Ligand binding assays
29 important questions on Ligand binding assays
What is a biopharmaceutical?
What kind of large molecules do we have?
- carbohydrates
- nucleic acids/ oligonucleotides (DNA, RNA, siRNA)
What is the aim of analysis of large molecules?
- PK
- imunogenicity
- PD
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What are the techniques that can be used for the analysis of large molecules?
- ECLIA: Electrochemiluminescence immuno assay (MSD)
- ELLA: Simple plex
- Singlulex: single molecule counting
- FACS: Flow cytometry
What is the basis structure of an immunoassay?
What is the secondary antibody?
What kind of enzyme conjugates are there?
- Alkaline phosphatase (AP)
- Beta-galactosidase
What is the difference between monoclonal antibodies and polyclonal antibodies?
polyclonal antibodies (antiserum): are obtained from the serum of animals that were immunized with a specific antigen. The antibody pool obtained from serum is the result of many b-cell clones each secreting one specific antibody.
from the serum antibodies can be extracted that can be used in heterogeneous immunoassays.
What are the sandwich immunoassays?
2) second antibody (with a chemical label) identifies the presence of the analyte
3) The antigen is sandwiched between 2 antibodies
4) This type of assay measures the amount of analyte in the sample by looking at the amount of labeled antibody that binds to the analyte on the solid support.
How is the quantification carried out?
What are the competitive binding immunoassays?
- This indirectly measures the amount of analyte in the sample by looking at the amount of labeled analyte that it displaces from the antibody.
Here the calibration curve functions the other way around. (It starts high and ends up low)
Why PK analysis?
What is a study of efficacy?
What kind of PK assays are there available for biologics?
- LC-MS/MS assays: limited part of unique primary sequence
- receptor/target binding assays: PK on location
- bioactivity/potency assays: activity-based PK profile
What are the most common methods for PK?
- chromatography assays (HPLC, LC-MS/MS)
There is more shift from immunoassays towards LC-MS/MS
LC-MS/MS for biologics may be more precise but it is not always sensitive.
What are the plusses and negatives of ligand binding assays?
- sensitive
- specific for structural conformation
- combination of epitope recognition
- idiotype specific (antibody drugs)
- specific assays for total, target-bound or free analyte
negative:
- relative variability/imprecision (no internal standard)
- dependent on availability (and changes) of critical reagents
- sensitive to structural changes
- influence of anti-drug antibodies
- time consuming assay optimization
What are the strengths and limitations of LC-MS/MS?
- quantitative assays
- good precision (use of internal standards)
- relatively fast assay optimization
negative:
- sensitivity
- focused on primary structure and signature peptides digestion
- limited association between concentration and activity
- sensitive to biotransformation of the parent drug
- analysis of free drug only
- production of internal standard.
How an immunoassay is developed? (required information)
- from preclinical studies you know a certain cmax. Then you could ask yourself if a dilution step is necessary or not.
- Determine the LLOQ next to the ULOQ to make sure that also the samples from the lowest dose cohort can be measured.
- You need to ask yourself do you need to transfer from a method or development? e.g. species transfer or a matrix transfer.
How an immunoassay is developed? (acquire reagents)
- you need to get all the other materials.
How an immunoassay is developed? (Assay "proof of concept" experiments)
What do you need to take into account if you want to optimize your assay?
- type of ELISA plate
- coating concentration
- blocking buffer
- composition of assay matrix
- incubation time
- sample diluent
- wash buffer
- concentration of detection antibody
- incubation time
- define the expected assay range
- linearity of dilution
- matrix issues (this occurs in patients and not in healthy patients usually)
- perform a pre-validation (5 different dilutions, 6 times in triplicates, 10 individual matrices, stock stability)
How the assay is validated?
- purpose (it's the purpose which defines the choice of technique, degree of validation claim of GLP etc)
- degree of validation (a limited validation is needed in case of exploratory biomarkers)
- regulatory requirement for validation
- GLP
What is the difference between partial/limited validation and complete validation?
complete validation: precision and accuracy: 6 runs in 3-fold at 5 levels. Next to this also the dilutional linearity, prozone, selectivity, and stability needs to be validated.
How do you validate precision and accuracy?
How do you validate the dilutional linearity and the prozone?
How do you validate selectivity?
How do you validate the stability?
long term frozen storage stability (usually until 1 year)
Where are the regulatory requirements coming from?
- EMA guideline on bionanalytical method validation
What are the GLP objectives?
- protect human health and the environment
- facilitate data recognition (animal welfare, harmonization, time and resource efficiency, avoidance of duplicative testing)
GLP is no guideline for scientific quality!
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