Luciferase Monoclonal Antibody

Structure and Function

Luciferase monoclonal antibodies are generated using hybridoma technology, where B cells from immunized mice are fused with myeloma cells. These antibodies bind specifically to epitopes on luciferase proteins, blocking or detecting their activity. Key structural features include:

• Target Specificity: Firefly luciferase (~60–65 kDa), Gaussia princeps luciferase (~20 kDa), or NanoLuc® (Nluc, ~19 kDa) .

• Host and Isotype: Primarily mouse-derived IgG (e.g., IgG2a for Gaussia luciferase) .

• Conjugation: Some antibodies are tagged with fluorophores (e.g., FITC) or linked to luciferase for bioluminescent assays .

Firefly Luciferase Antibodies

Notes:

• Firefly luciferase antibodies are critical for validating transgenic constructs in model organisms .

• Cross-reactivity with non-transgenic tissues is minimal .

Gaussia Princeps Luciferase Antibodies

Notes:

• Gaussia luciferase antibodies are smaller (~20 kDa) and often used in high-throughput screening .

NanoLuc® (Nluc) Antibodies

Notes:

• Nluc antibodies enable real-time monitoring of antigen binding in CAR-T cell therapy optimization .

Bioluminescent Assays

• Malibu-Glo Assay: Utilizes luciferase-fused scFvs (single-chain variable fragments) to screen antigen-binding domains. Benefits include:

  • Sensitivity: Detects low-expressed antigens without purification .

  • Speed: Reduces assay time from days to hours .

• AMCA (Automatic Magnet Chemiluminescence Immunoassay): NanoLuc-labeled antibodies improve SARS-CoV-2 detection limits by ~10-fold compared to traditional methods .

Antibody Engineering

• Photoconjugation: Protein G-NanoLuc fusions enable site-specific labeling of antibodies, preserving epitope binding. Applications include:

  • ELISA: Detects human IgG with luciferase-linked antibodies .

  • Immunostaining: Visualizes cell surface antigens in live cells .

Validation and Quality Control

• Western Blotting: Firefly luciferase antibodies reliably detect 60–65 kDa bands in transfected cell lysates .

• Immunohistochemistry: Tissue-specific expression profiling in Drosophila embryos or mammalian models .

Antibody Production

• Mouse-Derived Monoclonals: Five mAbs (DSHB-LUC-2, -3, -9, -16, -24) were generated against 6xHis-tagged firefly luciferase. Four showed strong WB signals, but only one worked in paraffin-embedded samples .

• Humanization: scFv-Nluc fusion proteins are optimized for CAR-T cell therapies, balancing expression and binding affinity .

Cross-Reactivity and Specificity

• Firefly Antibodies: No cross-reactivity with mouse heart, liver, or human fibroblasts .

• Gaussia Antibodies: Strict specificity for Gaussia princeps luciferase .

Emerging Techniques

• Tandem Fusions: Gx-Nluc constructs with multiple luciferase domains enhance bioluminescence intensity .

• Color Variants: Green/red-emitting NanoLuc variants enable multiplexed detection .