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Sandwich-Based Assay
Quantibody, like most arrays by RayBio®, utilizes a sandwich-based direct ELISA assay for detection. In this assay, capture antibodies are arrayed onto a glass support. The sample is then incubated with the array. Alexa Fluor 555/Streptavidin conjugated antibodies specific to the cytokines of interest are added and incubated with the array. From here, detection can be proceed through use of a laser scanner.
One standard glass slide is spotted with 16 wells of identical cytokine antibody arrays. Each antibody, together with the positive and negative control is arrayed in quadruplicate. One slide can thus be used for processing 16 samples. For cytokine quantification, the array specific cytokine standards are provided whose concentrations have been predetermined. The serial dilution of the cytokine standard will be assayed along side the samples to generate a sixpoint standard curve, leaving 10 wells for experimental samples.
Each item in the Quantibody product line has been tested to have similar or better detection sensitivity than traditional ELISA. Reproducibility and sensitivity is bolstered by use of a photobleaching resistant Alexa dye. Moreover, because we use an open format glass slide as our substrate, dozens or even hundreds of antibodies can be arrayed on the same array. Our custom quantibody array allows users to choose their own panels for cytokine detection. Raybiotech has tested and used the highest affinity antibodies possible on the market and each pair has been especially optimized for maximum sensitivity.
Sensitivity depends on the binding affinity of the antibody-antigen complex, which can vary for each cytokine. Raybiotech tests for the highest possible affinity, and each pair has been especially optimized for maximum sensitivity. Moreover, for the system itself, our Quantibody products have been tested to have similar or better detection sensitivity than traditional ELISA.
Quantibody arrays can accomodate many different type of samples: cell culture supernatant, serum, plasma, tissue lysate, and other body fluids. Typically, all that’s necessary is 50-100µl per sample for one array. The following parameters can be used for samples volume: 50-100µl of cell culture supernatant, 50-100μl of original or diluted serum or plasma; or 20-200µg of protein for cell lysates and tissue lysates. For those samples in which a high response for one cytokine is expected, while a low response is expected for another, we recommend testing each sample in stepwise dilutions.
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