MonoRab™ Anti-scFv antibodies

Highlights

Versatile Applicability

• Uniquely tailored to detect scFv from diverse sources, connection sequences, linkers, and forms of scFv-based bispecific antibodies and CAR-T cells

High Sensitivity

• Delivers accurate flow cytometry readings for CAR-T cell positivity at low titers, with the capability to trace even the smallest scFv concentrations in solutions

Non-binding blocking

• Designed to bind scFv without disrupting its target antigen interaction, preserving the functionality essential for effective immunotherapies

Selection Guide

*Anti-scFv antibody Cocktail (Min X) has no cross-reactivity to human IgG, mouse IgG, goat IgG and rabbit IgG.

Application Data

• Characterization of CAR-T cells by flow cytometry

  GenScript's Anti-scFv antibody exhibits ultra-high specificity for scFv-based CAR-T cells. Experimental results show that Anti-scFv antibodies at different dilution ratios can accurately detect the positive rate of CAR-T cells, and the antibody shows extremely low non-specific binding to untransduced T cells, therefore it can be used for the characterization of CAR-T cells.

  

  Figure 2. Characterization of scFv-based CAR-T cells by Anti-scFv antibodies

  The same CAR-T cells were detected using MonoRab™ Rabbit Anti-scFv Cocktail [iFluor 647] (GenScript, A02288) at different dilutions (1:50-1:500). The untransduced T cells and concentration-matched Rabbit IgG [iFluor 647] (GenScript, A02026) were used as controls.

• Comparison of scFv-based CAR-T cell characterization tools

  Flow cytometry results show that Anti-scFv antibody has better cell clustering effect and higher positive rate than Protein L and Anti-Fab antibodies in CAR-T cell characterization.

  

  Figure 3. Comparison of scFv-based CAR-T cell characterization tools

  Live pan-CD3+ T cells were isolated from human PBMCs and engineered to express an scFv-based CAR. MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282, 10μg/mL) and concentration-matched FITC-Labeled Recombinant Protein L and Alexa Fluor^® 647 AffiniPure Goat Anti-Human IgG, F(ab‘)2 were used to characterize the CAR-T cells. The signal of A02282 was amplified using Alexa Fluor^® 488 -Labeled Goat Anti-Rabbit IgG.

• Detect the binding of scFv to its target protein by ELISA

  Anti-scFv antibodies can be used to detect the binding ability of human scFv, murine scFv and bispecific antibodies to their target proteins.

  

  Figure 4. The binding curve between scFv/bispecific antibody and its target antigen was detected using MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) in an ELISA assay.

  In this ELISA assay, the target antigen was coated at a concentration of 1 μg/mL, and scFv was utilized within a concentration range from 1.37 ng/mL to 1000 ng/mL. Subsequently, A02282 was used as a detection antibody at a concentration of 1 μg/mL, and the signal was amplified using Mouse Anti-Rabbit IgG Fc Antibody (14H9H10) [HRP], mAb (GenScript, A01856) at a dilution of 1:20,000.

  Human scFv#1: anti-VEGF scFv; Antigen#1: VEGF; Mouse scFv#2: anti-BCMA scFv; Antigen#2: BCMA; Bispecific Antibody#3: scFv based anti-CD19×anti-CD3 bispecific antibody; Antigen#3: CD3.

• Detect the binding of scFv to its target cells by flow cytometry

  Flow cytometry results show that the Anti-scFv antibody exhibits a high specific signal for cells bound to scFv and an extremely low non-specific signal for cells not bound to scFv, thus it can be used to detect the binding ability of scFv to its target cells.

  

  Figure 5. Anti-scFv antibody detects the binding activity of scFv to its target cells

  MonoRab™ Rabbit Anti-scFv Cocktail [FITC] (GenScript, A02284) was used to analyze the binding ability of anti-CD20 antibodies to Raji cells. The blue solid line represents unstained cells, the red solid line represents cells with only A02284 added, and the green solid line represents cells with anti-CD20 scFv and A02284 added.

Product performance

• Versatile Applicability

  ELISA results show that MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) can specifically recognize scFv from various sources, various connection orders (VH-VL or VL-VH), various linkers, and various forms of scFv-based bispecific antibodies, such as BiTE, IgG(H)-(scFv)2, (scFv)2-(H)IgG, scFv-Fab-Fc, etc.

  

  Figure 6. Binding curves of anti-scFv antibodies with scFvs of different sequences and bispecific antibodies detected by ELISA

  scFv from different sources, different connection orders (VH-VL or VL-VH), different linkers or different forms of bispecific antibodies are coated on the plate at a concentration of 1 μg/mL;

  The concentration of A02282 ranges from 0.017 ng/mL to 1000 ng/mL; Mouse Anti-Rabbit IgG Fc Antibody (14H9H10) [HRP], mAb (GenScript, A01856) is used as the secondary antibody, and the dilution ratio is 1:20000.

• High Affinity

  The results of the Bio-Layer Interferometry (BLI) technology show that MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) exhibits high affinity to scFvs with various sources, various connection orders (VH-VL or VL-VH), various linkers, and various forms of bispecific antibodies based on scFv.

  

  Figure 7. BLI detects the affinity of Anti-scFv antibodies to different sequence scFv and bispecific antibodies

• High Sensitivity

  Western Blot results show that MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) exhibits high sensitivity to scFvs from different sources and bispecific antibody BiTE.

  

  Figure 8. Western Blot detects the sensitivity of Anti-scFv antibodies to scFvs from different sources and bispecific antibodies

  Western blot analysis of human scFv, mouse scFv-Fc and bispecific antibody (BiTE) at different concentrations was performed using MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282, 0.1 µg/mL). The signal was detected using Mouse Anti-Rabbit IgG Fc Antibody (14H9H10)[HRP], mAb, (GenScript, A01856, 1:20,000).

• High Consistency

  Three batches of MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) showed consistent binding curves to scFv, with CV<10% at each concentration point.

  

  Figure 9. Detection of batch-to-batch stability of MonoRab™ Rabbit Anti-scFv Cocktail by ELISA

  The scFv coating concentration was 1 μg/mL; MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) was diluted 3-fold starting from 111 ng/mL; Mouse Anti-Rabbit IgG Fc Antibody (14H9H10) [HRP], mAb (GenScript, A01856) was used as a detection antibody at a dilution ratio of 1:20000.

• Non-binding blocking

  Competition ELISA results showed that Anti-scFv antibody did not affect the binding of scFv to its target antigen after binding to scFv.

  

  Figure 10. MonoRab™ Rabbit Anti-scFv Cocktail (GenScript, A02282) exhibited non-inhibitory properties for the binding between scFv/bispecific antibody and its target antigen.

  In this ELISA assay, the target antigen was coated at a concentration of 1 μg/mL, and scFv was mixed with A02282 (with dilutions starting from 3.33 μg/mL) before being added to the antigen-coated plates. Subsequently, THE™ His Tag Antibody [HRP], mAb, Mouse (GenScript, A00612) was utilized as the detection antibody at a dilution of 1:5000.

  Human scFv#1: anti-VEGF scFv; Antigen#1: VEGF; Mouse scFv#2: anti-BCMA scFv; Antigen#2: BCMA; Bispecific Antibody#3: scFv based anti-CD19×anti-CD3 bispecific antibody; Antigen#3: CD3.

Science of VHH and scFv

• VHH and scFv Applications

• Advantages and Features

• Full Offerings

Single-chain fragment variable (scFv) and nanobody (also known as VHH) are both genetically engineered antibodies. Due to their small molecular weight, strong penetration, short half-life and low immunogenicity, these antibodies play an important role and have broad application prospects in disease prevention, diagnosis and treatment.

Bottlenecks of existing scFv and VHH research tools and reagents

scFv and VHH have broad applications and are attracting increasing interest from research institutions and pharmaceutical companies. However, they lack Fc fragments, making Fc-based detection and purification methods inapplicable. Additionally, pharmaceutical companies avoid adding tag proteins like Flag and His to scFv or VHH to prevent potential risks, meaning tag-based strategies are also unsuitable. For scFv and VHH without tags, current detection and purification methods face several challenges:

• Universal detection methods for scFv, such as Anti-Fab antibodies and Protein L, are not specifically developed for scFv, leading to compatibility issues, high background values, and low detection rates.
• No efficient universal detection and purification reagent exists for VHH.
• Idiotypic detection tools like anti-idiotypic antibodies and antigens are impractical for early-stage screening due to their long development cycles, high costs, and low applicability.

How GenScript Supports scFv and VHH Research with Advanced Solutions?

To support scFv and VHH drug research, GenScript offers a comprehensive solution with tools and reagents for every research stage. This includes antibody discovery, purification, functional identification, lentiviral transduction titer determination, CAR-T cell characterization and separation, and pharmacokinetic detection. Our goal is to provide complete support for researchers to ensure success in scFv and VHH research.

Figure 11. Overall solution for scFv and VHH

Advantages of scFv and VHH Applications

VHH nanobodies and single-chain fragment variables (scFv) are genetically engineered antibodies with advantages such as small size, strong penetration, short half-life, low immunogenicity, and easy engineering. They excel over full-length antibodies in cell therapy, bispecific antibody construction, solid tumor treatment, and drug delivery. Additionally, they are widely used in viral infection, biological monitoring, biosensors, and basic research. As research progresses, scFv and VHH are expected to play an increasingly significant role in medicine, agriculture, and industry.

Figure 12. Application of scFv and VHH

Feature Comparison of scFv and VHH

Nature	scFv	VHH
Size	28-35 KDa	12-15 KDa
Blood half-life	＜1h	≪ 1h
Source	Mice, rats, etc.	Camelidae
Development time	Shorter	Longer
Water Solubility	Good water solubility (+)	Good water solubility (+++)
Gather	Easy to aggregate	Almost no gathering
Stability	Moderate stability (+)	Excellent stability (++)
Resistant to extreme environments	Not resistant to high temperatures or extreme pH	Stable at high temperatures and extreme pH
Tissue penetration	Strong tissue penetration (++)	Better tissue penetration (+++)
Affinity	High affinity (+)	Higher affinity (++)
Cost of production	Low	Lower
Engineering Transformation	Easier to modify (+)	Very easy to modify (+++)
Epitope diversity	Consistent with traditional antibodies	New epitopes available
Hidden epitope recognition	Difficult to identify hidden epitopes	Can identify hidden epitopes
Expression system	Prokaryotes, yeast, mammals	Prokaryotes, yeast, mammals

*Note: “+” represents the degree of the descriptive item. The more “+” there are, the higher the degree is.

Table 1. Comparison of scFv and VHH structures and physicochemical properties²

Product List

Product Type		Species Specificity	Unconjugated	Conjugated
				HRP	Biotin	iFluor 488	iFluor 555	iFluor 647	PE	FITC
Anti-VHH antibody	Anti-Camelid VHH,mAb	Llama, Camel, Alpaca	A01860	A01861	A01995	A01862	A01863	A01994	A02227	A02226
	Anti-Humanized VHH, mAb	Humanized, Llama, Camel, Alpaca	A02165	A02167	A02166	A02168	A02169	A02170	A02171	A02172
	Anti-Camelid VHH, mAb Cocktail	Humanized, Llama, Camel, Alpaca	A02014	A02016	A02015	A02021	A02020	A02019	A02018	A02017
Anti-scFv antibody	Anti-scFv Antibody Cocktail	Humanized, Mouse	A02282	A02289	A02283	A02286	A02287	A02288	A02285	A02284
	Anti-scFv Antibody Cocktail (Min X)*	Humanized, Mouse		A02306	A02303	A02304		A02305	A02315
GS Linker Antibody								A02311	A02314
Application
Flow cytometry			✔		✔	✔	✔	✔	✔	✔
Western blot / Dot blot			✔	✔	✔
ELISA			✔	✔	✔
Immunohistochemistry			✔	✔	✔
Immunofluorescence			✔		✔	✔	✔	✔	✔	✔
Cell sorting			✔		✔	✔	✔	✔	✔	✔

*Anti-scFv antibody Cocktail (Min X) has no cross-reactivity to human IgG, mouse IgG, goat IgG and rabbit IgG.

Anti-VHH and anti-scFv antibody derivatives

Product Number	Product Name
L00905	MonoRab™ Anti-Camelid VHH Affinity Resin
L00946	MonoRab™ Rabbit Anti-Camelid VHH Antibody Plate (Clear, 8×12 strip)
L00951	MonoRab™ Anti-Humanized VHH Affinity Resin FF
L01008	MonoRab™ Rabbit Anti-VHH Microbeads
L01033*	VHH Affinity Ligand ELISA kit
L01034	MonoRab™ Anti-VHH Affinity Magnetic Beads

*The L01033 VHH Affinity Ligand ELISA kit already contains the CaptureSelect™ AAVX standard. If you want to detect Capto AVB、CaptureSelect™ AAV8 or CaptureSelect™ KappaXP，you need to purchase the AVB Affinity Ligand Standard(GenScript, Z03794)、AAV8 Affinity Ligand Standard(GenScript, Z03795) or KappaXP Affinity Ligand Standard (GenScript, Z03796) separately.

Other products

Product	Unconjugated	Conjugated
		HRP	Biotin	iFluor 488	iFluor 555	iFluor 647	PE	FITC
Mouse Anti-Human IgG Fab Antibody (12H3C4A6), mAb		A01855
protein L		M00098	M00097	M00921		M00922		M00920
Mouse Anti-Human IgG Fc Antibody (50B4A9), mAb	A02257	A01854
Streptavidin	Z02043	M00091
MonoRab™ Rabbit IgG Control (Whole Molecule), mAb	A02022			A02028	A02027	A02026	A02025
Human Kappa Light Chain (2F1C1), mAb, Mouse	V06801
Human Lambda Light Chain (2D54), mAb, Mouse	V06901
MonoRab™ Biotin Antibody (53C8), mAb, Rabbit	V90201			A02248		A02249	A02250

Reference

• 1. Monnier P , Vigouroux R , Tassew N .In Vivo Applications of Single Chain Fv (Variable Domain) (scFv) Fragments[J].Antibodies, 2013, 2(4):193-208.DOI:10.3390/antib2020193.
• 2. Asaadi Y , Jouneghani F F , Janani S ,et al.A comprehensive comparison between camelid nanobodies and single chain variable fragments[J].Biomarker Research, 2021, 9(1):87-.DOI:10.1186/s40364-021-00332-6.

Product list

Resource

GenScript’s scFv and VHH Handbook

Comprehensive Applications of Single-Chain Antibodies and Nanobodies for CAR-T Therapy and Antibody Drug Research

Free Download