Antibody Validation Guidelines

Antibodies are typically raised against native proteins (partial or full length) or short peptide sequences specific to their target protein. However, verifying that an antibody binds exclusively to its intended target without cross-reactivity is crucial for experimental success and scientific reproducibility.

1. Modern Validation Framework: The 5 Pillars

Aligned with international consensus guidelines (such as those established by the International Working Group for Antibody Validation - IWGAV), antibody validation relies on five core conceptual pillars to demonstrate specificity:

2. Peptide-Raised Antibodies & Immunogen Validation

When antibodies are generated using synthetic peptides, bioinformatic screening ensures target specificity before production:

• Sequence BLAST Alignment: Immunogen peptide sequences are queried against the UniProt database using BLAST to verify unique target alignment.
• Cross-Reactivity Thresholds: Non-target proteins sharing 85% or greater sequence identity with the immunogen peptide may cause off-target binding and require experimental screening.
• Epitope Region Mapping: When exact immunogen sequences are proprietary, evaluating the surrounding amino acid region helps predict potential isoform or species cross-reactivity.
• Biorbyt Sequence Assurance: Biorbyt immunogens are systematically checked against UniProt databases to minimize off-target sequence homology.

3. Application-Specific Validation Standards

Because target protein conformation varies between experimental techniques (e.g., denatured in SDS-PAGE vs. native in flow cytometry vs. fixed in IHC-P), validation must be performed specifically within the intended application:

• Western Blot (WB): Verification of correct molecular weight bands, assessment of non-specific binding, and evaluation in relevant positive and negative cell/tissue lysates.
• Immunohistochemistry (IHC-P): Validation in paraffin-embedded sections using appropriate antigen retrieval protocols, tissue-specific positive/negative controls, and blocking controls.
• Data Transparency: Raw, uncropped validation images should be accessible for evaluated species and applications, showing both positive binding and negative control samples.

4. Biorbyt Quality Commitments

Biorbyt continuously enhances product documentation and testing standards across our portfolio:

• Multi-Species Testing: Antibodies are tested in key biological models including Mouse, Rat, and Human tissue lysates and histology sections where feasible.
• Transparent Validation Images: Western blot data is displayed in raw form to show clean target detection alongside non-reactivity in negative controls.
• Recombinant Antibody Focus: Expanding our range of recombinant monoclonal antibodies to ensure high lot-to-lot consistency and eliminate genetic drift.