through Microarrays

The Ziplex® System has been shown to compare favorably to five commercially available gene expression analysis platforms while offering significant benefits from automation such as ease of use, fast hybridization times, as well as the ability to perform routine assays. In each case, the Ziplex produced expression signals that were either equal or better than those produced on global expression platforms in terms of reproducibility and sensitivity while significantly reducing both the time and complexity of the assays. This performance, along with the design of appropriate TipChip probes for well defined expression signatures, makes the Ziplex an ideal tool for the verification of differential gene expression results obtained from whole genome analysis. For a detailed comparison of the Ziplex System to other systems and an illustration of the migration of data from a global expression platform to Ziplex, download the references below.

Literature on Microarray Analysis Development by Axela

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