Affinity Measurement Platform
Based on Octet and Biacore-T2000 platform, Alpha Lifetech can provide reliable affinity determination services. We can provide affinity determination for multiple samples, such as antibodies, cells, proteins, small molecules etc. There are various affinity determination methods, including surface plasmon resonance (SPR) and bio-layer interference (BLI) affinity determination.
Alpha Lifetech has a professional and accurate affinity determination platform that combines ELISA with affinity determination, providing professional, efficient, rapid, and precise affinity determination results for worldwide customers. We can offer two methods for customers to choose from: Rapid Affinity Determination and Precise Affinity Determination. Rapid affinity determination is a single-concentration determination, while precise affinity determination can measure the affinity of different concentrations. Applied to the study of interactions between various small molecule compounds, peptides, proteins, oligonucleotides, and oligomers, as well as lipids, bacteriophages, viruses, and cells. The affinity measurement platform can lay a foundation for affinity drug testing and screening, antibody discovery, and facilitate subsequent research.
Introduction to Binding Affinity
Affinity is important in evaluating intermolecular interactions, affinity drug assays, and drug screening. Intermolecular interactions can be described by equations: L + R = LR, where L represents free ligands, R represents unbound receptors, and LR represents bound ligand-receptor complexes. Binding reactions define intermolecular interactions, where dynamic exchange occurs between bound and unbound states during the reaction until equilibrium is reached. This can be described by the two rate constants, Kon (binding rate constant) and Koff (dissociation rate constant), of the reaction. The Kd value, which is the reciprocal of the binding constant (Ka), is Koff/Kon, an important constant for reaction affinity. Therefore, the tighter the binding between two molecules, the higher the affinity. The smaller the Kd value, the vice versa. This equation can be represented as an S-shaped curve on a semi-logarithmic graph, with ligand concentration on the x-axis (on the logarithmic scale axis) and fractional boundary on the y-axis. The dashed line represents the ligand concentration at a Kd (1 nM) of 0.5 binding fraction.

Fig 1: Sigmoidal binding curve of varying concentrations of ligand bound to cell surface receptor. (Reference source: Hunter SA, Cochran JR. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.)
Methods for Determining Affinity
ELISA binding affinity assay
The widely used technique for studying antibody affinity is based on the ELISA method, which is characterized by its convenience, speed, simplicity, high sensitivity, and strong specificity. It can use a small amount of reagents (i.e., antibodies and antigens) and measure antibody affinity without the need for purification reagents. By immobilizing the antigen on a solid surface and detecting it using a primary antibody, the labeled secondary antibody reacts with the primary antibody to read and analyze the data in an enzyme-linked immunosorbent assay (ELISA) reader.

Fig 2: ELISA-like assays to evaluate the binding of the designed peptides to their targets. ( Reference source: Hajikarimlou, Maryam, et al., 2022. A computational approach to rapidly design peptides that detect SARS-CoV-2 surface protein S.)
Surface Plasmon Resonance (SPR) binding affinity assay
SPR technology mainly detects changes in refractive index. With the help of traditional optical phenomena and the resonance phenomenon of light, a biosensing analysis technology for the interaction between biomolecules can be constructed to detect the interaction between ligands and analytes on biosensing chips. Specific signals of binding and interaction between biomolecules can be obtained by monitoring the dynamic changes in SPR angles during biological reactions.

Fig 3: Surface plasmon resonance (SPR) analysis of H10/AGR2 binding. (Reference source: Garri, Carolina, et al., 2018. Identification, characterization, and application of a new peptide against anterior gradient homolog 2 (AGR2).)
Bio Layer Interference (BLI) binding affinity assay
Biofilm interference technology is a label-free, real-time monitoring optical detection technique mainly used for comprehensive quantitative analysis of intermolecular interactions and protein concentration determination. This technology uses a probe-based biosensor to directly detect changes in the thickness of the biofilm on the sample. By detecting the displacement changes of the interference spectrum, the binding and dissociation between biomolecules interacting on the sensor surface are detected, and the real-time displacement (nm) of the interference spectrum is displayed.

Fig 4: Biolayer interferometry (BLI) assay between aLDRG and chitinoligosaccharides. (Reference source: Li, Bing, 2023. Hallmarks of Comparative Transcriptome between Rhizomorphs and Hyphae of Armillaria sp. 541 Participating in Fungal Symbiosis with Emphasis on LysM Domains.)
Comparison of BLI and SPR Technology
Technology Name | BLI (Bio-Layer Interferometry) | SPR (Surface Plasmon Resonance) |
---|---|---|
Principle | Measures changes in the interference pattern of reflected light on the sensor surface, detecting molecular interactions via changes in optical thickness on the bio-layer. It provides a real-time binding curve (direct measurement). | Measures molecular interactions by detecting signal changes in the refractive index near the sensor chip’s surface (occurs when light interacts with the gold and glass interface, causing changes in the refractive index). Data is reflected as changes in the resonance angle (indirect measurement). |
Manufacturer | Sartorius | GE |
Instrument | ForteBio Biosensors | Open SPR Instrument |
System | ForteBio Octet System (for molecular interaction analysis) | TraceDrawer (developed by Ridgeview Instruments, Sweden) |
Advantages |
1. Broad sample compatibility, excellent stability, especially for detecting small molecules (specific requirements for sample purity and buffer conditions are less stringent). SSA chips are cost-effective for binding studies.
2. Faster throughput and shorter experimental times compared to SPR.
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1. Longer development history, offering higher sensitivity in comparison to BLI.
2. Greater precision and robustness for specific applications, such as detecting rare or valuable proteins with higher affinity and specificity data.
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Disadvantages | 1. Data precision slightly lower compared to SPR.
2. Requires careful maintenance of the instrument.
3. SSA chip cost is relatively high.
|
1. Buffer conditions for detecting very small molecules can be demanding, increasing the risk of detection failure.
2. Chips are generally more expensive than those used in BLI.
3. Sample evaporation during extended experiments can be an issue.
|
Chip Type | SSA Chip | NTA Chip |
Scope of Affinity Measurement
Affinity determination can include antigen-antibody (strong antigen-antibody, weak antigen-antibody), protein-protein, protein-peptide, protein-small molecule, and protein DNA/RNA (aptamer). When measuring KD, it is necessary to know one of the molar concentrations. When small molecules bind, one molecular weight cannot be less than 150 daltons.
Type | Scope | Precautions |
---|---|---|
1. Antigen-Antibody | 10^-6 to 10^-12 | The Kd values of most antibodies are in the range of 10^-6-10^-7 to 10^-9. It is generally believed that high-affinity antibodies are within 10^-9, while high-affinity. Antibodies are within 10^-12. |
2. Protein - Small Molecules | 10^-4 to 10^-5 |
The KD of small molecules and proteins is between 10^-4 and 10^-5, while 10^-3 and 10^-7 are normal and cannot reach 10^-10.
Covalent small molecules may reach 10^-10.
|
3. Avidin-Biotin | 10^-14 | Affinity can easily undergo non-specific binding, and streptavidin or deglycosylated affinity can be used. |
4. DNA-Protein | 10^-8 to 10^-10 | High quality and complete DNA; be careful to prevent the influence of electrophoresis. |
Sample requirements for affinity determination
Sample | Requirements |
---|---|
1. Large Molecule Sample | Protein > 50 µg, antibody > 100 µg, biotinylated protein > 200 µg; protein without biotin > 2 mg, purity requirement > 90%, buffer solution: PBS, HEPBS. Cannot contain imidazole groups; quality control is required. |
2. Small Molecule Sample | Quantity>1mg,powder or liquid, liquid needs to be soluble in water or DMSO. Try not to contain glycerol, imidazole, trehalose or other salts; try to avoid reagents with amino groups such as Tris in the buffer, generally PBS, HEPPS, etc. without organic reagents. |


Multiple sample analysis
Alpha Lifetech can provide affinity assays for various samples, including antibodies, cells, proteins, and other biomolecules.


Mature technology platform
We have advanced technologies such as spr binding assay, bli binding assay, and elisa binding assay.


Flexible project selection
Customers can choose between rapid affinity determination and precise affinity determination.


High-precision results
Our professional technical team can ensure efficient, accurate, and reliable affinity determination results.
Case StudyCASE
BLI rapid affinity assay antibody and aptamer affinity assay
Capture biotinylated aptamers with SA probe specificity and dissolve them. Dissolve and dilute the sample to a fixed concentration, solidify the probe-specific captured Target 1-5 aptamers, and after signal saturation, bind to the sample. Then add them to a 96-well plate.


Fig 5: Distribution of detection positions for 96-well plate samples. B refers to the buffer that is used for balancing and dissociation of sensors. L: Biotin Target 1-5 aptamers, 221: Sample.
Pay attention to drilling to ensure the stability and accuracy of the data, and set the corresponding program to obtain the results:

Fig 6: Interaction fitting diagram between Target 1, 2, and 3 aptamers and samples. (CH 1 \ 3 \ 5 represents the signal and data of the interaction between the solidified Target 1 \ 2 \3 aptamers probe and the sample.)

Fig 7: Interaction fitting diagram between Target 4 and 5 aptamers and samples. (CH 1 \ 3 represents the signal and data of the interaction between the solidified Target 4 \ 5 aptamer probe and the sample.)
results show
The following results were obtained: The affinity of the Target 1 adapter to the sample after fitting was 9.41 ^ -8; the affinity of the Target 2 adapter to the sample after fitting is 8.32 ^ -8; the affinity of the Target 3 adapter to the sample after fitting is 8.64 ^ -8; the affinity of the Target 4 adapter to the sample after fitting is 3.70 ^ -8; the affinity of the Target 5 adapter to the sample after fitting is 3.01 ^ -8.
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