# Development of Highly Multiplex Antigen Specificity Assays

> **NIH NIH U24** · JOHNS HOPKINS UNIVERSITY · 2020 · $271,389

## Abstract

PROJECT SUMMARY
 There is an increasing unmet need for immunoassays that yield a maximal amount of information from a
minimal amount of patient sample. One way to address this challenge is to develop very highly multiplexed,
sample sparing assays. This proposal presents new methods that integrate synthetic biology and next
generation DNA sequencing ("NGS") to characterize the specificities of humoral and cellular immune
responses using genetically encoded antigen libraries. The multi-PI project builds upon a solid foundation,
which has been established in recent years, for developing highly multiplex, sample sparing immunoassays.
The platforms described in this proposal will be developed and tested on a new antigen library that
encompasses the proteomes of all viruses known to infect humans ("the human virome"). Proof-of-concept
studies confirm both the quality of this library, and its utility for developing sample sparing, highly multiplex
antigen specificity assays. The following three Specific Aims have been developed to broadly analyze human
immune responses to viruses, using an absolute minimal amount of sample.
Specific Aim 1. Development of minimal human virome serologic assays
Two novel sample sparing serologic assays are proposed: 1) a 384-well, simplified bacteriophage-NGS based
assay, and 2) a rapid cytometric Luminex bead array assay. These technologies will comprehensively
characterize anti-viral antibodies at low cost, and require less than a single microliter of blood.
Specific Aim 2. Development of a minimal antigen library screening assay for cytotoxic T lymphocytes
A new platform for profiling CD8+ cytotoxic T lymphocyte (CTL) specificities has been devised. The system
employs lentiviral delivery of a genetically encoded antigen library to present MHC I-peptide complexes on
autologous antigen presenting cells, followed by phenotypic library enrichment and NGS analysis.
Specific Aim 3. Development of a minimal antigen library screening assay for T helper cells
A new platform for profiling CD4+ T helper (Th) cell specificities has been devised. The system employs
lentiviral delivery of a genetically encoded antigen library to present MHC II-peptide complexes on autologous
antigen presenting cells, followed by phenotypic library enrichment and NGS analysis.

## Key facts

- **NIH application ID:** 10145138
- **Project number:** 3U24AI118633-05S1
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** STEPHEN J ELLEDGE
- **Activity code:** U24 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $271,389
- **Award type:** 3
- **Project period:** 2020-06-10 → 2021-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10145138

## Citation

> US National Institutes of Health, RePORTER application 10145138, Development of Highly Multiplex Antigen Specificity Assays (3U24AI118633-05S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10145138. Licensed CC0.

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