Tracking humoral responses using self assembling protein microarrays

Abstract

The humoral immune response is a highly specific and adaptive sensor for changes in the body's protein milieu, which responds to novel structures of both foreign and self antigens. Although immunoglobulins represent a major component of human serum and are vital to survival, little is known about the response specificity and determinants that govern the human immunome. Historically, antigen-specific humoral immunity has been investigated using individually-produced and purified target proteins, a labor-intensive process that has limited the number of antigens that have been studied. Here, we present the development of methods for applying self-assembling protein microarrays and a related method for producing 96-well formatted macroarrays for monitoring the humoral response at the proteome scale. Using plasmids encoding full-length cDNAs for over 850 human proteins and 1700 pathogen proteins, we demonstrate that these microarrays are highly sensitive, specific, reproducible, and can simultaneously measure immunity to thousands of proteins without a priori protein purification. Using this approach, we demonstrate the detection of humoral immunity to known and novel self-antigens, cancer antigens, autoimmune antigens, as well as pathogen-derived antigens. This represents a powerful and versatile tool for monitoring the immunome in health and disease.

Protein arrays for biomarker discovery in lupus

“…The NAPPA array is constructed by printing a plasmid that carries the cDNA of a GST-tagged protein, alongside an anti-GST capture antibody, resulting in a functional microarray that takes advantage of in situ protein production/capture via an in vitro expression system [33]. Regardless of the protein's type or size, NAPPA arrays have been found to express approximately 95% of their proteins, and have been used in the detection of autoantibodies to IA2 and GAD65 in type 1 diabetes, p53 in ovarian and breast cancer, BCL2 in prostate cancer, and ML-IAP in melanoma [33][34][35]. These recent successful biomarker detections in lupus and other pathologies, and from a variety of antigen array systems, indeed show both rapid progress and considerable room for technology transfer and further advances.…”

Use of a Single-Chain Antibody Library for Ovarian Cancer Biomarker Discovery

“…Target Identification Using NAPPA-To identify the targets that bind to the top performing 19 scFv, we incubated each on high density protein microarrays (35). Briefly, NAPPAs were activated by incubating with the transcription-translation-coupled rabbit reticulocyte lysate expression system (Promega Corp., Madison, WI) for 90 min at 30°C followed by a 30-min incubation at 15°C to allow binding of the GST-tagged proteins to their anti-GST capture antibodies.…”

“…scFv Target Identification-The 19 ovarian cancer-specific scFv that exhibited the greatest ability to distinguish cases from controls were incubated on a NAPPA (35,37) to identify their protein targets. These arrays encode over 7000 human proteins distributed over three arrays.…”

A Contra Capture Protein Array Platform for Studying Post-translationally Modified (PTM) Auto-antigenomes

“…ELISA-We adapted rapid ELISA (45) to allow citrullination of target antigen before assessing its sero-reactivity. HaloTag ligand coated 96 well plates (Promega) were preblocked with superblock (Thermo Scientific) overnight.…”