An ultra-high-density protein microarray for high throughput single-tier serological detection of Lyme disease

Abstract

Current serological immunoassays have inherent limitations for certain infectious diseases such as Lyme disease, a bacterial infection caused by Borrelia burgdorferi in North America. Here we report a novel method of manufacturing high-density multiplexed protein microarrays with the capacity to detect low levels of antibodies accurately from small blood volumes in a fully automated system. A panel of multiple serological markers for Lyme disease are measured using a protein microarray system, Lyme Immunochip, in a single step but interpreted adhering to the standard two-tiered testing algorithm (enzyme immunoassay followed by Western blot). Furthermore, an enhanced IgM assay was supplemented to improve the test's detection sensitivity for early Lyme disease. With a training cohort (n = 40) and a blinded validation cohort (n = 90) acquired from CDC, the Lyme Immunochip identified a higher proportion of Lyme disease patients than the two-tiered testing (82.4% vs 70.6% in the training set, 66.7% vs 60.0% in the validation set, respectively). Additionally, the Immunochip improved sensitivity to 100% while having a lower specificity of 95.2% using a set of investigational antigens which are being further evaluated with a large cohort of blinded samples from the CDC and Columbia University. This universal microarray platform provides an unprecedented opportunity to resolve a broad range of issues with diagnostic tests, including multiplexing, workflow simplicity, and reduced turnaround time and cost.

Figure 1

Microarray platform architecture. (A) The silicon wafer substrate is (B) functionalized with a copolymer composite of poly(lactic acid) and poly(L-lysine) and (C) subsequently immobilized with protein probes. OspC (PDB ID: 1GGQ) is shown as an example. (D) Each probe-immobilized wafer substrate is diced into microchips using a stealth dicing process. (E) The diced microchips are picked and placed onto carrier tapes using a standard die sorting system. (F) The carrier tapes are loaded onto a high throughput surface mount technology (SMT) component placement system. (G) Individual microchips are picked and placed onto 24-pillar plates with each pillar containing a layout of 44 microchips.

Figure 2

(A) Standard two-tiered testing algorithm recommended by CDC. (B) Vibrant Lyme Immunochip testing algorithm. (C) Different bands on a typical Western blot / Immunoblot are tested one protein per microchip using multiple microchips per pillar.

Figure 3

Heat map of antibody-binding intensities in the training and validation sets of clinical samples. A positivity cutoff for each marker was defined as greater than 10 chemiluminescent units (yellow or red). Samples additionally captured in the Enhanced IgM assay have been compared side by side in the supplemental material. All three assays detect antibodies to the markers presented on the right y-axis of the heat map. These markers are not repeatedly labeled for the Standard IgM Assay and Standard IgG Assay. Chemiluminescent units for each sample are also shown in the Supplemental Data File.