Evaluation of biological sample preparation for immunosignature-based diagnostics

Abstract

To address the need for a universal system to assess health status, we previously described a method termed "immunosignaturing" which splays the entire humoral antibody repertoire across a peptide microarray. Two important issues relative to the potential broad use of immunosignatures are sample preparation and stability. In the present study, we compared the immunosignatures developed from serum, plasma, saliva, and antibodies eluted from blood dried onto filter paper. We found that serum and plasma provide identical immunosignatures. Immunosignatures derived from dried blood also correlated well with those from nondried serum from the same individual. Immunosignatures derived from dried blood were capable of distinguishing naïve mice from those infected with influenza virus. Saliva was applied to the arrays, and the IgA immunosignature correlated strongly with that from dried blood. Finally, we demonstrate that dried blood retains immunosignature information even when exposed to high temperature. This work expands the potential diagnostic uses for immunosignatures. These features suggest that different forms of archival samples can be used for diagnosis development and that in prospective studies samples can be easily procured.

Fig 1

Comparison of serum and plasma from a single donor. Sequential blood draws were collected from a single donor and used to probe the array. Raw fluorescence intensities are plotted. To illustrate the relation of individual data points between the plasma and serum, the individual data points are colored on the basis of the plasma (vertical axis). The color used to plot data points represents the relation of the data point to the median intensity (yellow spots). Red represents features three times above the median value, and blue represents features three times below the median. Data plotted are from a single donor and are representative of two independent donor samples (data for the second sample are not shown).

Fig 2

Analysis of serum protein recovery and immunoglobulin reactivity observed in dried whole-blood specimens. (A) Total protein recovery from Whatman 903 protein saver cards. The total protein content of the 1:100-diluted human serum samples was measured with the NanoDrop spectrophotometer. Data presented are the average of four separate dried-blood spots from the same donor and are representative of recoveries from different donors (data not shown). (B) Scatter plot showing the correlation between the immunosignatures of the fresh mouse whole-blood sample (x axis) and dried mouse whole-blood sample on collection paper (y axis) (R² = 0.832) represented by raw fluorescence intensities (RFIs). The black dot indicates the immunizing peptide. The color used to plot data points represents the relation of the data point to the median intensity of the eluted dried-blood spot (yellow spots). Red represents features three times above the median value, and blue represents features three times below the median. Diagonal green lines represent the values that are 1.3-fold different between the two samples. Data presented are representative of three separate peptide-KLH conjugates.

Fig 3

Comparison of influenza virus-infected and naïve murine immunosignatures recovered from dried-blood spots. Whole blood from convalescent-phase mice that were either infected with influenza virus A/PR/8/34 or mock infected with PBS were collected on 903 protein saver cards. The resulting dried-blood spots were than processed in the immunosignature assay. The resulting immunosignatures are plotted in a volcano plot to illustrate the fold change difference between samples and the significance of the difference. Each array feature is plotted using its fold change between conditions (x axis, log₂ scale) against its P value significance (y axis, −log₁₀ scale). The vertical lines represent the minimal detectable fold change cutoff (1.3-fold), and the horizontal line represents the P value cutoff of 0.005. Black features meet both cutoffs, and the gray features do not meet the cutoffs. Data presented represent convalescent-phase sera from one infection experiment using a pool of blood from three mice under each condition.

Fig 4

Comparison of submandibular saliva to dried-blood spots for IgA (A) and IgG (B). Saliva samples were compared to dried-blood spots collected at the same time from a single human donor. Raw intensities are plotted, and color represents the median intensity, where yellow dots represent features at the median for the saliva samples. Saliva is plotted on the vertical axis, and the dried-blood spot is plotted on the x axis. Data plotted are from a single human donor and are representative of four individual donors. RFI, raw fluorescence intensity.