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Thunder-Link® PLUS Conjugation System in a Multiplexed Study

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Introduction

RNA expression and protein sequencing assay (REAP-seq) is a tool that increases the throughput and simultaneous measurement of gene and protein expression levels in single cells with DNA-conjugated antibodies and droplet microfluidics. REAP-seq can be performed in single cells to identify and characterize novel or rare cell types, to provide insights into underlying mechanisms of cellular development, and to assess the response to therapeutic interventions.

In this study, the authors conjugated antibodies (normalized to 1mg/ml) to DNA barcodes (oligonucleotides of 65–66 bp)* using the Thunder-Link® PLUS Conjugation System, to target an average antibody / oligo ratio of 1:3. Using REAP-seq, the authors were able to quantify a range of proteins with 82 barcoded (conjugated) antibodies (AbBs) and >20,000 genes in a single workflow.

REAP-seq protein measurements in peripheral blood mononuclear cells (PBMCs) stained with a mixture of 45 AbBs, were compared with flow cytometry and showed good agreement. In a further comparison with flow cytometry, protein and mRNA expression of canonical markers for PBMCs (monocytes, B-cells, T-cells, and natural killer cells) assessed the specificity and sensitivity of REAP-seq protein assays and identified four major cell types (CD4+ and CD8+ T-cells, CD14+ monocytes, and CD20+ B-cells).

*Each oligonucleotide consisted of three parts: 1) 33-bp Nextera Read 1 sequence that was used as a primer for amplification and sequencing, 2) a unique 8-bp antibody barcode, and 3) 24- to 25-bp poly (dA) sequence that binds to the poly(dT) primer containing the cell barcode.

Results

In initial studies, the authors ‘benchmarked’ REAP-seq using peripheral blood mononuclear cells (PBMCs) stained with a mixture of 45 AbBs, followed by magnetic enrichment for CD3+ T-cells, CD19+ B-cells and CD11b+ myeloid cells. These PBMCS were subsequently colored with magnetic beads enriched for CD3+, CD19+ and CD11b+ and analysed by REAP-seq. This identified all three cell populations and provided a positive control for assessing the sensitivity and specificity of REAP-seq mRNA and protein measurements for canonical markers. Furthermore, to confirm that the protein assay has no effect on mRNA measurements, single cell RNA-seq alone was performed on PBMCs as a control.

The authors also compared REAP-seq proteomic measurements with flow cytometry and showed good agreement in the relative abundances of the four major cell types identified – namely CD4+ and CD8+ T-cells, CD14+ monocytes, and CD20+ B-cells.

AbBs for PBMC canonical markers (CD56, CD158e1, CD19, CD33, CD11b and CD155) also showed higher sensitivity at the protein level compared to mRNA, in keeping with the fact that protein levels cannot be directly inferred from mRNA levels, and that it is proteins –  not mRNA – that are the primary target for drugs.

Other Experiments Within This Study

The authors also used REAP-seq and a panel of 80 AbBs to assess the costimulatory effects of an agonist monoclonal antibody to CD27 (aCD27) on human CD8+ lymphocytes from three donors, as it has been shown to possess preclinical antitumor effects, and to identify and characterize an unknown cell type.

When only protein expression cluster data was considered, there was clear separation between cells from different treatment conditions, which was not seen with mRNA expression cluster data. Amongst several changes in protein and mRNA expression, REAP-seq was able to detect differential expression of unexpected genes that would not have been included in a pre-specified antibody panel for applications such as flow cytometry.

REAP-seq was also used to identify the unknown cell population. Across donors, 17 differentially expressed (but shared) proteins showed a pattern that was suggestive of common myeloid progenitors (CMPs).

Summary and Conclusion

The authors report on the outcomes of REAP-seq studies using Expedeon’s Thunder-Link® PLUS Conjugation System to directly conjugate monoclonal antibodies to DNA barcodes (AbBs,). By using Thunder-Link® PLUS Conjugation System the authors were able to conjugate using a quick and simple method, overcoming time-consuming and lengthy protocols associated with standard conjugation methods.

The AbBs were subsequently used in all REAP-seq studies reported. Preliminary REAP-seq studies in PBMCs, using a mixture of 45 conjugated AbBs, not only confirmed alignment with flow cytometry studies on the same cell population, but also that there was no effect on mRNA measurements. REAP-seq also identified changes in protein and mRNA expression across different aCD27 treatment conditions and was also able to identify a common myeloid progenitor, although further studies would be required to fully elucidate the exact cell type.

The REAP-seq approach minimizes steric hindrance and potential crosstalk by using unidirectional chemistry to create a stable, covalent bond between the antibody and aminated DNA barcode. Minimizing steric hindrance is an important feature to consider for increasing the throughput of this protein assay and in potentially extending this approach to incorporate intracellular labeling. The authors were also able to demonstrate the scalability of REAP-seq through the conjugation of up to 82 antibodies to unique DNA barcodes.

Use of Thunder-Link® PLUS Conjugation System to Facilitate the Simultaneous Measurement of Proteins and mRNAs in Single Cells.

In this study the authors relied on the Thunder-Link® PLUS Conjugation System to form stable antibody DNA barcode conjugations between 82 monoclonal antibodies (mAbs) and DNA barcodes for use in REAP-seq studies. The Thunder-Link® PLUS Conjugation System offers several advantages:

  • Quick and easy to use – Save time, no specialist knowledge required
  • High levels of antibody and oligo recovery – Save precious reagents
  • Use your own oligo and antibody, at your desired ratio – Flexible
  • Freeze dried – Ships at ambient temperature, long shelf-life
  • Stringently QC tested – Consistent high quality, excellent batch-to-batch reproducibility
  • Unidirectional chemistry – No risk of cross-linking
  • Covalent bond – Highly stable conjugates
  • Suitable for single-stranded oligos of 10–120 bases, double-stranded oligos up to 80 base pairs – Covers the majority of sequences
  • Linking chemistry works at both 5’ and 3’ end – Provides ability to combine with other modifications
  • Post-conjugation clean-up step – No interference from unbound oligos
  • Positive control antibody and oligo included – Enables confirmation of protocol success
  • A wide range of target proteins – Also applicable to antibody fragments and small proteins.

All the above are key for developing sensitive and specific antibody / oligonucleotide  conjugates for use in applications such as immuno-PCR, Proximity Ligation Assay and Electrochemical Proximity Assay.

Reference

Peterson, VM., Zhang, KX., Kumar, N., Wong, J., Li, L., Wilson, DC., Moore, R., McClanahan, TK., Sadekova, S., and Klappenbach, JA. Multiplexed quantification of proteins and transcripts in single cells. Nat Biotechnol. 2017 Oct;35(10):936–939. doi: 10.1038/nbt.3973. Epub 2017 Aug 30.