Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue

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Abstract

The ability to simultaneously quantify multiple signaling molecule protein levels from microscopic neural tissue samples would be of great benefit to deciphering how they affect brain function. This follows from evidence that indicates signaling molecules can be pleiotropic and can have complex interactive behavior that is regionally and cellularly heterogeneous. Multiplexed examination of tissue proteins has been exceedingly difficult because of the absence of available techniques. This void now has been removed by the commercial availability of bead-based immunoassays for targeted proteins that allow analyses of up to 100 (6–150 kDa) proteins from as little as 12 μl. Thus far used only for sera (human and mouse) and culture media, we demonstrate here that sensitive (as low as 2 pg/ml), wide-ranging (up to 2–32 000 pg/ml), accurate (8% intra-assay covariance) and reliable (4–7% inter-assay covariance) measurements can be made of nine exemplary cytokines (e.g., IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF-α) simultaneously not only from rat serum but, for the first time, also brain tissue. Furthermore, we describe animal handling procedures that minimize stress as determined by serum glucocorticoid levels since they can influence cytokine expression.

Introduction

Simultaneous measurement of multiple proteins can provide improved insight to brain function because of the inherent capacity for this experimental strategy to resolve complex interactions among signaling molecules (del Zoppo et al., 2000). Furthermore, application of such experimental strategies to brain should ideally be feasible on samples of exceedingly small size. This conclusion follows from the notion that brain shows marked structural and functional heterogeneity that in part may result from cell-specific (or region-specific) production, expression, and subsequent effects of various proteins including ligands and ligand receptors. Interleukin-1 (IL-1) function within brain is a prime example of such complex interaction and potential local specificity.

IL-1 is a prototypic inflammatory cytokine with widespread impact on neural function in health (Lynch, 2002, Schneider et al., 1998) and disease (Patel et al., 2003, Rothwell and Luheshi, 2000) that is produced both systemically (Dinarello, 1996) and centrally (Breder et al., 1988). The effects of IL-1 (including those in brain) are sometimes inferred from the behavior of related mRNA species (Dinarello, 1996) since cytokines are typically produced upon need (Oppenheim and Feldmann, 2001). However, newly synthesized IL-1β mRNA is not always transcribed to protein, making measurement of protein change more critical for deciphering function of this inflammatory mediator (Dinarello, 1996). Furthermore, IL-1 family cytokines consist of six principle, related family members, i.e., three receptor ligands (IL-1α, IL-1β and IL-1 receptor antagonist (IL-1Ra)), two receptor subtypes (IL-1RI and IL-1RII) and an accessory protein (IL-1AcP) (Dinarello, 1996). These IL-1 family inflammatory mediators show cell-specific patterns of production, expression and release since ligands are made principally by glia (and some neurons) while astrocytes and neurons express the signal transducing IL-1 receptor, IL-1RI (Ban et al., 1991, Ban et al., 1993, Fontana et al., 1982, Giulian et al., 1986, Lechan et al., 1990). In addition, neurons (and glia) can be differentially distributed between and within various brain regions (Dombrowski et al., 2001). Thus to most accurately characterize the net effects of IL-1 on brain function, not only should simultaneous measurements be made of six key IL-1 family cytokine proteins, but they should be made from sample volumes small enough to account for potential regional variations of the related variables.

Other evidences of the potential for complex interactions among signaling molecules in brain exist. For example, injury from brain disease can result in temporally and spatially distinct expression patterns of specific cytokines (Jankowsky and Patterson, 1999, Jean Harry et al., 2003), which can influence their own expression as well as that of counterparts (Oppenheim and Feldmann, 2001). Additionally, other classes of signaling molecules such as glucocorticoids (Dinkel et al., 2003, Goujon et al., 1997, John and Buckingham, 2003, Nadeau and Rivest, 2003) and prostaglandins (del Zoppo et al., 2000, Hori et al., 2000, Vane et al., 1998) can either reduce or promote inflammatory cytokine responses, respectively. Finally, similar scenarios of spatially and temporally distinct, yet interactive and thus complex, behavior can be expected for other intracellular, autocrine, paracrine and endocrine signaling molecule systems. However, because of the prior absence of easily available experimental techniques, few studies have centered on experimental strategies that included simultaneous measurements of multiple signaling molecules.

Recent availability of commercial instruments based on bead-based immunoassay technology (Dasso et al., 2002, de Jager et al., 2003, Dunbar et al., 2003, Earley et al., 2002, Kellar and Iannone, 2002, Kellar et al., 2001, Prabhakar et al., 2002, Vignali, 2000) is likely to help remove this void. For example, the Bio-Plex™ Suspension Array system (Bio-Rad; Hercules, CA) is an easy-to-use and flexible unit capable of simultaneously analyzing up to 100 (6–150 kDa) proteins from as little as 12 μl of sample, which has been done for analyses of serum (de Jager et al., 2003). The system is based on using spectrally addressed 5.5 μm diameter polystyrene beads that serve as the solid phase for this capture sandwich assay. For protein analyses, a monoclonal antibody directed against a desired analyte is covalently coupled to the dyed beads. The conjugated beads are then allowed to react with the sample containing an unknown amount of targeted protein (or a standard solution containing a known amount of the same protein—for calibration as well as positive controls). Next, a biotinylated antibody that is specific for the protein of interest is added to the reaction. This results in the formation of a sandwich of antibodies around the targeted protein. Then, the reaction mixture is detected by the addition of streptavidin–phycoerythrin, which binds to the biotinylated detection antibodies. Finally, the reaction mixture is read using specially designed hardware and software. To date however, measurements have only been reported for sera and culture media using bead-based immunoassays.

Here we present experimental strategies for animal preparation that minimize spurious activation of inflammatory cytokine expression from stress. Furthermore, we detail protocols for easy-to-use methods that confirm the utility of bead-based immunoassays to provide sensitive, accurate and reproducible measurement of nine exemplary cytokine proteins from rat serum. Importantly these methods, for the first time, are extended to similar measurements from brain tissue.

Section snippets

Materials and methods

Catalog numbers are given where specific products are essential.

Serum corticosterone

Corticosterone can influence expression of cytokines with brain (Dinkel et al., 2003, Goujon et al., 1997, John and Buckingham, 2003, Nadeau and Rivest, 2003) and so can be expected to be a sensitive and general reflection of animal status when serum and tissues are harvested for cytokine analyses. We initially used a standard strategy for processing brain tissue for immunohistochemical analyses of inflammatory markers (Caggiano and Kraig, 1996, Caggiano and Kraig, 1998, Caggiano et al., 1996).

Discussion

We demonstrate methods for simultaneous sensitive, accurate and reliable means for measurement of at least nine exemplary (e.g., cytokine) proteins from rat serum and brain tissue using bead-based immunoassay technology. Our results for serum indicate that seven of nine analytes (i.e., IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10 and TNF-α) showed a sensitivity and range of 2–4 to 16 000–32 000 pg/ml while IFN-γ and GM-CSF, respectively, showed a sensitivity and range of 2 to 4000–8000 pg/ml. Tissue

Conclusions

Bead-based assays can be highly versatile to specific experimental design. For example, a nine-cytokine, commercially available kit was chosen here to be exemplary of the capacity for multiplexed quantification proteins. However, evaluation of user-determined protein targets can be achieved by creating appropriate specific probes sets (de Jager et al., 2003) using commercially available bead coupling kits (e.g., Amine Coupling Kit (#171-406001) from Bio-Rad). Furthermore, bead-based assays can

Acknowledgements

This work was supported by grants from the National Institute of Neurological Disorders and Stroke (NS-19108 and NS-045923) as well grants from the American Heart Association (Bugher award to RPK and SDG to PEK). The authors would like to extend their gratitude to Jim Stejskal for his assistance and technical expertise with serum collection and corticosterone assays and Marcia Kraig for her continued assistance with this work.

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