Case Studies
Case 1: Kinetic screening of a single chain fragment variable antibody (scFv)
Aim: To quickly screen specific antibodies against synthetic peptides
Assay Format: Immobilized peptides, selected antibodies as the analyte
SPRi Assay and Data Analysis:
| Array Chip | Interaction Type | Injection | Analyte/ MW | Amount Injected | Flow Rate |
|---|---|---|---|---|---|
| Peptide | Peptide - Antibody | Multiple | scFv 28 kDa | 2µl 30nM | 4µl/s |
| ka (1/M•s) | kd (1/s) | Affinityt KD (M) | Associationt Duration (s) | Dissociationt Duration (s) |
|---|---|---|---|---|
| 5.02E+04 | 9.92E-04 | 1.98E-08 | 230 | 300 |
Affinity comparison between binding scFv antibodies
SPR competition assay between a selected pair of scFvs: TM20 and TM23. All injections were at a concentration of 96.43nM. There was some binding competition observed when TM20 was injected and followed by TM23, but competition was not observed in the reverse injection order.
Summary: scFv antibodies were selected against peptides and the kinetics of the interactions were calculated. A binding competition assay was then performed between the scFvs that exhibited the strongest binding to the arrayed peptides. Experimental data suggested that the two scFvs shared similar binding regions to the immobilized peptides.
Case 2: Captured glycan using antibody and lectin
Aim: To determine and identify glycoproteins from a complex sample
Assay Format: Immobilized antibodies, cell lysate as analyte, and lectins as secondary analyte
SPRi Assay and Data Analysis:
| Array Chip | Interaction Type | Injection | Analyte/ MW | Amount Injected | Flow Rate |
|---|---|---|---|---|---|
| Antibody | Antibody - Protein - Lectin | Multiple | Variable | 2µl 30nM | 4µl/s |
Summary: Antibodies arrayed on the SPRi chip first specifically capture protein from complex samples, such as cell lysate. Then, common lectins were injected over the captured protein. This two-step assay can determine the specific glycoform of the captured protein and provide additional characterization in a high-throughput format.
Case 3: Serum Glyco-Profiling
Aim: To profile glycoproteins in serum, and to discover glycan biomarkers
Assay Format: Immobilized lectins, serum as analyte, and antibody as an optional secondary analyte
SPRi Assay and Data Analysis:
| Array Chip | Interaction Type | Injection | Analyte/ MW | Amount Injected | Flow Rate |
|---|---|---|---|---|---|
| Lectin | Glycoprotein | Multiple | Serum | 2µl | 4µl/s |
| ka (1/M•s) | kd (1/s) | Affinityt KD (M) | Associationt Duration (s) | Dissociationt Duration (s) |
|---|---|---|---|---|
| 2.3E+05 | 1.0E-04 | 4.34E-08 | 230 | 300 |
The kinetic data in the table above is calculated from just one immobilized lectin interacting with injected glycoproteins.
Glycosylated proteins in cancer sera captured by multiple lectins
Identification of serum biomarkers using a specific antibody: Transferrin was identified as a component of the complex serum by a secondary injection of anti-transferrin (left figure). Kinetic binding of transferrin to two lectins (AAA, DSL) and anti-transferrin is also compared and displayed (right figure).
Summary: The high-throughput comparison of serum glycoproteins can facilitate the development of new fields of glycoprotein biomarker discovery. Aberrant glycosylation is a hallmark of cancers, and serum biomarkers offer great diagnostic potential due to minimal invasion during sample collection. If a glycoprotein is diverse in its glyco-profile, it can be more easily detected by a sandwich assay, utilizing lectins and its respective antibody. This is because multivalent binding strengthens the affinity between glycans on the glycoprotein and lectin molecules. Extensive study of these glycan structures can enhance the specificity and accuracy of discovered glyco-biomarkers.