Enzymes are common pharmaceutical targets as they catalyze important physiological processes. Measuring enzyme kinetics via the Michaelis-Menten equation requires rapidly mixing enzymes and substrates to measure initial reaction rates. Various detection methods exist, including spectrophotometry, fluorimetry, radioactivity, and coupled reactions. Stopped flow is often used to rapidly mix reactants for kinetic analysis. Careful assay design is needed for each specific enzyme reaction.
Radioimmunoassay allows for the measurement of wide range of materials of clinical and biological importance. This technique has a significant impact on medical diagnosis due to the ease with which the tests can be carried out, while assuring precision, specificity and sensitivity.
The radioimmunoassay technique, as the name implies, achieves sensitivity through the use of radionuclides and specificity that is uniquely associated with immunochemical reactions. It can detect substance from a range of Nano gram(ng) to Pico gram(pg).
Bioassay (commonly used shorthand for biological assay), or biological standardization is a type of scientific experiment. A bioassay involves the use of a live animal (in vivo) or tissue (in vitro) to determine the biological activity of a substance, such as a hormone or drug. Bioassays are typically conducted to measure the effects of a substance on a living organism and are essential in the development of new drugs and in monitoring environmental pollutants. Both are procedures by which the potency or the nature of a substance is estimated by studying its effects on living matter. A bioassay can also be used to determine the concentration of a particular constitution of a mixture.
An Over view on Bioassay, structure & principles, types & methods of bioassay. Also mention of other assay's like biotechnology, microbio assay, immunoassay etc.
Radioimmunoassay allows for the measurement of wide range of materials of clinical and biological importance. This technique has a significant impact on medical diagnosis due to the ease with which the tests can be carried out, while assuring precision, specificity and sensitivity.
The radioimmunoassay technique, as the name implies, achieves sensitivity through the use of radionuclides and specificity that is uniquely associated with immunochemical reactions. It can detect substance from a range of Nano gram(ng) to Pico gram(pg).
Bioassay (commonly used shorthand for biological assay), or biological standardization is a type of scientific experiment. A bioassay involves the use of a live animal (in vivo) or tissue (in vitro) to determine the biological activity of a substance, such as a hormone or drug. Bioassays are typically conducted to measure the effects of a substance on a living organism and are essential in the development of new drugs and in monitoring environmental pollutants. Both are procedures by which the potency or the nature of a substance is estimated by studying its effects on living matter. A bioassay can also be used to determine the concentration of a particular constitution of a mixture.
An Over view on Bioassay, structure & principles, types & methods of bioassay. Also mention of other assay's like biotechnology, microbio assay, immunoassay etc.
This presentation deals with the in-depth analysis of various cardiac stimulants & depressants both directly & indirectly acting on frog's heart. Also, includes a nice quiz, a good exercise for the grey cells of the brain at the end of the presentation.
Photosensitization is a treatment involving the interaction of the two nontoxic factors, photoactive compound and visible light, which in the presence of oxygen results in the selective destruction of the target cell.
Different micro‐organisms, such as multidrug‐resistant bacteria, yeasts, microfungi and viruses, are susceptible to this treatment.
Endotoxin Testing is performed to ensure that injectable preparations and medical devices are free from pyrogens and safe for human use.
Pyrogens constitute a heterogeneous group of fever causing substances which comprise both microbial and non-microbial substances. The most potent and most widely known are the endotoxins or lipopolysaccharides (LPS), which are cell wall components of gram-negative bacteria. Gram-positive bacteria are also sources of pyrogens, in particular lipoteichoic acid (LTA), as are particles from yeasts and viruses. Non-microbial pyrogens often emanate from production environments. Small particles of packaging materials are a typical example.
ROLE OF IMMUNOASSAY FOR DETECTION OF NARCOTIC DRUG.pptxTereena1
This prestention contains details of different techniques of immunoassays and its details and their use in detection of narcotic drugs such as opiuim and cannabis
INTRODUCTION
In drug development, preclinical development, also named preclinical studies and nonclinical studies, is a stage of research that begins before clinical trials (testing in humans) can begin, and during which important feasibility, iterative testing and drug safety data are collected, typically in laboratory animals.
Preclinical pharmacology and toxicology are essential elements of the drug discovery and development process and are critical in enabling the translation of findings from the laboratory and the clinic. The drug discovery process is complex and involves numerous iterative steps designed to optimize the pharmacological and drug-like properties of a candidate molecule, a New Chemical Entity (NCE), and minimize the potential for side effects and toxicities. Key concepts addressed in this record include: compound identification; lead optimization; pharmaceutical profiling; the use of animal models to predict efficacy and safety and toxicological assessment as these relate to the regulatory requirements for Phase I trial initiation. Commentary is also provided on the current challenges associated with translational medicine as it applies to the effective evolution of candidate NCEs into viable clinical candidates.
The main goals of preclinical studies are to determine a starting, safe dose for first-in-human study and assess potential toxicity of the product, which typically include new medical devices, prescription drugs, and diagnostics. On average, only one in every 5,000 compounds that enters drug discovery to the stage of preclinical development becomes an approved drug.
pathogen inactivation of cellular components.pptxDrShinyKajal
Chemical inactivation
Photo-inactivation
Solvent-detergent Plasma
Photosensitizers
Methylene Blue light treatment
Psoralen Ultraviolet Light Treatment
Riboflavin Light Treatment
INTERCEPT System
Mirasol system
Platelet and plasma Pathogen Inactivation
FRALE and azridine compounds
The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and
offering a wide range of dental certified courses in different formats.for more details please visit
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This presentations will help you to understand a modification of PCR i.e. Real Time PCR. What are the components of a real time pcr and its methodology and its applications.
Pyrogrn Testing & MLT in pharmaceutical products.pptx
Bioassay development part 2
1. Principles of Drug Discovery &
Development
Bioassay development
B19FE – Semester 2
8 Lectures
Dr Colin Rickman
(c.rickman@hw.ac.uk)
2. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 2
Enzymes as pharmaceutical targets
• Enzymes catalyse the conversion of a substrate in to a
product.
• This is essential for almost all physiological processes and
may be the causative agent of a pathological state.
• Enzymes are also excellent targets for treatment of
infections.
– The enzymes present in the pathogen may not be present in the host.
– If present their amino acid sequence may have diverged sufficiently during
evolution to provide a therapeutic window.
3. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 3
Enzyme catalysis - Michaelis-Menten
• For reactions which can be described by the simple steps above, the
rate is given by the Michaelis-Menten equation.
• Under steady state conditions the rate of formation and breakdown of
the enzyme-substrate complex is equal.
• The efficiency of enzymes can vary from very low to limited only by
diffusion (catalytically perfect).
4. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 4
Enzyme catalysis – Measurement of the initial rate
• To measure enzymatic rates requires the accurate measurement of an
enzyme catalysed reaction under conditions where [ES] is constant.
• In a time course of reaction this occurs early on, before substrate is
consumed sufficiently to alter the rate of ES formation.
• Depending on the catalytic rate of the enzyme this can be very fast.
• To measure this value accurately requires rapid reagent mixing and
detection.
5. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 5
Enzyme catalysis – Measurement of the initial rate
• Suitable detection of reaction products.
– Simultaneous recoding of reaction products during experiment.
– Ability to stop reaction and measure products offline.
• Rapid initiation of the reaction.
– Rapid mixing of enzyme and reagents.
– Ability to pre-mix enzyme and reagents before triggering start of reaction.
6. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 6
Methods for detection of reaction products -
Spectrophotometry
• Many substances absorb light in
the ultraviolet or visible regions
of the spectrum.
• By shining a light of known
wavelength through a solution
the concentration can be
measured.
• This is calculated using the
Beer-Lambert law:
7. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 7
Methods for detection of reaction products -
Spectrophotometry
• This can be measured in a
cuvette-based or plate-based
spectrophotometer.
• Choice depends on the method
used and the volume of
information required.
8. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 8
Methods for detection of reaction products -
Spectrophotometry
• Almost all spectrophotometers
work using a monochromator.
• This splits white light in to a
spatially separated spectrum of
UV or visible light.
• By moving the slit position and
width a small range of
wavelengths can be sampled.
9. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 9
Methods for detection of reaction products -
Spectrophotometry
• This approach can be used
wherever the substrate and
product differ substantially in
their absorption at a specific
wavelength.
• An example of this is alcohol
dehydrogenase.
• The NADH product absorbs light
at 340 nm in comparison to
NAD+.
• The sensitivity is limited by the
path length (normally 1cm or
less for a cuvette) and the
extinction coefficient.
10. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 10
Methods for detection of reaction products -
Spectrophotometry
• The advantage of spectrophotometry is it can be monitored
over time without altering the reaction conditions.
• It can also be adapted to high throughput screening.
• However, a small extinction coefficient will severely limit the
sensitivity of this approach.
• A disadvantage is that other components of the reaction
may also have significant absorbance at the required
wavelength.
– This decreases the overall sensitivity of the assay.
11. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 11
Methods for detection of reaction products -
Spectrofluorimetry
12. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 12
Methods for detection of reaction products -
Spectrofluorimetry
• Fluorescence is defined as the “Emission of radiation, generally
light, from a material during illumination by radiation of a higher
frequency”.
• The difference in wavelength between the absorbed and emitted
photon is referred to as the Stokes shift.
13. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 13
Methods for detection of reaction products -
Spectrofluorimetry
• This shift in wavelength is utilised
in a spectrofluorimeter.
• By blocking the excitation
wavelength with a filter the
emission is detected against a
very low level of background
signal.
– c.f. spectrophotometry where the aim
is to detect a decrease in intensity of a
bright light.
• This makes spectrofluorimetry a
highly sensitive technique
(theoretically ~100 times more
sensitive than spectrophotometry).
14. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 14
Methods for detection of reaction products -
Spectrofluorimetry
• Can be used whenever the
substrate or product differ in their
fluorescent properties.
• An example of a highly fluorescent
drug is quinine used to treat
malaria.
• Quinine blocks the
biocrystallisation of heme in to
hemozoin inside the parasite.
• This results in build-up of toxic
heme leading to their death.
• It is also the flavouring using in
tonic water.
Quinine
15. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 15
Methods for detection of reaction products -
Spectrofluorimetry
16. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 16
Methods for detection of reaction products -
Spectrofluorimetry
• The advantage of spectrofluorimetry is it can be monitored
over time without altering the reaction conditions.
• It can also be adapted to high throughput screening.
• It is also theoretically more sensitive than
spectrophotometry.
• However, it is only useful if either the substrate or product
are fluorescent
– Although it may be possible to engineer them to be.
17. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 17
Methods for detection of reaction products – Coupled
assays - Chemiluminescence
• If the reaction substrate or
products are not naturally
chromophores/fluorophores (and
then can’t be engineered to be
so) a coupled reaction can be
used.
• This feeds the reaction products
from the test enzyme in to a
subsequent reaction generating
a detectable output.
• A simple example of this is the
use of the luciferase enzyme
from fire flies.
18. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 18
Methods for detection of reaction products – Coupled
assays - Chemiluminescence
• Luciferase catalyses the
reaction of luciferin with oxygen
to form oxyluciferin and light.
• This process requires ATP.
• If the enzyme reaction to be
assayed produces ATP then it
can be coupled to the luciferase
reaction and light output
measured.
• Alternative coupling assays can
make use of NADH production
and assayed using
spectrophotometry.
19. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 19
Methods for detection of reaction products -
Radioactivity
• Radioactive detection of products is the most sensitive
approach available (~106 times more sensitive than
spectrophotometry).
• Radioactive 3H or 14C can be incorporated in to the
substrate during synthesis.
• However it cannot be detected simultaneously during the
reaction.
– The reaction must be stopped for detection.
• The product and substrate must be separated to allow
specific detection.
– Normally both product and substrate are radioactive.
20. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 20
Methods for detection of reaction products -
Radioactivity
• Separation can be achieved by
thin layer chromatography or
electrophoresis followed by
scintillation counting.
• Alternatively, separation by
HPLC can be combined with
simultaneous detection of
radioactive components.
• If the product and substrate are
sufficiently different in their
chemical properties they may be
simply separated using a filter
pad.
Isotope Half-life Max energy of
emission (MeV)
14C 5730 yr 0.156
3H 12.35 yr 0.0186
32P 14.3 days 1.709
21. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 21
Enzyme catalysis – Measurement of the initial rate
• Suitable detection of reaction products.
– Simultaneous recoding of reaction products during experiment.
– Ability to stop reaction and measure products offline.
• Rapid initiation of the reaction.
– Rapid mixing of enzyme and reagents.
– Ability to pre-mix enzyme and reagents before triggering start of reaction.
22. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 22
Rapid initiation of the reaction – Continuous flow
• Two syringes (containing enzyme and substrate) are compressed at a
constant rate.
• They mix thoroughly and pass down the flow tube.
• The flow rate must be sufficiently high to ensure a turbulent flow.
– For a 1 mm diameter tube a flow rate in excess of 2 m s-1 is required.
• The advantage is a dead time as low as 10 µs, however, large amounts
of substrate and enzyme are consumed.
23. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 23
Rapid initiation of the reaction – Stopped flow
24. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 24
Rapid initiation of the reaction – Stopped flow
• The reaction mix in stopped flow fills the stopping syringe which once
filled to the required level sends a trigger to the detector and stops the
flow.
• This allows recording with a dead time of 0.5 ms and observations over
several minutes.
• In comparison to the continuous flow approach, stopped flow requires
only 100 – 400 µl.
• Both continuous flow and stopped flow are ideal for spectrometric
recording.
25. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 25
Rapid initiation of the reaction – Quenched flow
• The quenched flow technique is an adpatation of the continuous flow
approach which does not require simultaneous detection during the
experiment.
• The quencher can contain an acid or denaturant to stop the reaction
after a period of time determined by the flow rate and l.
• The minimum dead time is approximately 5 ms with a maximum
recording time of around 150 ms.
• The quenched reaction can then be analysed (often radioactivity).
26. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 26
Rapid initiation of the reaction – Flash photolysis
• Flash photolysis requires the use of caged compounds which may be
the main substrate or a required co-factor (more common).
• A pulsed laser source (340 nm) provides the energy to break the caged
compound releasing the active molecule (in this case ATP).
• This allows pre-mixing of enzyme and substrate minimising dead time.
• However, this approach is limited by the availability of caged
compounds.
27. B19FE (Semester 2) Principles of Drug Discovery & Development – Bioassay Development 27
Enzymes as pharmaceutical targets - Summary
• Enzymes are a common therapeutic target permitting the
regulation of a biochemical process.
• Michaelis-Menten kinetics provides a standardised means
of measuring enzyme kinetics and the influence of targets
on this reaction.
• Spectrophotometry, spectrofluorimetry, radioactivity and
coupled reactions are the principle means of detection.
• Stopped flow is the most popular means for the rapid
mixing and measurement of initial rates and velocities.
• Every assay needs to be specifically designed for the
enzyme reaction to be assayed.