The Mighty Antibody Series 1: IHC Antibodies - The Golden Navigator for DSP Spatial Biology

Principle of DSP Spatial Biology

The DSP spatial proteomics platform uses probes as the means for target detection and quantification. Each probe consists of three parts:

1. An antibody that binds the target protein.
2. A UV-photocleavable linker.
3. A tagging nucleotide sequence, known as the DSP barcode.
   IHC antibodies can be labeled with DSP barcodes using kits like the GeoMx DSP Protein Barcoding Kit.

Figure 1 DSP Barcode Schematic

DSP Workflow:

1. Staining: Tissue sections are stained using IHC methods with fluorescently labeled probes (for morphology) and quantification reagents (antibodies labeled with DSP barcodes).
2. Region Selection (ROI/AOI): Whole slide imaging is performed to define Regions of Interest (ROI), or further select Areas of Illumination (AOI) within ROIs based on morphological markers.
3. UV Cleavage & Collection: UV light is used to cleave the linkers within the selected ROI/AOI, releasing the DSP barcodes for collection.
4. Barcode Collection: The oligonucleotides are collected into a 96-well plate (each well corresponds to a selected ROI/AOI on the tissue section).
5. Sequencing & Analysis: The collected oligonucleotide barcodes are amplified via PCR for library construction, sequencing adapters are added, and the libraries are sequenced on a high-throughput sequencing platform, followed by downstream data analysis.

Figure 2 Experimental Workflow Schematic

Antibodies validated for IHC applications play the role of "navigators" in DSP spatial proteomics. They are the cornerstone enabling DSP technology to identify, localize, and quantify target proteins. A high-quality IHC antibody library provides a powerful "toolbox" for DSP spatial proteomics.

• As a pre-experimental tool: They help researchers determine the precise spatial regions (ROIs) requiring DSP spatial proteomic analysis.
• When used directly as DSP probes: They form the core of the DSP spatial proteomics technology itself, directly used to detect and quantify specific proteins within the selected areas.

Antibodies suitable for DSP must also meet the following requirements:

• Core Requirement 1: Extremely High Specificity
  Antibodies performing excellently in IHC/IF are strong candidates for DSP applications. Starter's proprietary recombinant rabbit monoclonal antibody technology leverages the richer immune repertoire of rabbits to generate a large number of highly specific antibodies.
• Core Requirement 2: No Cross-Reactivity
  The antibody must bind only to its target protein and not exhibit cross-reactivity with other proteins or other antibodies within the panel. Starter antibodies undergo rigorous validation across multiple species and various tissue sample types to ensure no cross-reactivity.
• Core Requirement 3: Affinity and Sensitivity
  Antibodies need sufficiently high affinity to effectively bind their target protein in complex samples like Formalin-Fixed Paraffin-Embedded (FFPE) tissues. (e.g., antibodies must recognize epitopes exposed after formalin cross-linking and antigen retrieval). They must also provide high signal-to-noise, generating detectable signals even for lowly expressed targets while maintaining very low background. Starter focuses significantly on recombinant rabbit mAbs. Rabbit mAbs typically exhibit equilibrium dissociation constant (KD) values in the picomolar range, whereas most mouse mAbs are in the nanomolar range, indicating the higher sensitivity of rabbit mAbs.
• Core Requirement 4: Low Aggregation Tendency
  Antibodies must remain stable and not aggregate after being labeled with oligonucleotide tags and throughout the experimental process.
• Core Requirement 5: Compatibility with Oligonucleotide Labeling
  This is a specific requirement for DSP technology. Antibodies need to be covalently conjugated to one or more oligonucleotide sequences without compromising their binding capability. Retained activity post-labeling: The labeling process (typically via conjugation to lysine or cysteine residues on the antibody) must not disrupt the antigen-binding site. Starter can provide high-concentration antibodies without carrier proteins, supporting oligonucleotide conjugation.

 Figure 3 DSP Protein Barcoding Labeling Requirements

• Core Requirement 6: Batch-to-Batch Consistency
  Antibodies from different lots must perform consistently after labeling. Starter's recombinant rabbit mAbs, with the preserved antibody plasmid, allow for more stable long-term storage and support high batch-to-batch consistency.

Starter provides a large number of high-quality IHC antibodies, offering limitless possibilities for DSP proteomics.

Validation Data

• S-RMab® TTF-1 Recombinant Rabbit mAb, PBS Only (SDT-R097) CD163 Recombinant Rabbit mAb 

S-RMab® Ki67 Recombinant Rabbit mAb  

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