MILLIPLEX^® Cytokine Panels: Alternate Sample Types

Many studies indicate the relevance of immune factors in ocular disease, kidney disease, neonatal immunity, neurodegenerative disease, and periodontal disease, among others. Multiplex immunoassays are a valuable research tool used to broadly survey multiple analytes in a single sample to advance discoveries in various diseases. The MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A is used for simultaneous measurement of pg/mL levels of 48 human immune factors and is fully verified in serum, plasma, and cell/tissue culture supernatants. To demonstrate the utility of the MILLIPLEX^® multiplex cytokine panels in various sample types used across research fields, human tears, urine, milk, and saliva were evaluated. Additionally, to characterize neuroinflammation during progressive stages of neurological disease, cerebrospinal fluid (CSF) cytokines were measured in samples from mild cognitive impairment (MCI), Alzheimer’s Disease (AD), and healthy controls. This article highlights the utility of a MILLIPLEX^® human cytokine multiplex panel for use in multiple alternate sample types.

• Sample Preparation and Demographics
• Immunoassays and Data Analysis
• Analyte Detectability in Healthy Samples
• Analyte Concentrations in Healthy Samples
• Cytokine Analysis in Neurodegenerative Disease
• Summary
• Related Cytokine Multiplex Panels
• Request Information
• References

Sample Preparation and Demographics

Milk, tear, saliva, and urine samples were obtained from healthy donors from BioIVT, Westbury, NY. The milk and tear samples were immediately aliquoted and stored at -80 °C following donation. Urine and saliva were obtained and filtered (0.2 μm) before being aliquoted and stored at –80 °C. CSF samples were obtained from Discovery Life Sciences, Huntsville, AL, from patients with AD, MCI, and healthy individuals. For AD and healthy serum, the blood was allowed to clot for 30 minutes before centrifugation for 10 minutes at 1,000 x g. The serum was removed and either assayed immediately or aliquoted and stored at –80 °C. Frozen samples were thawed completely, vortexed, and centrifuged prior to use, to remove particulates. Samples were run neat. The donor information for each sample type is detailed in Table 1.

Immunoassays and Data Analysis

The multiplex immunoassay was performed as outlined in the protocol in 96-well plates using MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A. Serum samples were diluted 1:100 in assay buffer (L-AB) for testing RANTES concentration. All samples were tested neat, otherwise. The assay was read on a Luminex^® 200™ instrument and data was acquired via xPONENT^® v. 4.3 software. Data analysis was performed using the Belysa^® Immunoassay Curve Fitting Software. Figures were prepared in GraphPad Prism and Microsoft Excel.

Analyte Detectability in Healthy Samples

The human tear samples showed the highest number of detectable samples in the MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A, with 18 analytes showing 90–100% reading over the limit of detection (LOD) in the kit and 21 analytes with 80–89% detectability (Table 2). Human milk samples showed 18 analytes, saliva samples with 12 analytes, and CSF with 13 analytes at 80% or higher sample detectability. Urine samples were detectable in 80% or more samples for 6 analytes.

Analyte Concentrations in Healthy Samples

Analyte concentration varied depending on the sample type (Figure 1). All analytes in MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A were detectable in at least one of the samples. Some of the analytes had notably high concentrations in one or more of the sample types. Although none of the sample values exceeded the standard curve ranges, further testing may be required to find the appropriate sample dilution for samples to fall on the linear part of the standard curve.

Table 3 notes seven analytes where further sample dilution is recommended in the specified sample type.

Cytokine Analysis in Neurodegenerative Disease

Neuroinflammation may play a role in AD and other neurodegenerative diseases, highlighting the utility of broadly profiling cytokines in CSF samples. Distinctly different responses were observed for CSF samples obtained from individuals with AD, MCI, and healthy individuals (Figure 2A). Significantly elevated levels of IL-9 were noted in AD and MCI CSF compared to CSF from healthy individuals (p < 0.005). Additionally, MDC was elevated in MCI CSF compared to healthy donor CSF (p < 0.005). Likewise, noteworthy differences were detectable between serum samples from healthy donors and AD patients (Figure 2B). A significant increase in MCP‑1 (p < 0.005) in AD serum was observed, as well as decreased concentrations of sCD40L (p < 0.0005) and PDGF-AA (p < 0.005).

Summary

The data presented above highlights the capability of MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A for analysis of multiple biological fluids including urine, tears, milk, saliva, and CSF, in addition to the already extensively verified serum, plasma, and cell/tissue culture supernatants. Many scientific publications also underscore the advantages of multi-analyte profiling using MILLIPLEX^® kits.^1–6 For the purpose of this data, the biological fluids from healthy donors were tested neat. Researchers should determine the appropriate sample dilution for their sample type and disease state of interest.

For Research Use Only. Not For Use In Diagnostic Procedures.

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