The advantage of using multiple techniques to quantify targets

At BioLegend we understand the need to validate scientific results. When it comes time to report the content of a specific target, it may be necessary to use multiple techniques to either verify a trend, or confirm absolute or relative numeric values. 

BioLegend offers a wide variety of reagents for life science research and diagnostic applications. Antibodies are at the center of our portfolio, and we’re constantly seeking to validate our reagents for novel applications as well as existing technology. For example, we offer MaxPar® Ready Antibodies, specifically formulated for optimal conjugation with metal labeling kits for use with CyTOF®. Likewise, our MojoSort™ magnetic cell separation reagents are compatible with existing magnetic separation columns, in addition to stand alone magnets.

Recently, we have partnered with NanoString to provide antibodies in their applications. Specifically, the NanoString® 3D Biology™ Technology, which simultaneously profiles and quantitates DNA, RNA, and proteins. The data provided by this system further validates the specificity and quality of our antibodies.

Figure 1. Illustration of NanoString protein detection system.

Nanostring’s nCounter system uses molecular "barcodes" and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction. Each color-coded barcode is attached to a single target-specific probe corresponding to a nucleic acid of interest. To detect proteins in specific panels, BioLegend’s antibodies have been conjugated to NanoString’s nucleic acid barcodes.

Figure 2. CD3 PE/Cy5 titration on normal human peripheral blood lymphocytes ranging from 1.0 - 0.015 µg/test. Isotype control and clone UCHT1 are shown as indicated.

BioLegend’s antibody validation process involves testing and qualification at all stages of development, including hybridoma clone verification, applications testing, and quality control. For monoclonal antibodies (not IgM), antibody purity is always >95% and guaranteed to be low in aggregates. Additional testing includes verification of the isotype by ELISA. Once an antibody reaches the application testing stage, clone specifications need to be met to advance the product. The specifications for flow cytometry testing, which is most relevant for NanoString’s applications, involves titration of the antibody and staining on positive and negative cell populations to confirm specificity.

In addition to BioLegend’s stringent QC process (visit www.biolegend.com/qc for more information), all DNA probe-antibody conjugates are validated in biological samples. An important step is to make sure flow cytometry signal and nCounter signal correlate well.

Figure 3. Correlations in protein signal fold-change between flow cytometry and the nCounter® Vantage 3D™ assay. Ten proteins in three samples types with varying expression were assayed. Sample A expressed all proteins at similar levels, while Samples B and C expressed the same proteins at variable levels. Fold-change expression for Sample B vs. A (green) and Sample C vs. A (orange) are plotted. Flow cytometry data were averaged across 3 replicates; nCounter data were averaged across 6 replicates.

Verifying the reagents with biological samples is crucial as well, and this analysis can highlight the differences in RNA vs protein kinetics.

Figure 4. Human PBMC were stimulated with PMA and profiled by flow cytometry for protein analysis and the Vantage 3D RNA:Protein Immune Cell Profiling Assay for simultaneous RNA and protein analysis (input of 50,000 PBMC). Data from both platforms showed high correlation. The addition of RNA profiling from the same sample on the nCounter platform also highlights key concordance and disconcordance between the two analytes. Grey lines indicate untreated cells, green lines indicate treated cells.

BioLegend’s innovative approach to scientific discovery with highly specific novel antibodies is a great match for current complex research as well as future novel technologies. Providing reagents suitable in a wide diversity of applications is how we enable discovery. 

To learn more about BioLegend reagents by applications visit the advanced search. 

To learn more about NanoString products visit http://www.nanostring.com/ .