纳米流式
随着研究持续深入与纳米流式检测技术的不断升级，传统抗体因尺寸较大、空间位阻强，已难以满足超微颗粒的标记需求。
Proteintech现已开发出一系列适用于纳米流式检测的抗体
scFv单链抗体（single-chain variable fragment）是由抗体重链可变区（VH）和轻链可变区（VL）通过一段柔性连接肽（linker）连接而成，具有与完整抗体分子相同的抗原结合特异性。scFv不含Fc片段，分子量小（25-30 kDa），穿透力强，易于改造。
scFv结构
完整抗体结构
scFv单链抗体特点：
-
极小分子量
仅为完整抗体的1/6 -
高穿透性
更有效地穿透组织样本，适用于肿瘤和感染性疾病等的靶向治疗
-
无Fc段
能避免Fc受体引起的非特异性结合 -
易于改造
基因工程理想载体
Proteintech的scFv单链抗体带有His标签以便于纯化和验证，在C端预留了半胱氨酸位点，可供个性化标记。Proteintech提供预标记的scFv抗体，采用AccuBright™ 定点标记技术实现对抗体与生物素或荧光标记物的定点和定量标记，确保不同批次的信号强度一致。这些预标记的scFv抗体用于流式和免疫荧光检测，能够在拥挤的细胞环境中以及用于纳米级应用时发挥更出色的表现。
scFv单链抗体案例展示：
scFv单链抗体检测数据：
1x10^6 human PBMCs were surface stained with 0.25 ug Anti-Human CD9 Rabbit scFv Antibody (SF98095-1-RR, Clone: 240830E6) or Rabbit IgG Isotype Control RecAb (98136-1-RR, Clone: 240953C9), and PE Anti-GGGGS Linker Rabbit Recombinant Antibody. Cells were then stained with CoraLite® Plus 647 Anti-Human CD3 (OKT3) Mouse IgG2a Recombinant Antibody. Cells were incubated with FC Receptor Block prior to staining. Cells were not fixed.
1x10^6 human PBMCs were surface stained with 0.25 ug CD19 Recombinant Antibody (SF98560-1-RR, Clone: 250499A2) or 0.25 ug Rabbit scFv Isotype Control (SF98136-1-RR, Clone: 240953C9), and PE Anti-6*His, His-Tag Rabbit RecAb (PE-FcA98450, Clone:251107A1). Cells were then stained withCoraLite® Plus 647 Anti-Human CD3. Cells were not fixed. Lymphocytes were gated.
1x10^6 mouse splenocytes were surface stained with 0.25 ug Mouse CD47 Recombinant Antibody (SF98456-3-RR, Clone: 242850C4) (red) or 0.25 ug Rabbit scFv Isotype Control (SF98136-1-RR, Clone: 240953C9) (blue), and PE Anti-6*His, His-Tag Rabbit RecAb (PE-FcA98450, Clone: 251107A1). Cells were not fixed.
Biolayer interferometry (BLl) kinetic assays of SF65666-1-TR against Mouse CD8a were performed. The affinity constant is 1.41 nM.
源于羊驼的纳米抗体（VHH）
由一个重链可变区组成的羊驼单域抗体，是目前可以得到的具有完整 功能的稳定可结合抗原的最小单位。分子量仅 15 kDa，是常规 IgG 抗体分 子量的 1/10。
纳米抗体VHH特点：
-
分子量小：
分子量仅 15 kDa，是传统 IgG 抗体（150 kDa）的 1/10，或 Fab 片段（50 kDa）的 1/3 -
应用广泛：
免疫沉淀、免疫荧光、超分辨率成像等
-
稳定性高：
热稳定性高，可常温运输 -
多重验证：
序列明确，结构清晰；应用验证
-
亲和力高：
表位结合能力强，不受空间环境影响
纳米抗体VHH检测数据：
BLI analysis of the interaction between varying concentrations of anti-CD16 VHH and immobilised, biotinylated CD16A. Fit indicates fitting of data to a 1:1 binding model. Binding curves are similar for CD16B.
1X10^6 HEK CTLA4 transient transfected cells (red) and HEK parental cells were surface stained with 0.5 µg CoraLite® Plus 647 conjugated CTLA-4 VHH (CL647-ctl). Cells were not fixed.
Human PBMCs were stimulated with CD3/CD28 T Cell Activation Beads (KMS310) and after 3 days the magnetic beads were removed. Left: 1x10^6 stimulated cells were surface stained with 2ug CoraLite® Plus 555 conjugated PD-1 VHH (CL555-pd1) and CL750 CD3 (CL750-65151). Right: 1x10^6 stimulated cells were surface stained with 2ug CoraLite® Plus 555 conjugated PD-1 VHH (CL555-pd1) (red) or unstained (blue). 1X10^6 unstimulated cells were surface stained with 2ug CoraLite® Plus 555 conjugated PD-1 VHH (CL555-pd1) (black dashed). Cells were treated with FC Receptor Block prior to staining. Cells were not fixed. Lymphocytes were gated.
Left: 1x10^6 PHA-treated (3d) human PBMCs were surface stained with 0.125ug CoraLite® Plus 555 conjugated PD-L1 VHH (CL555-pdlo) and CL750 CD3 (CL750-65151). Right: 1x10^6 PHA-treated (3d) human PBMCs were surface stained with 0.125ug CoraLite® Plus 555 conjugated PD-L1 VHH (CL555-pdlo) (red) or unstained (blue). 1X10^6 untreated human PBMCs were surface stained with 0.125ug CoraLite® Plus 555 conjugated PD-L1 VHH (CL555-pdlo) (black dashed). Cells were treated with FC Receptor Block prior to staining. Cells were not fixed. Lymphocytes were gated.
scFv单链抗体（single-chain variable fragment）是由抗体重链可变区（VH）和轻链可变区（VL）通过一段柔性连接肽（linker）连接而成；纳米抗体 (VHH)由一个重链可变区组成的羊驼单域抗体，是目前可以得到的具有完整功能的稳定可结合抗原的最小单位。
| scFv | VHH | |
|---|---|---|
| 分子量 | 30-35 KDa | 12-15 KDa |
| 结构组成 | VH、VL、linker | 一个重链可变区（VHH） |
| 亲和力 | 高亲和力 | 高亲和力 |
| 组织穿透性 | 高穿透性 | 高穿透性 |
| 恶劣条件下的稳定性 | 稳定性低，易受环境影响 | 稳定性高 |
| 应用 | CAR-T、ADC药物等 | 超分辨率成像、表面等离子体共振等 |
