Nanobodies, often referred to as single-domain antibodies (sdAb/VHH), are naturally occurring heavy-chain-only antibodies that lack light chains. They are present in cartilaginous fish, including sharks and camelids like llamas.
Due to their exceptional qualities, which include high specificity, high stability, low immunogenicity, strong affinity, high tissue penetration, and ease of modification, they are frequently utilized in tumor immunotherapy, cell therapy, and other research disciplines. In light of this, research institutions and pharmaceutical companies are paying more and more attention to nanobodies as potential next-generation therapeutic antibodies.
Utilizing the phage antibody library approach, Sino Biological has independently built a platform for the production of nanobodies and has produced numerous successful nanobody candidates.
In addition to antigen preparation, alpaca immunization, library creation, nanobody screening, expression, purification, and humanization services, Sino Biological also provides one-stop bespoke nanobody services. This means that customers have access to full-service R&D and manufacturing support for nanobody technology.
Service highlights
- Multiple screening methods: solid-phase, liquid-phase, cell panning
- Supernatant screening: FACS, ELISA
- Directly obtaining sequences for engineering
- Flexible screening strategies: subtractive, competitive, and cross-screening
- Numerous identification methods are available, including ELISA, FACS, SPR/BLI, IHC, IF, WB, IP, and Cell assay.
Technical route of developing nanobodies
The nanobody development process involves several stages, including alpaca immunization, phage library construction, nanobody screening, expression, purification, and validation.
Following alpaca immunization, B lymphocytes are extracted from peripheral blood, and total RNA is isolated and reverse-transcribed into cDNA. This cDNA serves as a template for PCR amplification to generate diverse nanobody gene fragments, which are then inserted into vectors to create nanobody phage libraries.
Multiple rounds of panning are conducted to select antigen-specific nanobodies, which are then sequenced, expressed, and validated. Additionally, we offer in vitro efficacy evaluation services, including assessments of nanobody druggability and biological activity.
Nanobody development by phage display technique. Image Credit: Sino Biological Inc.
Service details for nanobody production
Timeline: 4–5 months
- Antigen preparation
- Antigen is provided by the clients
- Alternatively, clients can use the antigen production service
- Immunization and serum titer test
- Pre-immune bleed
- Alpaca immunization
- Serum titer test
- Final bleed
- Library construction and screening
- Phage library construction and evaluation
- Phage library screening and clone identification
- Antibody production and purification
- Vector construction
- Transient transfection of HEK293/CHO cells
- Protein A purification
- QC analysis
- SDS-PAGE and UV analysis
- ELISA validation
- FACS analysis (optional)
- SPR/BLI analysis (optional)
- Cell-based assays (optional)
- Deliverables
- Purified antibodies
- Sequence of the positive clones
- Expression vector
- CoA
Other nanobody services
- Nanobody Humanization Service
- Timeline: 3–4 weeks
- 100 % success rate
- > 95 % humanization level
- Affinity validated by ELISA/SPR/BLI
- HTP VHH Production Services
- As fast as 2 weeks
- Monovalent, bivalent, multivalent VHH, VHH-Fc fused forms
- Multi-analysis: SDS-PAGE, SEC-HPLC, endotoxin, WB, ELISA, SPR/BLI, FACS, etc.
- Cell-Free VHH Expression Service
- As fast as 5 days
- Over 99 % success rate
- Comparable activity to proteins expressed in mammalian cells
- Challenging antibody synthesis
Case study of nanobody development
Sino Biological has successfully screened positive clones against drug target A using the nanobody platform. These clones outperform the positive control antibodies in terms of activity and binding affinity.
Competitive activity (IC50). Image Credit: Sino Biological Inc.
Cell-binding Activity (EC50). Image Credit: Sino Biological Inc.
IC50 and EC50 Data. Source: Sino Biological Inc.
| Sample ID |
IC50 (μg/mL) |
EC50 (μg/mL) |
| Positive Control 1 |
0.14426 |
0.38 |
| Positive Control 2 |
0.1419 |
0.33 |
| Clone 1 |
0.08344 |
0.23 |
| Clone 2 |
0.10723 |
0.51 |
Positive Control 1: KD=6.60E-09M. Image Credit: Sino Biological Inc.
Positive Control 2: KD=6.37E-10M. Image Credit: Sino Biological Inc.
Clone 1: KD=1.44E-09M. Image Credit: Sino Biological Inc.
Clone 2: KD=3.42E-09M. Image Credit: Sino Biological Inc.
Learn More Here!