Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

Abstract

Background: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification.

Results: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies.

Conclusion: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

Figure 1

Schematic layout of antibody microarray slide and RCA immunoassay. At the far left is an illustration of the 1" × 3" slide platform containing sixteen individual sample wells with an etched barcode. Within each of the wells, a 16 × 16 configuration of printed capture antibodies is arrayed. Each of the capture antibodies is capable of binding analytes from applied samples and undergoing RCA signal amplification. Finally, the fluorescently labeled signal, detected through conventional laser scanning, is quantified.

Figure 2

An example of raw data quality and outlier removal. (panel a, top) Raw data (37 analytes) from array 4 containing all sample replicates shown on an MvA plot (a typical microarray data plot of the log ratio vs. the log difference for each pair of intensities. See: Dudoit, S., Yang, Y. H, Callow, M. J., and Speed, T. P. (2002) Statistica Sinica 12, 111–140). The dashed lines indicate a 99% confidence interval around the data and outliers of this interval are shown in red, black or magenta. (panel b, bottom) Redacted data with 1% of outlier data removed (all points outside of the displayed confidence interval).

Figure 3

Pair wise scatter plots between three replicates of a sample. Each replicate was assayed on a different slide. Solid lines represent linear regression fits. Regression equation is indicated within legend box along with the individual slide barcodes for this particular assay. R² value of the fit is indicated in the title. Both X and Y-axes indicate mean fluorescence signal Log₂(MFI).

Figure 4

Effect of Normalization. The top panel reveals the raw data, shown as the Log₂(MFI), for the analyte Monokine induced by interferon gamma (MIG) across three replicates with 11 patient samples (numbered on the X-axis). The bottom panel reflects the impact of normalization in reducing variation in intensity within each sample and hence the replicate MFI CV.

Figure 5

Variance Decomposition. Example of a variance decomposition analysis performed on the analytes for array 1 in a client study. The X-axis corresponds to analyte name and y-axis corresponds variance. Red blocks reflect platform variation, while green and blue blocks represent inter-patient and inter-treatment variance respectively.

Figure 6

LLQ/ULQ. (Left) Plot of 3 replicate points from a 15-point titration series of IL-8. The LLQ is indicated by the green vertical line and the ULQ indicated by the rightmost black vertical line. The zero point was removed from the curve fitting procedure since the data undergoes a log transformation. The right panel reveals sample values that fell within and above dynamic range of assay. Here, the majority of tested points for IL-8 fell within the LLQ/ULQ dynamic range.

Figure 7

New array validation. Stress testing at 50X MDL analyte concentration. The pink line reveals the specific MFI signal for each analyte at 50X MDL in the presence of all detectors (n × n). The blue line shows the signal for each analyte under conditions where all analytes are added at 50X MDL along with all detector antibodies minus the cognate detector antibody (n × (n-1)) to reveal non-specific signal contributed by non-cognate detectors.