BD^® AbSeq Immune Discovery Panel

Similar performance between the IDP versus freshly mixed BD^® AbSeq Antibodies. Following isolation from whole blood, peripheral blood mononuclear cells (PBMC) were split into resting (untreated) and activated (treated with CD3/CD28 for 24 hours) groups.

A 1:1 mixture of the resting and activated cells were stained with either the IDP or a freshly prepared mixture of the same AbSeq specificities. Equal number of cells were then loaded onto the BD Rhapsody™ Cartridges and AbSeq and WTA libraries were generated and sequenced (n = 2 individual experiments for this study). Data were analyzed using SeqGeq™ Software and Dataview Software.

A. UMAP demonstrated strong overlap in the cell groups identified between the IDP and fresh BD^® AbSeq Antibody-stained samples.

B. The total number of AbSeq molecules detected by the IDP and fresh BD^® AbSeq Antibody mixture showed a high correlation with R² greater than 0.98.

Performance of all 30 AbSeq specificities included in the IDP. PBMCs were activated and prepared as described in the figure above. After staining, cells were captured on the BD Rhapsody™ System and AbSeq and WTA libraries were generated and sequenced. To obtain over 80% sequencing saturation, the libraries were sequenced at 20,000 reads/cell for WTA and 30,000 reads/cell for AbSeq using the Illumina™ NextSeq™ High-Output Kit. Data were analyzed using SeqGeq™ Software and Dataview Software. We repeated the above experiments with at least two different donors. The representative figures from one donor are shown here.

A. Cell annotation of UMAP of resting + activated PBMCs resolved by the IDP antibodies and the WTA mRNA profile.

B. Heat maps of each AbSeq clone from IDP on UMAP from (A) showing the specificity of AbSeq detection for individual cell type.

The IDP is a flexible backbone panel and accommodates additional AbSeq specificities. Three BD® AbSeq Antibodies were added and mixed with the reconstituted IDP pellet (n = 2).

A. The IDP performance was not impacted by drop-ins as shown by high correlation (R² over 0.99 with or without drop-ins).

B. The IDP specificities of CD8[SK1] and CD45RA (top row) were assessed against the specificity of drop-ins CD8 [RPA-T8], CD45RO and CD38 (bottom row) and are shown in UMAP. Drop-in for CD38 detected cell types that are expected to be positive for CD38. Drop-in clone for CD8 (RPA-T8) showed a staining pattern very similar to the IDP clone (SK1) suggesting the high specificity of drop-in antibody as well as the compatibility of two clones for the same antigen. The contrasting expression pattern of CD45RO (drop-in) compared to CD45RA (IDP) further confirmed that adding the AbSeq specificities to the IDP had no adverse impact on experimental outcomes.

The IDP is designed to work with RNA and multiplexing assays

A. WTA and AbSeq libraries from IDP-stained cells (1:1 mixture of resting and activated PBMCs) were generated and sequenced. To illustrate the power of multiomic analysis, we analyzed the WTA data only (mRNA analysis) and compared with WTA + AbSeq data (mRNA and protein analysis). UMAP coordinates and unbiased clustering (Phenograph) using only WTA (mRNA) data are shown on the left, while coordinates and annotations using WTA + AbSeq (mRNA and protein) data are shown on the right. With a multiomics approach, additional cell types were revealed offering deeper biological insights.

B. To test the compatibility of the BD^® Single-Cell Multiplexing Kit (SMK) and the IDP, we performed cell staining with the SMK and the IDP together and generated WTA, AbSeq and SMK libraries for sequencing. The expression of markers in the IDP was then compared to data generated in the absence of the SMK. These data showed that addition of the SMK does not impact the IDP as demonstrated by high correlation (R2 >0.99) between the IDP + WTA versus IDP + WTA + SMK.