Sanne Hoeks

Cytokine assays 91 6 tertiary structure as these are vital in their interactive ability and biological activity. Protein array shelf life is relatively short due to protein denaturation and difficulties still exist in finding and isolating capture molecules for the wide range of proteins within the human genome. 52 A complex balance between quantifying amounts of bound protein, maintaining sensitivity and reducing background noise is difficult to obtain especially due to the low affinity or low specificity of capture agents. Most poignant however, is the inability of the chip to provide a complete view of the proteome with abundant proteins overpowering the detection of less abundant proteins whose levels are also key in therapeutic analysis. 53 High-performance liquid chromatography Unlike protein microarrays, high-performance liquid chromatography (HPLC) does not identify cytokines using protein–protein interactions but rather, as compounds with specific weights, hydrophobicity, protonating abilities, ligand affinity and ion exchange. 54 In short, HPLC uses the specific chemistry of each compound as a method of identification and separation. This method allows the quantification and purification of compounds by loading a sample onto a separation column containing solid particles under pressure. The sample is then separated into individual compounds according to their interaction with the column particles. The separation is in itself influenced by the liquid solvent condition and the chemical interactions between sample and solvent. HPLC has successfully been used to purify and separate cytokines such as IL-1 derived from various cells such as macrophage and epidermal cells. 54 However, even with the ability to achieve better separation than ordinary liquid chromatography, HPLC is a less than optimal method of analysis. Some disadvantages of this process include a high cost and complexity, the coelution of compounds with similar structure and polarity, 55 the irreversible absorption of compounds which then remain undetected and the low sensitivity of the apparatus to certain compounds as a result of the speed of the process. 56 Sandwich antibody assays It is known that several parameters must be met in order for a protocol to be optimal for cytokine biomarker discovery. As seen in bioassays, protein microarrays and HPLC, parameters are often either suboptimal or conflicting within an assay. Brining each of these factors close to or within optimal range will, however, give way to the perfect assay. There are two types of sandwich antibody assays, those that are plate-based and those that are bead-based. Plate-based assays such as Sandwich Enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery electrochemiluminescence (MSD) as well as bead-based assays such as multiplex immunoassays (MIA) are currently on the forefront of achieving the parameter goals required for cytokine biomarker discovery. In essence, these sandwich assays work with the principle of sandwiching a cytokine between two specific antibodies that intern bind to two none competing epitopes of that cytokine. 46 For bead based analysis, the antibodies are