Searching for biomarkers: humoral response profiling with luciferase immunoprecipitation systems

Abstract

B-cell-mediated humoral responses are triggered in many human diseases, including autoimmune diseases, cancer, and neurologic and infectious diseases. However, the full exploitation of the information contained within a patient's antibody repertoire for diagnosis, monitoring and even disease prediction has been limited due to the poor diagnostic performance of many immunoassay formats. We have developed luciferase immunoprecipitation systems (LIPS) that harnesses light-emitting proteins to generate high-definition antibody profiles that are optimal for both diagnostics and biomarker discovery. Here, we describe the results and implications from a range of LIPS-antibody profiling studies performed in our laboratory. These include highly sensitive diagnostics for domestic and global pathogens, insights into infection-related diseases, discovery of new biomarkers for human diseases, subcategorization of symptoms and identification of pathogenic autoantibodies against self-proteins. These investigations highlight the types of humoral response profiles associated with different diseases, provide new information related to disease pathogenesis and offer a framework for incorporating LIPS antibody profiling into global health initiatives and disease monitoring.

Figure 1. Anti-K8.1 and anti-v-cyclin antibodies in uninfected controls and KS patients

Shown are antibody titer results from LIPS screening of 36 uninfected normal controls (CTRL) and 35 KS patients. Each symbol represents the average antibody titer from duplicate testing from an individual patient or control. The geometric mean antibody titer and 95% CI for anti-K8.1 lytic antibodies anti-v-cyclin latent antibody in light units (LU) are plotted on the Y-axis using a log₁₀ scale.

Figure 2. Autoantibody biomarker detection by LIPS

(A) Autoantibodies detected by LIPS often show a wide range of titers. Autoantibody titers are determined in a control normal volunteer (NV) group as a reference scale. Antibody titer values for each antigen-antibody measurement greater than the control mean plus 3 SD were color-coded to signify the relative number of standard deviations above these cut-off values. In some autoimmune diseases antibody responses ranging from the mean plus 3 standard deviations (SD) to over 2000 standard deviations greater than control subjects can be detected. (B) A heatmap of autoantibody responses in Sjögren's syndrome (SjS). The heatmap shows the heterogeneity of responses and antibody titers to the different antigens in selected SjS patients, whereby each row in the heatmap represents one patient. Of note, approximately 15% of the SjS cohort examined by LIPS did not show statistical response to any of the autoantigens tested [21].

Figure 3. Anti-cytokine autoantibodies detected by LIPS in thymoma patients with opportunistic infection correlate with immunodeficiency

(A) Heatmap analysis of anti-cytokine autoantibody profiles in thymoma patients. Anti-cytokine autoantibody titers to 5 informative cytokines are shown for each of five thymoma patients with and twelve thymoma patients without opportunistic infection (OI). Each row in the heatmap represents one patient from a total of 17 thymoma patients analyzed. The titer values greater than the mean of the 30 normal volunteers plus 3 standard deviations were color-coded from green to black to signify the relative number of standard deviations above these reference values. (B) Anti-IL-12 p35 autoantibodies correlate with OI. A decision tree algorithm using rapid miner (www.rapidminer.com) identified IL-12 p35 as the most informative anti-cytokine response for distinguishing thymoma patient with and without OI.