Small “Life” Makes Sense (7) | Detection Antibodies for Fusion Proteins, Single-Chain Variable Fragments (scFv), and Bispecific Antibody Flexible Linkers (G4S)n
Release Date:
2024-06-27 17:23
Last year, Hycells launched its blockbuster, universal CAR positivity assay antibody—Anti-G4S linker—which quickly won widespread recognition and collaboration from a growing customer base thanks to its ease of use and high precision. Anti-G4S linker is a universally applicable CAR positivity assay reagent that has been rigorously validated by numerous customers; by eliminating unnecessary and time-consuming steps, it accelerates the drug development process. It is suitable for detecting CARs with G4S linkers targeting any antigen ((G4S)n, where n ≥ 2), as well as for assays involving various antibody types that incorporate (G4S)n (where n ≥ 2). Moreover, it exhibits robust resistance to interference, remaining unaffected by antigen cross-reactivity and delivering more accurate in vivo CAR proportion measurements. Its exceptional sensitivity ensures outstanding detection performance even at very high or very low concentrations.
Building on the successful development of the Anti-G4S linker product and the positive feedback from customer applications, Hycells has leveraged its existing technology service platform to innovate and expand into diverse application scenarios, further developing the new G4S-HRP product. We are dedicated to providing essential antibody tools for the screening, characterization, and target engagement studies of fusion proteins bearing the G4S linker, single-chain variable fragments (scFvs), and bispecific antibody drug development platforms.
| Item number | Product Name | Specifications |
| GS-ARHR20 | Anti-(G4S)n (B02H1)mAb(HRP) | 20 μL |
| GS-ARHR100 | Anti-(G4S)n (B02H1)mAb(HRP) | 100 μL |
| GS-ARHR200 | Anti-(G4S)n (B02H1)mAb(HRP) | 200 μL |
| GS-ARHR1000 | Anti-(G4S)n (B02H1)mAb(HRP) | 1ml |
Specificity: This product is specific for scFvs in different species (humanized, mouse) and different orders (VH-linker-VL, VL-linker-VH). It can also recognize various forms of scFv-based bispecific antibodies, such as DART, BiTE, TandAbs, IgG(H)-scFv2, scFv-Fab-Fc, etc.
Host Species: Rabbit
Conjugate: Horseradish peroxidase (HRP)
Concentration: 0.5 mg/mL
Storage Instructions: Store at –20°C and protect from prolonged exposure to light. This product is stable for 1 year upon receipt when handled and stored as instructed.
Purification: Protein A affinity column
Isotype: Rabbit IgG1
Clonality: Monoclonal antibody cocktail
Background on the Flexible Linker (G4S)n
01/ Single-chain antibody (scFv)
A complete antibody consists of two heavy chains (H) and two light chains (L). Through genetic engineering, it is possible to engineer the antibody so that it expresses only the variable regions. By using a synthetically designed linker gene to join the heavy-chain variable region (VH) with the light-chain variable region (VL), a recombinant gene is generated; the antibody expressed from this recombinant gene is referred to as a single-chain variable fragment (scFv). Structurally, the N-terminus of the heavy chain can be linked to the C-terminus of the light chain, or vice versa. The linker typically comprises 15–25 amino acids and is usually composed of glycine (Gly) and serine (Ser), i.e., a (G4S)n linker, which exhibits moderate flexibility and protease resistance. The primary function of the linker is to connect VH and VL while maintaining sufficient flexibility, ensuring that even after the functional domains of VH and VL have folded, they can still pair up to form a monovalent antigen-binding site. scFvs possess several advantageous properties, including small molecular size, strong tissue penetration, and high specificity. They play a critical role in targeted therapy, medical imaging diagnostics, and biosensing applications. Importantly, in the field of cell therapy, scFvs can serve as the antigen-recognition domain of CAR-T cells, thereby determining the targeting specificity of these cells.
02/ Recombinant fusion protein
Through analysis of natural multi-domain protein linkers, researchers have identified numerous candidate linkers suitable for protein fusion. In addition, many empirically designed linkers with diverse sequences and conformations have been created for the construction of recombinant fusion proteins. Currently, commonly used classical linkers can be categorized into three types: flexible linkers, rigid linkers, and cleavable linkers.
Take Lilly’s blockbuster drug, dulaglutide, a fusion protein, as an example: it consists of two glucagon-like peptide-1 (GLP-1) molecules linked via a flexible (G4S)n linker to the Fc region of human IgG4, thereby avoiding drug inactivation that would otherwise result from Fc fusion.
Among them, the most commonly used flexible linkers are primarily composed of Gly and Ser, namely the (G4S)n linker. By adjusting the value of n, the linker length can be optimized to achieve appropriate separation of protein functional domains or to maintain essential interdomain interactions.
03 / Bispecific antibody
Take the BiTE-format bispecific antibody as an example: Amgen’s BiTE bispecific antibody technology platform is a format developed by Micromet (which was later acquired by Amgen), in which two single-chain antibodies are linked together via a (G4S)n linker, with each single-chain antibody capable of recognizing a distinct antigen. A bispecific BiTE antibody targeting both CD19 and CD3 has the following structural configuration:
In summary, the G4S HRP product is suitable for PK and WB assays of single-chain antibodies (scFVs), fusion proteins, and bispecific antibody drugs that contain the (G4S)n linker structure, serving as an important antibody tool for screening, detection, and targeted research.
G4S HRP Product Advantages:
No.1 High Applicability
No. 2 High Affinity
No. 3 High Sensitivity
No. 4 High Batch-to-Batch Stability
No. 5 Non-specific signal
G4S HRP product combined with activity assay data
Ab1 Format Ab2 Format
| Sample | EC50 (μg/mL) | Top |
| Ab1-Hycells | 0.5461 | 2.783 |
| Ab2-Hycells | 0.06573 | 3.147 |
G4S HRP—PK Application Testing
Anti-human Fc mAb system
| Sample | EC50 (μg/mL) | Top |
| Ab1-Casein | 0.5461 | 2.783 |
| Ab1-Monkey Plasma | 0.5328 | 2.923 |
| Sample | EC50 (μg/mL) | Top |
| Ab2-Casein | 0.06169 | 2.725 |
| Ab2-Monkey Plasma | 0.1182 | 2.794 |
Conclusion: A universal assay system that uses anti-human Fc antibody-coated beads to capture the target antibodies in serum is suitable for PK analysis of target antibodies in monkey serum.
Antigen system
| Sample | EC50 (μg/mL) | Top |
| Ab2-Casein | 0.06415 | 2.545 |
| Ab2-Human Plasma | 0.05497 | 2.624 |
| Ab2-Monkey Plasma | 0.06468 | 2.655 |
Conclusion: The system in which capture antigen is used to immobilize the antibody of interest in serum is suitable for PK assays of the antibody of interest in human or monkey serum.
For more details on Hycells Bio’s G4S-HRP products, please contact our sales representative. You can also scan the QR code below to request a sample kit. Contact: Mr. Xie. 15201775322 。
Applications are welcome.
Long-press the QR code to apply for a trial.
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