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Antibodies are specialized protein molecules produced by immune B cells upon antigen exposure, capable of binding antigens with exceptional specificity. Owing to their high affinity and target selectivity, antibodies serve as indispensable tools across biomedical research, diagnostics, and therapeutic development.
Immunoglobulin G (IgG), the archetypal antibody, comprises two identical heavy chains and two identical light chains. The light chain contains one variable (VL) and one constant (CL) domain, while the heavy chain features one variable (VH) and three constant domains (CH1, CH2, CH3). The antigen-binding site is formed by paired VH and VL domains, where sequence hypervariability dictates antigen specificity. The conserved Fc region (CH2-CH3) mediates immune effector functions including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
Heavy-chain antibodies (HCAbs), naturally occurring in camelids and cartilaginous fish, consist solely of two heavy chains. These lack CH1 domains but retain CH2-CH3 regions and a single variable domain (sdAb). With dimensions of 2.5 nm × 4 nm and a molecular weight of ~15 kDa (merely 10% of IgG), sdAbs retain full antigen-binding capacity while offering transformative advantages:
Figure 1. Schematic of camelid serum antibodies: includes conventional IgG1 with two heavy and two light chains, and heavy-chain-only antibodies (HCAbs) IgG2 and IgG3, which lack light chains and contain sdAb domains. IgG2 has a longer hinge than IgG3. (Muyldermans S., 2013)
The common method for obtaining single-domain antibodies is to immunize llamas with antigens, allowing antibody maturation through the in vivo immune system. Peripheral blood B lymphocytes are isolated from llamas, RNA is extracted, reverse transcribed to obtain cDNA, and cDNA is used as substrate for PCR amplification to obtain diversified sdAb gene fragments. These diversified fragments are then linked to phagemids to construct phage libraries. Appropriate candidate sdAbs are screened from the llama antibody library through phage display and validated. The entire process mainly includes llama immunization, phage library construction, antibody screening, expression purification, and validation stages.
1. Antigen Preparation:
2. Immunization:
3. Serum Monitoring:
4. Lymphocyte Harvest:
Critical Parameters:
| Factor | Optimization |
| Llama Selection | Healthy, immunologically naïve adults |
| Antigen Purity | ≥90% with native conformation |
| Immunization Interval | 1–2 weeks for optimal response |
1. RNA Extraction: TRIzol-chloroform method → isopropanol precipitation
2. cDNA Synthesis: Oligo(dT) + random hexamer priming
3. Nested PCR Amplification:
4. Phagemid Ligation: Clone into phage display vector (e.g., pHEN2)
5. Electroporation: Transform TG1 E. coli; assess diversity (ideal library size: >10⁹ CFU)
6. Phage Rescue:
Quality Control Metrics:
1. Antigen Immobilization: Coat immunotubes with 50 µg antigen (4°C, ON)
2. Blocking: 3% BSA in PBS (RT, 2 hr)
3. Phage Selection:
4. Elution: Trypsin (0.25 mg/mL; RT, 30 min) → neutralize with AEBSF
5. Amplification: Infect TG1 cells → plate on ampicillin-glucose agar
6. Validation:
Technical Refinements:
Expression Systems:
| Platform | Advantages | Limitations |
| E. coli | Rapid expression (24–48 hr), low cost | Inclusion bodies; cytoplasmic reduction |
| Pichia pastoris | Secretory expression; high yield (>1 g/L); oxidative folding | Longer timeline; glycosylation risk |
Purification Workflow:
Validation Assays:
Single domain antibodies (sdAbs) represent a paradigm shift in biologics development. Their unique biophysical properties—small size, robust stability, deep tissue penetration, and modular engineering—position them as next-generation tools for previously undruggable targets. By implementing the optimized protocols detailed herein, researchers can harness this transformative technology to accelerate therapeutic discovery.
Creative Biogene harnesses the full potential of single-domain antibody (sdAb) technology to accelerate the development of innovative biologics. Through optimized immunization strategies, advanced phage display systems, and scalable expression platforms, our sdAb development services support everything from discovery to production.
Creative Biogene's End-to-End sdAb Development Platform Includes:
With robust technical expertise and a customer-centric approach, Creative Biogene is your reliable partner in sdAb-based therapeutic and diagnostic development. Contact us today to learn how our customizable services can power your next breakthrough.
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