As more and more discoveries are made which lend understanding to the role of proteins in disease and health, quantification of these proteins in tissues and fluids becomes increasingly important and valuable. One approach that is desirable because of its simple, high-throughput and inexpensive nature, involves arraying antibodies on a 96-well plate in a multiplexed ELISA format (Quansys Array). This protocol allows reaction detection by imaging with a digital camera, which is often cheaper than the detection system utilized in other assay formats.
Alpha Innotech produces the Fluorchem Imager line, which offers multiple gel imaging systems. The product line is extensive and there is a camera to meet almost any need. Each of the systems in this line is comprised of a darkbox and cooled CCD camera, which is controlled by and outputs images to, a separate computer. The darkbox provides many features that are useful for gel imaging, such as a filter wheel and various illumination and excitation sources (eg. epi- and transillumination), all of which are controllable from the box or the computer.
Several imagers from this line have been tested for use in imaging the Quansys Array. There are both 12 and 16 bit cameras that can be chosen based on dynamic range requirements. The 12-bit camera actually outputs the data in 16-bits, such that the dynamic range appears to be equal to the 16-bit camera. For applications with very bright samples, the 16-bit camera may be a better choice as the larger bin capacity should reduce “bleeding”. For the micro-ELISA assay used in testing, the 12-bit cameras showed excellent dynamic range and acceptable bleeding. While the 16 bit camera is a slower full frame camera, the 12-bit camera is an interline CCD which offers real time focusing which is a definite plus if much adjustment will be made to the sample or camera.
The choices of resolution offered by the 12-bit cameras in the Fluorchem line include 2 Megapixel (Fluorchem) and 4 Megapixel (Fluorchem SP). The Fluorchem is a newly available model and so it was not tested. Its predecessor, the Fluorchem 8900, actually has slightly fewer pixels indicating that this resolution is sufficient for nine analyte arrays, while the Fluorchem SP was necessary for higher density arrays. The highest density tested was a twenty-five analyte array, for which the Fluorchem SP worked very well.
All of the cameras tested showed excellent sensitivity, which is important when assaying low concentration analytes. While the quantum efficiency of these cameras is lower than some of the competitors at the target wavelength, the sensitivity was sufficient to quantify picogram levels of analytes in the case of some cytokines. This is facilitated by longer exposures that are made possible because the cameras are cooled from –28 C to –46 C (depending on the camera).
The problem encountered with this system, when used as an array imager, is that the light falloff (vignetting) towards the outside of the plate is excessive. This greatly reduces the accuracy of a quantitative assay. With the help of Alpha Innotech, lenses were found which reduced the effect, but more work could be done to further improve the uniformity.
Adam Whitney
Technician
Antibody Array R&D
Spendlove Research Foundation
Utah, USA