Bioanalytical chemistry of cytokines--a review

Abstract

Cytokines are bioactive proteins produced by many different cells of the immune system. Due to their role in different inflammatory disease states and maintaining homeostasis, there is enormous clinical interest in the quantitation of cytokines. The typical standard methods for quantitation of cytokines are immunoassay-based techniques including enzyme-linked immusorbent assays (ELISA) and bead-based immunoassays read by either standard or modified flow cytometers. A review of recent developments in analytical methods for measurements of cytokine proteins is provided. This review briefly covers cytokine biology and the analysis challenges associated with measurement of these biomarker proteins for understanding both health and disease. New techniques applied to immunoassay-based assays are presented along with the uses of aptamers, electrochemistry, mass spectrometry, optical resonator-based methods. Methods used for elucidating the release of cytokines from single cells as well as in vivo collection methods are described.

Keywords: Aptamers; Cytokines; Immunoassay; Microfluidics; Optical resonators; Review.

Figure 1

An abbreviated version of the cytokine network highlighting the molecular communication between different cell types in the immune system.

Figure 2

Overview of cytokine properties, detection needs and the different methods that have been used to meet these detection challenges.

Figure 3

Overview of bead-based immunoassays. Different color-coded beads with dyes that fluoresce either red or green are used. The instrument measures the bead color intensity and the mean fluorescence intensity of the labeled detection antibody which is typically labeled with a streptavidin/phycoerythrin (PE) conjugate.

Figure 4

Overview of immunoassay improvements. Note that aequorin requires calcium activation to achieve its photo-protein state. Additionally, several steps are included in the electrochemiluminescence. These steps are outlined in the text and in other review articles describing these techniques.

Figure 5

Capillary electrophoresis with immunoaffinity capture. The example electrophorogram is reprinted with permission from: Detection of cerebral spinal fluid-associated chemokines in birth traumatized premature babies by chip-based immunoaffinity CE, T.M. Phillips and E. Wellner, Electrophoresis. 2013 34(11):1530–8. John Wiley & Sons. doi: 10.1002/elps.201200634.

Figure 6

Example of a programmable biochip. A) Capture antibody is immobilized to porous agarose. Detection antibodies are labeled with appropriate quantum dots to allow spectral discrimination. B) Agarose beads are driven into wells through a pressurized system. C) The design features of the chip are shown with the fluid lines. D) A modeling analysis of the overall pressures in the chip. Reprinted with permission from: JV Jokerst, J Chou, JP Camp, J Wong, A Lennart, AA Pollard, PN Floriano, N Christodoulides, GW Simmons, Y Zhou, MF Ali, JT McDevitt, Location of Biomarkers and Reagents within Agarose Beads of a Programmable Bionano-chip. Small. 2011, 7(5): 613–624. John Wiley & Sons, Inc. doi:10.1002/smll.201002089

Figure 7

Aptamer-based FRET assay for IFN-γ. Reprinted with permission from: N Tuleuova; CN Jones; J Yan; E Ramanculov; Y Yokobayashi; A Revzin; Anal. Chem. 2010, 82, 1851–1857. DOI: 10.1021/ac9025237 Copyright © 2010 American Chemical Society

Figure 8

Example of a multiplexed electrochemical-based immunoassay. The magnetic beads are coated with horseradish peroxidase (HRP) that reacts with hydrogen peroxide and hydroquinone (HQ) to give a product that is measured at −0.3V vs. Ag/AgCl. The bottom picture (B) shows the multiplexed channels. Reprinted with permission from: R Malhotra; V Patel; BV Chikkaveeraiah; BS Munge; SC Cheong; RB Zain; MT Abraham; DK Dey; JS Gutkind; JF Rusling; Anal. Chem. 2012, 84, 6249–6255. DOI: 10.1021/ac301392g Copyright © 2012 American Chemical Society

Figure 9

Schematic illustrating the Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) approach. The targeted peptides from the cytokines are listed in black. The peptide sequence denoted in red (GSGC) is added to allow for a more immunogenic response during antibody production. Polyclonal antibodies are retrieved and immobilized for affinity chromatography capture followed by RPLC-separation and MS/MS detection.

Figure 10

Example of whispering gallery mode. Glass beads that exhibit a resonance are immobilized with capture antibodies. As the beads come into resonance with the incident wavelength, the increase in intensity can be observed. Analyte addition to the beads causes this resonance wavelength to shift. The change in the wavelength is proportional to analyte concentration. The photograph of the beads in resonance are reprinted with permission from: HA Huckabay, SM Wildgen, RC Dunn, Label-free detection of ovarian cancer biomarkers using whispering gallery mode imaging. Biosensors and Bioelectronics, Volume 45, 2013, 223 – 229. © Elsevier. dx.doi.org/10.1016/j.bios.2013.01.072.