Fusion Protein ELISA
The extension of a molecule’s half-life offers additional benefits for patients. IgG-Fc fusion therapeutics, by extending the half-life of the drug provide clinically relevant advantages regarding patient preference and adherence. In general, patients suffering from chronic diseases are more likely to adhere to medications that require less frequent, intermittent dosing regimens.
Fc-fusion proteins have proven to be a successful alternative to improve pharmacological properties of therapeutic drugs with low immunogenic potential. Overall, these drugs have an acceptable safety profile with adverse events that are generally specific to the mechanisms of action of each drug class. By utilizing and manipulating Fc-Fc receptor interaction, developers are able to improve pharmacokinetics, significantly increase serum half-life and selectively enhance or disable effector functions, all while maintaining drug efficacy. Combined with an increased knowledge of the complexity of antibody disposition, Fc-fusion has become an ideal platform for increasing half-life, thus reducing dosing frequency, reducing immunogenicity by providing optimal effects while avoiding undesired complications. Overall, advances in Fc-fusion technology have led to greater flexibility when developing therapeutics by selectively addressing the needs of various disease settings.
- Currently the widest range of ELISAs for detection of therapeutic fusion proteins
- Standards used are lyophilized to offer greater stability.
- These ELISA employ monoclonal antibodies in a dual capture sandwich assay format, ensuring exceptional specificity and sensitivity in drug detection, even at low concentrations.
- Calibrated against innovator drugs where available to ensure a high degree of accuracy.
- To ensure no matrix interference and reproducible dilutional linearity, we run several serum and plasma spiking experiments at various dilutions to optimize. Additionally, our team ensures that precision is maintained at CV <10%.
- Validated against ICH / EMA guidelines under the ISO 13485 certification.
- Where available, validation of the kit against international standards from NIBSC / WHO
- 96 well plates with break apart strips includes lyophilized standards for stability.
- Expiry period of a minimum 10 months.
Performance Characteristics and Validation Information:
Sensitivity: The limit of detection is defined as the lowest detectable concentration corresponding to a signal of Mean of ‘0’ standard plus 2* SD. Each kit mentions sensitivity in the datasheet.
Calibration: The Standards provided in the kit are also calibrated against commercially sourced innovator drugs and / or biosimilars. In case where no commercial innovator drugs are available, the kits have been validated using research grade fusion proteins recombinantly expressed.
Recovery and Matrix Effect: The kits have been optimized at specific dilution to ensure optimal noise:signal ratios. Please do not change the dilution of samples recommended in the IFUs. For any queries, please connect with us directly.
Precision: Precision is defined as the percent coefficient of variation (%CV) i.e. standard deviation divided by the mean and multiplied by 100. Assay precision was determined by both intra and inter assay reproducibility on two pools with low, medium, and high concentrations. While actual precision may vary from laboratory to laboratory and technician to technician, it is recommended that all operators achieve precision below these design goals before reporting results.
| Pool | Intra Assay %CV | Inter Assay %CV |
|---|---|---|
| Low | <10% | <10% |
| Medium | <5% | <5% |
| High | <5% | <5% |
Custom Assay Development
Validated as per bioassay guidelines and developed per your exact requirements, Krishgen offers custom assay development for your therapeutic targets. Our 20+ years of expertise and collaborative workflow allow for successful development with fast TAT. Talk to our custom services team today.
Related Research Areas
Related Resources
Data Reduction: We typically use linear graphs and 4PL (2nd order) as the data reduction method. Cubic spline is also recommended in our IFUs. Our typical graph shows the graph in 4PL and also indicates the R2 value with the trend line shown.
Fc-Fusion Proteins
Fc-fusion proteins have been successfully implemented in the treatment of many diseases. Advances in engineering and design of these therapeutic proteins have helped prolong the drug half-life, which in turn allows for longer dosing intervals (i.e. weekly or bi-weekly administration) and, thus, may improve patient adherence in a real world setting. In this review, we provide a brief summary of half-life extension technologies. Here, we focus on IgG-Fc fusion and the key roles that the Fc fragment plays in both physiology and drug therapy, and the potential to elicit immune responses in humans.
Overall, Fc-fusion proteins have proven to be a successful alternative to improve pharmacological properties of therapeutic drugs, with more convenient utilization in the real world and with low immunogenic potential.
The Fc fragment in therapeutics
IgG is the most commonly used antibody class for therapy, with a molecular weight of approximately 150 kDa. In Fc proteins, the peptide or protein of interest (a ligand, an extracellular domain of a soluble receptor, etc.) is fused with an Fc fragment primarily to benefit from half-life extension. Taking advantage of size increase and the natural recycling process of IgG, Fc proteins are thought to be protected from degradation by recycling IgG Fc-Rn fused proteins into circulation.
The choice of the IgG isotype is critical in therapeutics: IgG1, IgG2 and IgG4 are often preferred to IgG3 due to their longer half-lives of approximately three weeks. Also, IgG subtypes differ on their ability to exert effector functions depending on the binding affinity of IgG to FcγRs: IgG1 and IgG3 have the highest binding affinity and, therefore, are more cytotoxic. In contrast, the binding affinity of IgG4 is approximately 10-fold less than the affinity of IgG1 and IgG3, whereas the binding affinity of IgG2 is undetectable. The choice of either one or other IgG isotype as an Fc fusion partner will depend of the desired level of half-life extension and cytotoxic activity purchased for the final compound. Most of the approved therapeutic antibodies are indicated for the treatment of cancer and autoimmune diseases. They belong to the IgG1 subclass because of its potent ability to exert effector functions through high affinity binding to the Fc receptors, which is an important advantage for the treatment of those diseases. Inversely, the IgG2 and IgG4 subclasses are the preferred backbone of a therapeutic candidate when a lack of cellular activity is desired.
If you are unable to locate your desired ELISA kit on our website, or have a technical query, please drop us a message and our team will come back to you with availability. If we do not have it available, we will suggest a feasible custom development option.
Require a Custom Assay?
Krishgen can develop and manufacture to your specific requirements.
Whether you require a change in the assay range or sensitivity, want an assay validated for a specific matrix or even want an novel immunoassay developed, the Krishgen team can support you. Get a free technical consultation to learn about the feasibility and timeline of your new assay.