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Ihre Position: Home > Antibody drug application > SPR & BLI： Analysis Platforms for Protein Binding Activity
SPR & BLI： Analysis Platforms for Protein Binding Activity
Release time: 2022-03-10 Source: ACROBiosystems Read: 16356

SPR & BLI： Analysis Platforms for Protein Binding Activity
Ensuring the Quality of your Proteins from Start to Finish

INTRODUCTION
The quality control of recombinant protein products requires rigorous and detailed testing of structural, physical, and chemical properties, followed by biological activity validation of protein binding and functional ability.
One of the most common methods to detect protein-protein interactions and binding activity is Enzyme-Linked Immunosorbent Assay (ELISA) due, in part, to its cost-effectiveness and ease of use. However, using ELISA for bioactivity testing has several limitations, including: 1) its dependence on secondary antibodies and enzyme amplification effects makes it difficult to obtain accurate results when detecting the proteins with a similar label or weak activity. 2) ELISA is an end-point detection method and cannot be used to detect protein binding dynamics.
An important parameter in determining intermolecular interactions is affinity. Determining the affinity is crucial for understanding molecules and identifying biological processes, drug discovery, screening, etc. The evaluation of drug efficacy, the stability of biomacromolecules, and their complexes require comprehensive studies to explore dynamics of molecular mechanics, thermodynamics, thermal stability,
Including intermolecular binding, speed, strength, and the mechanism of binding.
ACROBiosystems is focused on our customers' needs when designing products. Our team always prioritizes customers' experimental application in protein product quality control, reasonable design of quality control programs and standards. Therefore we adopted advanced surface plasmon resonance (SPR) and biofilm interferometry (BLI) platforms to detect proteins' activity and affinity (where applicable). These platforms work as a production-quality control testing project to ensure that all of the protein products can meet the various applications of antibody-drug development.
Technical principles & advantages

Surface Plasmon Resonance（SPR）

Surface Plasmon Resonance (SPR) works via an incident light striking the interface of two media with different refractive indexes at a critical angle which may cause metal free-electron resonance. Due to this resonance, the electrons absorb the light energy, which greatly weakens the reflection light at a certain angle. The angle of incidence that makes the reflected light disappear completely within a certain angle is called the SPR angle. The range of SPR varies with the refractive index of the surface, which is in turn proportional to the mass of biomolecules bound to the metal surface. Therefore, the dynamic changes of SPR angle in biological reactions can be obtained by specific signals of the interaction between biomolecules.

Technical advantages：
Label-free detection
High sensitivity
Can measure a wide range of affinity
Real-time monitoring, accurate data analysis
SPR
Figure 1. SPR detection principle
SPR（Biacore 8K, Biacore T200）
Figure 2. SPR platform（Biacore 8K, Biacore T200）
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SPR has high sensitivity, and a wide detection range can be applied to the level below μ M. The response signal is very low or pM level ultra-high affinity detection (Figure 3-A &B).
Cat. No. ADB-Y19

Fig 3-A Anti-Adalim*mab Antibodies (mouse IgG1, Cat. No. ADB-Y19) captured on CM5 chip via anti-mouse antibodies surface can bind human adalim*mab with an affinity constant of 1.36 pM.            

Cat. No. CDA-H5284

Fig 3-B Human CD32a Protein, Strep Tag (Cat. No. CDA-H5284) immobilized on CM5 Chip can bind Rituximab with an affinity constant of 1.47 μM as determined in a SPR assay.            

Biofilm Interference Technology (BLI)

Biofilm Interference Technology (BLI) can achieve real-time monitoring of intermolecular interactions with biosensors. After light passes through the biofilm layer of the sensor, transmission and reflection will occur; the frequency of reflected light is affected by the thickness of the biofilm. Some frequencies of reflected light interfere constructively with the incident light, while others interfere destructively. The spectrometer detects these interference light waves and forms an interference spectrum, which is displayed as the interference spectrum's relative displacement intensity (nm). Therefore, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer will detect the displacement of the interference spectrum in real-time. This displacement directly reflects the thickness of the biosensor surface.

Technical advantages：
Label-free detection
High detection throughput and high sensitivity
No pretreatment is required for samples
Real-time monitoring, semi-automatic detection, can provide accurate dynamic data
BLI
Figure 4. Principle of BLI technology
BLI（ForteBio Octet 96e）
Figure 5. BLI platform（ForteBio Octet 96e）
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BLI has no special requirements for sample buffer, eliminating the sample pretreatment. The overall testing time is relatively short, which can respond to customer needs faster and simultaneously test multiple data (Figure 6).
Fig 6 Affinity test between RBD mutants, variants and Human ACE2 by BLI assay

Fig 6 Affinity test between RBD mutants, variants, and Human ACE2 by BLI assay            

Although the principles of SPR and BLI are different. In most cases, the level of test results is the same, which can better evaluate the reliability of the data (Figure 7-A &B)
Cat. No. PD1-H5257

Fig 7-A Human PD-1, Fc Tag (Cat. No. PD1-H5257) captured on CM5 chip via anti-human IgG Fc antibody, can bind Human PD-L1, His Tag (Cat. No. PD1-H5229) with an affinity constant of 3.6 μM as determined in an SPR assay (Biacore T200) (Routinely tested).            

Cat. No. PD1-H5257

Fig 7- B Loaded Human PD-1, Fc Tag, low endotoxin (Cat. No. PD1-H5257) on ProteinA Biosensor, can bind Human PD-L1, His Tag (Cat. No. PD1-H5229) with an affinity constant of 5.3 μM as determined in BLI assay (ForteBio Octet Red96e) (QC tested).            

The SPR and BLI platforms have different principles and have their advantages in detecting intermolecular forces. In general, SPR and BLI are label-independent and highly sensitive. They provide high-quality kinetic, affinity, specificity, and selectivity data for real-time monitoring of protein binding and dissociation. It can be used for detailed analysis of biomolecular interactions, ranging from quest for early drugs to development of quality control.
ACRO uses both platforms in concert, depending on the protein types and attributes. Our team can choose a single or two platforms quickly and effectively for activity verification.
Application case

ACROBiosystems is committed to providing high-quality recombinant proteins with highly comprehensive production and quality control platforms. Our team is committed to providing the most suitable solutions and resources to fit our customer's application, experimental design, and support protein verification. One way to do this is by providing detailed protocols at no extra cost to show the steps and details of detection and ultimately save time, materials, and efforts on our customer's experimental design and planning.

Biofilm Interference Technology (BLI)

ACROBiosystems detected all Fcγ receptors and FcRn using SPR and BLI platforms (Figure 8-A & B).
Cat. No. FCA-H52H1

Fig. 8-A Herc*ptin captured on Protein A Chip can bind Human CD64, His Tag (Cat. No. FCA-H52H1) with an affinity constant of 0.108 nM as determined in SPR assay (Biacore 8K) (Routinely tested).

Cat. No. FCA-H52H1

Fig. 8-B Loaded Herc*ptin on Protein A Biosensor, can bind Human CD64, His Tag (Cat. No. FCA-H52H1) with an affinity constant of 0.297 nM as determined in BLI assay (ForteBio Octet Red96e) (Routinely tested).

SPR or BLI as the QC test method ensures that each batch of receptor proteins' affinity activity and binding signal strength are highly consistent (Figure 9).
Cat. No. CDB-H5228

Figure 9. SPR verification, fixed to CM5 chip Human Fc gamma RIIB / CD32b Protein (Cat. number CDB-H5228) It can bind Ritux*mab with an affinity constant of 10 μM (Biacore T200). Protocol

Cat. No. CDB-H5228

Figure 10. Using the analysis of similarity scoring software, different batches of Human Fc gamma RIIB / CD32b (Cat. No. CDB-H5228). The similarity is more than 90%

In order to meet more of our customer needs, we established a method using antibodies as a fixed phase.
It was measured by Protein A capture or CM5 direct fixation of antibodies. The test results showed no significant difference in the affinity of the Fcy receptor and human IgG1 antibody detected by different methods. (Figure 11-A &B &C & D).
Cat. No. CDB-H5228

Fig. 11-A Immobilized Human CD32b/c, His Tag (Cat. No. CDB-H5228) on CM5 Chip via anti-His antibody can bind Ritux*mab with an affinity constant of 10.1 μM as determined in an SPR assay (Biacore T200) (QC tested).            

Cat. No. CDB-H5228

Fig. 11-B Ritux*mab immobilized on CM5 Chip can bind Human CD32b/c, His Tag (Cat. No. CDB-H5228) with an affinity constant of 4.09 μM as determined in SPR assay (Biacore 8K) (Routinely tested).            

Cat. No. CDB-H5228

Fig. 11-C Loaded Human CD32b/c, His Tag (Cat. No. CDB-H5228) on HIS1K Biosensor, can bind Ritux*mab with an affinity constant of 4.30 μM as determined in BLI assay (ForteBio Octet Red96e) (Routinely tested).            

Cat. No. CDB-H5228

Fig. 11-D Loaded Ritux*mab on FAB2G Biosensor, can bind Human CD32b/c, His Tag (Cat. No. CDB-H5228) with an affinity constant of 5.4 μM as determined in BLI assay (ForteBio Octet Red96e) (Routinely tested).            

Detection of the binding ability of antigen and antibody

The binding activity of BCMA protein and anti-BCMA monoclonal antibody was detected by SPR and BLI methods, respectively (Figure 12-A&B).
Cat. No. BCA-H522y

Fig 12-A Anti-Human BCMA MAb (human IgG1) captured on CM5 chip via Anti-Human IgG Fc antibodies surface, can bind Human BCMA, His Tag (Cat. No. BCA-H522y) with an affinity constant of 13.0 nM as determined in an SPR assay (Biacore T200) (Routinely tested).            

Cat. No. BCA-H522y

Fig 12-B Loaded Anti-Human BCMA MAb (human IgG1) on Protein A Biosensor, can bind Human BCMA, His Tag (Cat. No. BCA-H522y) with an affinity constant of 29.1 nM as determined in BLI assay (ForteBio Octet Red96e) (Routinely tested).            

The SPR method was used to detect the binding ability of CD3 antigen and CD3×BCMA bispecific antibody (Figure 13).
Cat. No. CDD-H52W1

Fig.13 Bispecific T-cell Engager (CD3 X BCMA) immobilized on CM5 Chip can bind Human CD3E&CD3D Heterodimer Protein, His Tag&Tag Free (Cat. No. CDD-H52W1) with an affinity constant of 31.8 nM as determined in an SPR assay (Biacore T200).

Cross-experiment with multiple species

The SPR method was used to verify Cynomolgus CD20 Full Length, His Tag (Cat. No. CD0-C52H8) bind Ritux*mab and Ofatumumab monoclonal antibodies (Figure 14-A & B).
Cat. No. CD0-C52H8

Fig 14-A Ritux*mab captured on CM5 chip via anti-human IgG Fc antibody can bind Cynomolgus CD20 Full Length, His Tag (Cat. No. CD0-C52H8) with an affinity constant of 5.48 nM as determined in an SPR assay (in the presence of DDM and CHS) (Biacore T200) (Routinely tested).            

Cat. No. CD0-C52H8

Fig 14-B Ofatumumab captured on CM5 chip via anti-human IgG Fc antibody can bind Cynomolgus CD20 Full Length, His Tag (Cat. No. CD0-C52H8) with an affinity constant of 3.51 nM as determined in an SPR assay (in the presence of DDM and CHS) (Biacore T200) (Routinely tested).            

The BLI method was used to verify Human CD47, His Tag (Cat. No. CD7-H5227) and Cynomolgus / Rhesus macaque CD47, His Tag (Cat. No. CD7-C52H1) Binding ability to anti-CD47 antibody (Figure 14-C&D).
Cat. No. CD7-H5227

Fig 14-C Loaded Anti-Human CD47 MAb (Human IgG4) on AHC Biosensor, can bind Human CD47, His Tag (Cat. No. CD7-H5227) with an affinity constant of 6.18 nM as determined in BLI assay (ForteBio Octet Red96e) (Routinely tested).            

Cat. No. CD7-C52H1

Fig 14-D Loaded Anti-Human CD47 MAb (Human IgG4) on AHC Biosensor, can bind Cynomolgus / Rhesus macaque CD47, His Tag (Cat. No. CD7-C52H1) with an affinity constant of 2.93 nM as determined in BLI assay (ForteBio Octet Red96e) (Routinely tested).            

Detection of Binding Ability of Proteins and Small Molecules

At ACROBiosystems, we strive to meet our customers' needs in different research directions, based on our extensive experience in SPR and BLI macromolecular affinity detection. Meanwhile, we develop various molecular interaction types for affinity detection, such as protein binding to small molecules (Figure 15).
Detection with SPR technology Human HSA Protein, His Tag (Cat. No. HSA-H5220) ability to bind to Naproxen Sodium（a small molecule analgesic and anti inflammatory drug）.
Cat. No. HSA-H5220

Fig 15 Human HSA Protein, His Tag (Cat. No. HSA-H5220) immobilized on CM5 Chip can bind Naproxen Sodium with an affinity constant of 41 μM as determined in an SPR assay (Biacore T200).

>>> Click to download more references about SPR/BLI affinity test cases

ACROBiosystems provides molecular interaction analysis and testing services for biomedical R & D customers based on the Biacore and Forte Bio Octet platforms. It can provide qualitative and quantitative analysis of protein, antibody, and Fab fragment interactions. The main services include antibody screening, characterization, consistency evaluation, and interaction between biological macromolecules, which contributes to the development of biomedicine.
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