Bioanalysis
Chapter 1 & 2 Bischoff 31/05/2021
Proteoforms = different proteins.
Blood has an overall protein conc of about 60 mg/ml. That means that for each ‘biomarker molecule’
there will be 108 molecules of other proteins in blood.
Qualitative bioanalysis = substance identification
Quantitative bioanalysis: substance conc or amount
Matrix = biological material in which a substance must be determined (e.g. blood, urine,
cerebrospinal fluid, bronchoalveolar lavage, hair, tissue)
Calibrator = blank matrix containing a known conc of the analyte
Internal standard = substance that resembles the analyte and corrects for method variability.
Sample preparation includes conc adjustment (enrichment or dilution), removal of interfering
compounds, stabilization of analytes, and adjustment of analyte properties.
Separation of molecules is needed because different molecules give the same or a similar response.
Separation increases the depth of analysis (e.g. in biomarker discovery), improves sensitivity,
improves selectivity, and makes a sample compatible with detection systems (e.g. mass
spectrometry).
Data analysis and biostatistics is used for identification, quantification, and in case of biomarkers for
discrimination of cases from controls, and diagnosis/prognosis.
Guide for selecting a bioanalytical method: goal of analysis > physico-chemical properties of the
analyte > biological matrix > conc range (required sensitivity) > required throughput > admissible cost
> available instrumentation and trained personnel.
Measuring an exogenous (xenobiotic) compound:
Target analyte is different from any molecule in the human body (e.g. a synthetic drug molecule). It
can be added to analyte-free authentic biological matrix to generate calibrators. It can likely be
synthesized in stable-isotope-labelled form and used as an internal standard for mass spectrometry.
Reference material is generally available.
Measuring an endogenous compound:
Target analyte is already in the human body (e.g. a protein biomarker). Analyte-free authentic
biological matrix is not available (resort to a surrogate matrix to generate calibrators or use the
standard addition method, which is tedious). It may be difficult to obtain a stable-isotope-labelled
analogue to serve as internal standard. Reference material is often not available or is not identical to
the endogenous analyte, which may in itself be heterogenous.
Liquid-liquid extraction (LLE):
Fraction of analyte in the organic phase vs fraction of analyte in the
aqueous phase > goal to have as many analytes in the organic phase as
possible.
P = Corg / Caq
V = Vorg / Vaq
Forg + faq = 1
Optimization of extraction field can be obtained by choice of organic
solvent, adjusting the pH of the aqueous phase, salting out (add salt to the
aqueous phase), or ion-pair extraction.
,Ion-pair extraction:
Advantages of LLE: easily performed with few steps, mechanism is easily understandable.
Disadvantages of LLE: limited choice of selectivity, large volumes of organic solvents, potential loss
during solvent evaporation, emulsions, analyte adsorption, difficult to automate.
Solid (/reversed) phase extraction (SPE): stationary phase hydrophilic, mobile phase hydrophobic
Conditioning of reversed-phase SPE material: hydrophobic tails go together since they don’t want to
touch water > conditioning to make available.
Ion-exchange SPE.
Chapter 3 & 4 Bischoff 01/06/2021 Ligand Binding Assays
Ligand binding assays are based on equilibrium binding, analysed with Scat chard analysis. A
microtiter plate format is used.
Sample > wash > block remaining plastic surface (albumin) > primary ab with enzyme > enzyme
substrate > detector.
Saturation binding curve: binding site occupation
, Immunoassays:
Most widely used ligand binding assay (LBA). It requires affinity binders (generally antibodies), and
drug-protein conjugates must be prepared to render low-molecular-weight pharmaceuticals
immunogenic. Antibodies recognize only a (small) part of the antigen/analyte (the epitope).
Generation of monoclonal antibodies: immunisation of mice > isolation of B cells from the spleen >
cultivation of myeloma cells > fusion of myeloma and B cells > separation of cell lines > screening of
suitable cell lines > in vivo/vitro multiplication > harvesting.
Receptor assay:
LBA that measures pharmacologically active molecules based on receptor binding, the production of
receptors is by recombinant DNA technology. Due to difficulty of purifying active receptors, they are
often used in semi-purified form.
- Soluble receptors: progesterone, intracellular, gene transcription
- Membrane-bound receptor: N-terminus extracellular domain, 7 TM transmembrane
domains, intracellular C-terminus
Detection principles:
- Radioactivity
- Colorimetric
- Enzyme multiplied immunoassay technique (EMIT)
- Chemiluminescence
- Fluorescence polarization
- Fluorescence resonance energy transfer
Radioisotopes:
Liquid scintillation counting, LC with radioactivity detection, or
autoradiography for detection. Quantification: 1 B1 = 1
disintegration/sec (Bq/mol, specific activity).
Competitive radio-receptor assay: multiple compounds, only 1 radioactive compound (tracer, cheap)
> compete with potential ligands.
Sandwich ELISA: primary ab > ligand > secondary ab (different epitopes) > third ab with enzyme >
substrate > detection.
Enzyme substrates:
- Colorimetric (e.g. alkaline phosphatase)
- Chemiluminescent (e.g. luminol, luciferin) > emission of light
- Fluorescent
Enzyme multiplied immunoassay technique (EMIT): no wash and separation
Chapter 1 & 2 Bischoff 31/05/2021
Proteoforms = different proteins.
Blood has an overall protein conc of about 60 mg/ml. That means that for each ‘biomarker molecule’
there will be 108 molecules of other proteins in blood.
Qualitative bioanalysis = substance identification
Quantitative bioanalysis: substance conc or amount
Matrix = biological material in which a substance must be determined (e.g. blood, urine,
cerebrospinal fluid, bronchoalveolar lavage, hair, tissue)
Calibrator = blank matrix containing a known conc of the analyte
Internal standard = substance that resembles the analyte and corrects for method variability.
Sample preparation includes conc adjustment (enrichment or dilution), removal of interfering
compounds, stabilization of analytes, and adjustment of analyte properties.
Separation of molecules is needed because different molecules give the same or a similar response.
Separation increases the depth of analysis (e.g. in biomarker discovery), improves sensitivity,
improves selectivity, and makes a sample compatible with detection systems (e.g. mass
spectrometry).
Data analysis and biostatistics is used for identification, quantification, and in case of biomarkers for
discrimination of cases from controls, and diagnosis/prognosis.
Guide for selecting a bioanalytical method: goal of analysis > physico-chemical properties of the
analyte > biological matrix > conc range (required sensitivity) > required throughput > admissible cost
> available instrumentation and trained personnel.
Measuring an exogenous (xenobiotic) compound:
Target analyte is different from any molecule in the human body (e.g. a synthetic drug molecule). It
can be added to analyte-free authentic biological matrix to generate calibrators. It can likely be
synthesized in stable-isotope-labelled form and used as an internal standard for mass spectrometry.
Reference material is generally available.
Measuring an endogenous compound:
Target analyte is already in the human body (e.g. a protein biomarker). Analyte-free authentic
biological matrix is not available (resort to a surrogate matrix to generate calibrators or use the
standard addition method, which is tedious). It may be difficult to obtain a stable-isotope-labelled
analogue to serve as internal standard. Reference material is often not available or is not identical to
the endogenous analyte, which may in itself be heterogenous.
Liquid-liquid extraction (LLE):
Fraction of analyte in the organic phase vs fraction of analyte in the
aqueous phase > goal to have as many analytes in the organic phase as
possible.
P = Corg / Caq
V = Vorg / Vaq
Forg + faq = 1
Optimization of extraction field can be obtained by choice of organic
solvent, adjusting the pH of the aqueous phase, salting out (add salt to the
aqueous phase), or ion-pair extraction.
,Ion-pair extraction:
Advantages of LLE: easily performed with few steps, mechanism is easily understandable.
Disadvantages of LLE: limited choice of selectivity, large volumes of organic solvents, potential loss
during solvent evaporation, emulsions, analyte adsorption, difficult to automate.
Solid (/reversed) phase extraction (SPE): stationary phase hydrophilic, mobile phase hydrophobic
Conditioning of reversed-phase SPE material: hydrophobic tails go together since they don’t want to
touch water > conditioning to make available.
Ion-exchange SPE.
Chapter 3 & 4 Bischoff 01/06/2021 Ligand Binding Assays
Ligand binding assays are based on equilibrium binding, analysed with Scat chard analysis. A
microtiter plate format is used.
Sample > wash > block remaining plastic surface (albumin) > primary ab with enzyme > enzyme
substrate > detector.
Saturation binding curve: binding site occupation
, Immunoassays:
Most widely used ligand binding assay (LBA). It requires affinity binders (generally antibodies), and
drug-protein conjugates must be prepared to render low-molecular-weight pharmaceuticals
immunogenic. Antibodies recognize only a (small) part of the antigen/analyte (the epitope).
Generation of monoclonal antibodies: immunisation of mice > isolation of B cells from the spleen >
cultivation of myeloma cells > fusion of myeloma and B cells > separation of cell lines > screening of
suitable cell lines > in vivo/vitro multiplication > harvesting.
Receptor assay:
LBA that measures pharmacologically active molecules based on receptor binding, the production of
receptors is by recombinant DNA technology. Due to difficulty of purifying active receptors, they are
often used in semi-purified form.
- Soluble receptors: progesterone, intracellular, gene transcription
- Membrane-bound receptor: N-terminus extracellular domain, 7 TM transmembrane
domains, intracellular C-terminus
Detection principles:
- Radioactivity
- Colorimetric
- Enzyme multiplied immunoassay technique (EMIT)
- Chemiluminescence
- Fluorescence polarization
- Fluorescence resonance energy transfer
Radioisotopes:
Liquid scintillation counting, LC with radioactivity detection, or
autoradiography for detection. Quantification: 1 B1 = 1
disintegration/sec (Bq/mol, specific activity).
Competitive radio-receptor assay: multiple compounds, only 1 radioactive compound (tracer, cheap)
> compete with potential ligands.
Sandwich ELISA: primary ab > ligand > secondary ab (different epitopes) > third ab with enzyme >
substrate > detection.
Enzyme substrates:
- Colorimetric (e.g. alkaline phosphatase)
- Chemiluminescent (e.g. luminol, luciferin) > emission of light
- Fluorescent
Enzyme multiplied immunoassay technique (EMIT): no wash and separation
