The protein-detection landscape has seen a number of advances in the last decade. Much of our knowledge of proteins has come from using ELISA, Western immunoblot, immunoprecipitation, and two-hybrid systems. Of those advances, the ability to simultaneously detect multiple proteins in a single sample has greatly enhanced our understanding of some of the complex protein interactions that occur in normal and disease states. The manifold benefits of multiplex protein detection are increasingly being appreciated by researchers.
Validation of genomic data is a major area where rapid multiplex protein detection is needed. Many researchers are now familiar with instances where alterations in mRNA do not equate to changes in protein levels. Some protein levels are increased by mRNA stability rather than increased mRNA transcription.
There are examples where a treatment or disease state triggers endoplasmic reticulum stress, which results in the blockage of translation of many proteins that are not related to protein folding, independent of mRNA upregulation. Others include instances where differential splicing and post-translational modifications are required for protein activity rather than mere protein presence, such as phosphorylation of Akt, or cleavage of interleukin-1 and tumor necrosis factor from inactive to active proteins. Proteomic mapping and biomarker discovery are other areas enhanced by multiplex protein detection.
Researchers’ demand for the obvious advantages of multiplex protein detection has fostered the entrance of multiple technologies and platforms into the research marketplace. Protein-detection technologies can be differentiated by the direct use of antibodies (e.g., ELISA, reverse-phase arrays) and those that do not use antibodies (e.g., mass spectrometry).
The systems that utilize antibodies are less expensive, and therefore, have obtained greater market adoption. Most antibody-utilizing technologies are derivatives of ELISA. Many are sophisticated technologies, however the high cost and need for high-maintenance equipment has limited their use. Fortunately, there are lower cost solutions that offer as good, if not better, quality in multiplex protein detection.
Quansys Biosciences has developed a multiplex protein array technology, Q-Plex™ multiplex ELISA, that enables high-quality low-cost multiplex protein detection. Using modern liquid-dispensing systems that are capable of precisely dispensing nanoliter volumes of liquid, multiple proteins are spotted down into the well of standard 96-well plates.
For sandwich-formatted ELISAs, the proteins spotted down are capture antibodies. The capture antibodies are separated from each other by their location on a Cartesian coordinate system allowing multiple ELISAs (multiplex ELISA) to be performed in a single well. The sandwich ELISA is performed with two cocktails, an antigen standard and biotinylated secondary or detection antibodies. A luminescent signal generated by infrared (IR) fluorescence or chemiluminescence is imaged by either an IR scanner or CMOS or CCD camera with the luminous intensity being proportional to the amount of antigen bound by the antibodies.
The use of chemiluminescent or IR fluorescent based detection systems in the Q-Plex ELISA enables most laboratories to use readily accessible multifunctional equipment negating the need to purchase additional expensive single-function equipment. The images are then analyzed with software to quantify the pixel values and perform regression analysis giving actual quantitative values for the unknown samples.