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.

A customizable multiplex protein microarray for antibody testing and its application for tick-borne and other infectious diseases

“…The microarray platform is advantageous over other existing gold standards for tick-borne diseases and was able to overcome several of their limitations. Average time for multitier testing for several tickborne pathogens could take several months whereas the microarray technology takes only about a day to perform [21]. The microarray detected 17 tick-borne and other infections along with Lyme disease with sensitivities and specificities listed in Table 3.…”

“…Wafers were functionalized as described previously [21,24] (https://tinyurl.com/mr9ctppy). Briefly, silicon wafers were exposed to an environment of pure oxygen for 2h followed by washing (deionized Water) and coating (1% (vol/vol) with 3-aminopropyltriethoxysilane (APTES) in N-methylpyrrolidone (NMP).…”

“…Serum samples were probed using 1:20 dilution on the pillar plate and incubated for 1h at room temperature followed by alternate washing and incubation as described previously [21].…”

“…It can detect a large number of antibodies against varied infectious agents at the same time. Detection of antibodies can be performed using low sample volumes with low cost and a fast turnaround time [21]. Given the flexible nature of the multiplex platform, we aimed to provide a multiplexed testing solution for Lyme, its co-infections, and other possible infections of interest.…”

“…The existing diagnostic assays possess various limitations that restrict their applicability in the diagnosis of these infections. The diagnosis of Lyme disease and other infections using several blot-based and single-plex ELISA tests remain rudimentary in terms of arriving at a diagnostic conclusion [21]. Additionally, blot-based assays may have overlapping proteins with similar mass requiring additional testing to tease out the specific antigen to which the antibody is bound.…”