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MABSOL® Biotinylated Proteins

Bring pre-labeled biotinylated proteins directly to your bench from ACRO Biosystems!


Introduction 

Biotin is commonly used as a protein tag to facilitate the detection, purification, and immobilization of the proteins. The bond between biotin and its binding partner Avid in (or Streptavidin) is unique in the following ways.

  • Strong (Ka of 1015 M-1)
  • Specific
  • Multi-moiety
  • Stable
  • Minimal interference

​With characteristics mentioned above, biotin-avidin (streptavidin) system is now considered a versatile independent technology in following applications. 


Application

ELISA

Biotinylated proteins can be used in ELISA as two-way antibodies for both capture and detection with high specificity and detection sensitivity.

FACS

Biotinylated proteins can be used along fluorophore-tagged SA to detect/isolate cells expressing particular surface markers.

Biopanning

It is a technique often used for the selection of phage displays during antibody drug development. Biotinylated proteins can be used with SA-coated magnetic beads/surface in biopanning with higher coating density and uniformed antigen presentation.

SPR

It is a standard method used by pharmaceutical researchers to study protein binding kinetics. Biotinylated proteins can be used along with Biacore Sensor Chip SA for SPR analysis with low baseline drift and low noise. 

lmmuno-capture and Enrichment

Biotinylated proteins can be used to isolate antibodies from plasma or other biological fluid for subsequent analyses with high sensitivity, and the processed sample can be easily analyzed in quantitative mass spectrometry.


Product Series

MABSOL® biotinylated protein collection includes two unique and complimentary product series, the PrecisionAvi series built upon the AviTag technology, and the UltraLys series produced using the in-house developed chemical labeling method. These products are made with every attention to details.

PrecisionAvi Series

An exclusive collection of ready-to-use AviTagTM biotinylated proteins

The products in this series are exclusively produced using the AviTagTM technology. Briefly, a unique 15 amino acid peptide, the Avi tag, is introduced into the recombinant protein during expression vctor construction. The single lysine residue in the Avi tag is enzymatically biotinylated by the E. coli biotin ligase BirA.

This single-point enzymatic labeling technique brings many advantages for commonly used binding assays.

  • The biotinylation only happens on the lysine residue of Avi tag.
  • NO interference with the target protein's natural binding activities.
  • The protein orientation is uniform when immobilized on an avidin-coated surface

UltraLys Series

A unique series of chemically labelled biotinylated proteins with ultra-sensitivity

The products in this series are produced using our in-house developed chemical labelling approach. The primary amines in the side chains of lysine residues and the N-terminus of protein are conjugated with biotins.

  • Higher detection sensitivity

In-house developed chemical labelling approach


Key Features

Closest to Natural Conformation and Modification

The Production of our recombinant proteins including the biotinylated proteins is carried out using our proprietary HEKMax® expression platform. As expression hosts, the human HEK293 cells have a variety of advantages compared to other cell types as summarised in the table below.

Expression
Systems

Folding Phosphorylation Proteolytic
Processing
Glycosylation
E. coli + + + N/A
Insect cell + ++ ++ Poor
Plant cell +++ +++ +++ Poor
CHO cell ++++ ++++ ++++ Non-human like
Human cell +++++ +++++ +++++ Human Authentic

High Bioactivity & Detection Sensitivity

The bioactivity of biotinylated proteins is determined both by the structure of the protein itself, and by the way how biotinylation is performed. For every single protein, we test multiple options of tags and biotinylation methods, and evaluate the products in a variety of binding assays. Only those with the best performance are selected for production. Figure 1 is an example of our internal evaluation experiments.

 

 

Fig. 1 Binding activity of different forms of biotinylated PD-1 evaluated in a functional ELISA against rhPD-Ll {Cat. No. PD1-H5258}.

Our biotinylated proteins demonstrate high bioactivity and superb detection sensitivity in different application. In functional ELISA, the biotinylated human BCMA has high binding activity with BAFF as showed in figure 2. Figure 3 shows that the biotinylated PD-1 can be well applied to Alpha LISA to detect the binding activity with its ligand PD-L2. The figure 4 shows that the biotinylated protein can be easily used for determining the affinity between protein and therapeutic antibody in SPR assay. Also, the biotinylated proteins are often used in cell based assay, such as cytotoxicity assay and evaluation of CAR expression (Fig. 5 & Fig. 10).

 

 

Fig. 2 - ELISA - Immobilised Human BAFF, Fc Tag (Cat. No. BAF-H4268} at 5 µg/mL {100 µL/well) can bind Biotinylated Human BCMA, Fc Tag (Cat.No.BC7-H82F0) with a linear range of 0.12-1.95 ng/mL. 

 

 

 

Fig. 3 - AlphaLISA -  Biotinylated Human PD-l(Cat. No. PD1-H82Fl)at 1 µg/mL (5 µL/well) can bind Human PD-L2 (Cat. No. PD2-H5220} with a linear range of 0.02-0.625 µg/mL. 

 

Fig. 4 - SPR- lmmobilised biotinylated human VEGF165 (Cat. No. VE5-H82Q0) on CMS Chip via streptavidin, can bind Avastin with an affinity constant of 0.417 nM as determined in SPR assay (Biacore T200}. 

 

Fig. 5 - Cell based assay - Recombinant human TNF-alpha (Cat. No. TNA-H82E3) induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor actinomycin D. The EC50 for this effect is 0.014-0.029 ng/ml. The result shows that the biotinylated human TNF-alpha is consistent with naked TNF-alpha in cytotoxicity assay.

Low Batch-to-Batch Variation

We routinely apply rigorous quality control measures to ensure consistent performance of our products. Newly produced products are subjected to side-by-side comparison with our internal standard in a variety of assays.

Only those within an acceptable margin of difference are allowed to be released. As demonstrated in figure 6, three different batches of biotinylated hTNF-alpha (Cat. No. TNA-H82E3} are tested and compared using a standard ELISA analysis against Adalimumab, the result shows that the batch variation among the tested samples is negligible. The batch-to-batch conformance is also good as displayed in cell based assay (Fig. 7). 

Fig. 6 In the above left ELISA analysis, three different Jots of biotinylated hTNF-alpha (Cat. No. TNA-H82E3) were used detect immobilized Adalimumab (0.5 µg/ml). The result showed that the batch variation among the tested samples is negligible.

Fig. 7 In the above right Recombinant biotinylated human TNF-alpha (Cat. No. TNA-H82E3} induces cytotoxicity effect on the WEH1-13VAR cells in the presence of the metabolic inhibitor aetinomycin D. The EC50 for this effect is 0.029-0.052 nglml.


Case Studies

SPR: Antibody Optimization with Biotinylated FcRn

The half-life and efficacy of a therapeutic antibody largely depends on its Fc fragment. To obtain candidate antibody with desired pharmaceutical properties, the interaction between Fc fragment and Fc receptors needs to be evaluated. SPR is a common assay used for determing binding affinity between Fc and Fc receptors. 

Stable and efficient coating with less baseline drift present a major challenge in this approach. As shown here, the combo of ACROBiosystems' AviTag™ biotin-labeled FcRn and GE'ssensorChip-SA delivers satisfactory result in a binding assay against Herceptin.

Fig. 8 lmmobi/ized Biotinylated Human FcRn, His Tag, Strep Tag (Cat. No. FCM-H82W4} on SA chip, can bind Herceptin with an affinity constant of 1.47 µMas determined in a SPR assay (Biacore T200}. 

 

lmmuno-capture with Biotinylated TNF-alpha

TNF-alpha antibody is widely used in treating autoimmune diseases. In order to optimize the administration, it's very important to monitor the serum concentration of TNF-alpha antibody. Our collaborator AbSciex has shown that, using ACROBiosystems's biotinylated TNF-alpha (Cat. No. TNA-H821R), TNF-alpha antibody in the serum could be easily detected by immune-capture coupled with quantitative MS/MS. 

Biotinylated TNF-alpha is first coupled to streptavidin coated magnetic beads, and then capture TNF-alpha antibody in the serum. After that, MS/MS is applied to quantify TNF-alpha antibody. lmmuno-capture with biotinylated TNF-alpha significantly increases the sensitivity by ten folds. 

Fig. 9 Chromatograms spiked Adalimumab in human plasma: blank plasma, 5, 50, and 500 ng/ml.

Evaluation of CAR expression with Biotinylated CD19

The following case study was provided by our in-house R&D team. The data showed that the binding of biotinylated human CD19 (Cat. No. CD9-H8259) to anti-CD19 scFv-modified cells was specifically mediated by anti-CD19-CAR and CD19 interaction.

Inhibitor Screening with Biotinylated PD-1

ACROBiosystems has developed a PD-1-PD-Ll inhibitor screening assay kit (Cat. No. EP-101) for rapid and high throughput screening of candidate inhibitory antibodies or small molecules of the PD-1 pathway. 

This inhibitor screening ELISA pair is designed to facilitate the identification and characterization of new PD-1 pathway inhibitors. The assay takes advantage of our in-house developed binding of biotinylated human PD-1 to immobilized human PD-Ll in a functional ELISA assay, and employs a simple colorimetric sandwich ELISA platform.

Briefly, we provide you with a human PD-1-Biotin protein, a human PD-Ll protein, an anti-PD-1 neutralizing antibody (as method verified Std.), and streptavidin-HRP reagent. Both biotinylated PD-1 and PD-Ll proteins are expressed in the HEK293 cells.

Product Lists

PrecisionAvi Series (AviTag™) Product List 

Molecule Cat. No.  Species Structure
2B4 2B4-H82F0  Human 2B4 (22-221)-Fc-Avi
2B4 2B4-H82E9 Human 2B4 (22-221)-Avi-His
4-lBB 41B-H82E3  Human lBB (24-186)-Avi-His
4-lBB 41B-H82F7 Human 4-lBB (24-186)-Fc-Avi
4-lBB Ligand 41L-H82F9 Human 4-lBB Ligand (50-254)-Fc-Avi
ANGPTL7 AN7-H82F3 Human ANGPTL7 (27-346)-mFc-Avi
APRIL APL-H82F5 Human Fc-Avi-APRIL (105-250)
B7-1 B71-H82F2 Human B7-1 (35-242)-Fc-Avi
B7-1 B71-H82E9 Human B7-1 (35-242)-Avi-His
B7-2 CD6-H82E2 Human B7-2(26-247)-Avi-His
B7-2 CD6-H82F5 Human B7-2 (26-247)-Fc-Avi
B7-H3 B73-H82E6 Human B7-H3 (29-245)-Avi-His
B7-H3 B73-H82F5 Human B7-H3 (29-245)-Fc-Avi
B7-H3 (41g) B7B-H82E8 Human B7-H3 (41g) (27-461)-His-Avi
B7-H3 (41g) B7B-H82F5 Human B7-H3 (41g) (27-461)-Fc-Avi
B7-H4 B74-H82E2 Human B7-H4(29-258)-Avi-His
B7-H5 B75-H82E1 Human B7-H5 (33-194)-Avi-His
B7-H5 B75-H82F3 Human B7-H5 (33-194)-Fc-Avi
B7-H6 B76-H82E5 Human B7-H6 (25-262)-His-Avi
B7-H7 B77-H82F5 Human B7-H7 (23-344)-Fc-Avi
BAFF  BAF-H82Q2 Human His-Avi-BAFF (134-285) 
BAFF BAF-H82F3 Human Avi-Fc-BAFF (134-285)
BAFFR BAR-M82F0 Mouse BAFFR (10-71)-Fc-Avi
BCMA BCA-H82E4 Human BCMA (1-54)-His-Avi
BCMA BC7-H82F0 Human BCMA(l-54)-Fc-Avi
BCMA BCA-M82F0 Mouse BCMA (1-49)-Fc-Avi
BCMA BCA-C82F4 Cynomolgus BCMA(l-53)-Fc-Avi
BTLA BTA-H82E6 Human BTLA (31-150)-His-Avi
BTLA BTA-H82F3 Human BTLA (31-150)-Fc-Avi
Molecule  Cat. No. Species Structure
BTLA BTA-H82F8 Human BTLA (31-134)-Fc-Avi
BTNlAl BT1-H82E6 Human BTNlAl (27-242)-His-Avi
BTN3Al BT1-H82F7 Human BTN3Al (30-254)-F-Avi
BTN3A2 BT2-H82E7 Human BTN3A2 (30-248)-His-Avi
CA125 CA5-H82F4 Human CA125 (12660-12923)-Fc-Avi
CBLB CBB-H81Q8 Human His-Avi-CBLB (39-426)
CD155 CD5-H82E3 Human CD155 (21-343)- His-Avi
CD155 CD5-H82F6 Human CD155 (21-343)-Fc-Avi
CD155 CD5-M82F7 Mouse CD155 (29-348)-Fc-Avi
CD200 OX2-H82F1 Human CD200 (31-232)-Fc-Avi
CD200 Rl CR2-H82F4 Human CD200 Rl (27-266)-Fc-Avi
CD23 CD3-H82Q5 Human His-Avi-CD23 (48-321)
CD27 TN7-H82F6 Human CD27 (21-192)-Fc-Avi
CD27 Ligand TN7-H82F4 Human Avi-Fc-CD27 Ligand (39-193)
CD27 Ligand CDL-M82Qb Mouse His-Avi-CD27 Ligand (47-195)
CD28 CD8-H82F2 Human CD28 (19-152)-Fc-Avi
CD3 epsilon CDE-H82E1 Human CD3E (23-126)-His-Avi
CD30 CD0-H82E6 Human CD30 (19-379)-Avi-His
CD38 CD8-H82E7 Human CD38 (43-300)-Avi-His
CD3E & CD3 CDD-H82W1 Human CD3E (23-126)-His-Avi
CD3D(22-105)
CD4 CD4-H82F3 Human CD4 (26-396)-Fc-Avi
CD4 CD4-H82E8 Human CD4 (26-396)-His-Avi 
CD40 CD0-H82E8 Human CD40 (21-193)-Avi-His
CD40 TN5-H82F9 Human CD40 (21-193)-Fc-Avi
CD40 Ligand CDL-H82F1 Human Avi-Fc-CD40 Ligand (113-261) 
CD47 CD7-H82E9 Human CD47 (19-139)-His-Avi
CD47 CD7-H82F6 Human CD47(19-139)-Fc-Avi
CD52 CD2-H82F3 Human CD52 (25-36)-Fc-Avi
CD84 CD4-H82E5 Human CD84 (22-225)-His-Avi
CD96 TAE-H82E3 Human CD96 (22-503)-His-Avi

Ultralys Series (Chemical labeling) Product List

Molecule Cat. No. Species Structure
B7-H4 B74-H8222 Human B7- H4 (29-258)-His
CD19 CD9-H8259 Human CD19 (20-291)-Fc
CD3E & CD3D CDD-H82W0 Human CD3E (23-126)-Fc-His
CD3D (22-105)-Fc-Flag
CD3 epsilon CDE-H8223 Human CD3 epsilon (23-126)-His
CX3CL1 CX1-H8221 Human CX3CL1 (25-341)-His
EphB4 EP4-H8229 Human EphB4 (16-539)-His
EpCAM EPM-H8223 Human EpCAM (24-265)-His
EpCAM EPM-H8254 Human EpCAM (24-265)-Fc
ErbB3 ER3-H8223 Human ErbB3 (20-643)-His
FcRn FCM-H8286 Human Fc GRT 24-297)-His
B2M (21-119)-Strep II 
FGF basic BFF-H8117 Human FGF basic (143-288)
Growth Hormone R GHR-H8222 Human Growth Hormone R (27-264)-His 
GM-CSF GMF-H8214 Human GM-CSF (18-144)
GPA33 GP3-H8224 Human GPA33 (22-235)-His
Her2 HE2-H822R Human Her2 (23-652)-His
IL-6 IL6-H8218 Human IL-6 (30-212)
Mesothelin MSN-H8223 Human Mesothelin (296-580)-His
Mesothelin MSN-H826X Human Fc-Mesothelin (296-580) 
Protein L RPL-P814R N/A His-Protein L
SOST SOT-H8245 Human His-SOST (24-213) 
TFPI TFI-H8226 Human TFPI (29-282)-His
Transferrin R TFR-H8243 Human His-Transferrin R (89-760) 
TNF-alpha TNA-H8211 Human TNF-alpha (77-233) 
VEGF165 VE5-H8210 Human VEGF165 (27-191)

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