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
Biotinylated proteins can be used in ELISA as two-way antibodies for both capture and detection with high specificity and detection sensitivity.
Biotinylated proteins can be used along fluorophore-tagged SA to detect/isolate cells expressing particular surface markers.
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 chemi
cal 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 produc
tion. 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) |