Performing High-Sensitivity Protein Biomarker Analysis

Quanterix’ SP-X imaging and analysis platform enables high-sensitivity analysis of protein biomarkers in a simple, efficient, and flexible manner. The Simoa Planar Array Homebrew Kit designed by Quanterix allows SP-X users to prepare planar array immunoassays for any analyte of their choice.

This article demonstrates the benefits of the Simoa Planar Array Homebrew Kit to screen and test a variety of antibodies and allow quick preparation of high-performance planar immunoassays.

Assay Principle

The kit offers all-inclusive assay plates spotted with an immobilized anchor antibody in each of the 96 microwells. The anchor antibody is highly individualized to the equipped Simoa Planar Array Homebrew Tag. The kit also offers all the materials required to carry out biotinylation of up to six detector antibodies and peptide Tag labeling of up to six capture antibodies. In the Simoa planar Homebrew assay format (see Figure 1), initially, peptide-tagged capture antibody attaches the plate-bound anchor.

The rest of the assay workflow follows the well-known sandwich immunoassay format. The antigen is interposed between the biotinylated detector and peptide-tagged capture antibodies. A chemiluminescent HRP substrate and Streptavidin-HRP are used to signal the formation of immunocomplex. This signal’s intensity is directly correlated to the analyte quantity in the Standard or sample of interest.

Formation of immunocomplexes via Simoa Planar Array Homebrew and standard sandwich immunoassay.

Figure 1. Formation of immunocomplexes via Simoa Planar Array Homebrew and standard sandwich immunoassay.

Results

Example 1. Comparison of Human IL-6 Assay Performance in Planar Homebrew and Standard Formats

This study compared a planar Homebrew human IL-6 assay’s performance with that of the Simoa planar array human IL-6 assay kit available in the market. In the Homebrew assay format, immobilized anchor antibody in a pre-spotted Homebrew plate was used to incubate tagged IL-6 capture antibody. Subsequently, the anchored IL-6 capture antibody was used to incubate the IL-6 calibrators.

In the standard assay format, IL-6 capture antibody pre-spotted in the plate was used to incubate IL-6 calibrators. For the remaining assay steps, the same biotinylated detector antibody reagent, Chemiluminescent substrate, and SA-HRP reagent were used.

Both IL-6 assay formats exhibited a similar limit of detection (6.8 fg/mL for Homebrew format and LOD = 9.7 fg/mL for standard format) and the same lower limit of quantification (LLOQ = 27.83 fg/mL).

Calibration curve of hIL-6 assay (0.028–114.0 pg/mL)

Figure 2. Calibration curve of hIL-6 assay (0.028–114.0 pg/mL)

Example 2. Successful Human IL-22 Antibody Pair Screening and Sample Detection Using Simoa Planar Homebrew Kit

The antibody screening data related to a recently created directly spotted human IL-22 planar array was compared with that of the same antibody pairs produced using the Simoa Planar Array Homebrew Kit to demonstrate its benefits for screening antibody pairs.

Planar Homebrew array or directly spotted standard array format was used to screen five monoclonal antibody candidates found to be specific for human IL-22 (see Table 1) in different capture/detect orientations.

Table 1. Human IL-22 antibodies used for screening

Antibody Candidate Host Species Monoclonal/Polyclonal
Ab1 Rat Mono
Ab2 Rat Mono
Ab3 Mouse Mono
Ab4 Rat Mono
Ab5 Mouse Mono

 

Table 2 illustrates signal-to-background ratios for human IL-22 standard (2 pg/mL) as an indicator of the performance of antibody pairs. As the table shows, in both Homebrew and standard assay formats, Ab4 as detector and Ab1 as capture represent the antibody pair that produces the highest signals (highlighted by green). These data testify the Planar Array Homebrew Kit’s value as an antibody screening tool.

Table 2. Signal to background ratio of 2 pg/mL hIL-22

  Standard format Homebrew format
Detect antibody Ab3 Ab4 Ab3 Ab4
Capture antibody Ab1 10.6 123.8 3.5 381.5
Ab2 1.2 1.0 0.8 1.0
Ab5 0.9 1.0 1.1 1.2

 

The benefits of the Simoa Planar Homebrew Kit were further demonstrated by using IL-22 Homebrew and standard Assay formats to detect endogenous human IL-22 in plasma and serum samples. Figure 3 illustrates the sample reading data, which signifies an extremely close correlation between the sample reading data produced using the Homebrew and standard planar array, where R2 = 0.97.

Human IL-22 sample detection correlation: Standard versus Homebrew (8 serum and 8 plasma samples were tested).

Figure 3. Human IL-22 sample detection correlation: Standard versus Homebrew (8 serum and 8 plasma samples were tested).

Example 3. Versatility of Planar Array Homebrew

The Simoa planar array Homebrew assay has been applied to successfully screen multiple antibody pairs for mouse and human analytes as well as to determine robust Homebrew immunoassays using antibodies from different species. Some examples are illustrated in Table 3.

Table 3. Antibody types successfully utilized with Simoa Planar Array Homebrew Kit to date.

Host Mono/poly Capture Detect
Mouse Monoclonal
Rabbit Monoclonal
Rat Monoclonal
Goat Monoclonal

 

Conclusion

The Simoa Planar Array Homebrew Kit enables the determination of the Quanterix planar array format’s high-sensitivity immunoassay performance without using specialized array printing equipment. The kit offers a simple, robust, and versatile workflow for screening antibody pairs and developing high-sensitivity immunoassays that target any analyte chosen by the end-user.

About Quanterix

Quanterix is on a mission to change the way in which healthcare is provided by giving researchers the ability to closely examine the continuum from health to disease. In order to make this vision a reality, we brought together the most experienced management team, renowned scientists, industry leading investors and expert advisors, to form a collaborative ecosystem, united through the common goal of advancing the science of precision health.


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Last updated: Aug 13, 2019 at 3:54 AM

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