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Summary of all the lectures of bioanalysis
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Summary of all the lectures of bioanalysis
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BioanalysisLecture 1
Introduction
Protein mixture of 7. We want to separate and identify using HPLS, gel electrophoresis, MALDI-TOF
and iso elective focussing
Medicine analysis. Calibration of the system. Extract the medicine by LLE or SPE. You have to make a
calibration line of the extraction.
Practical manual:
Study safety aspects, know the risks, use Pubchem
Repetitive and Gibson’s pipette know how to use
Know how to use precision glassware
Preparation must be documented in the lab journal
Preliminary: test beforehand online on nestor, then discussing in small groups discussing the
practical. Use your manual and lab journal during the discussion and do not use during the test.
Upload your lab journal on beforehand as a pdf.
Debriefing is also verbally and written
Fill in 2 pledges for the practical part and the exam.
Make a schedule for the practical days. As the preparation.
Protein: why a certain wavelength, why trypsin,
Medicine: ketoprofen is acid. Diazepam is neutral, other is basic. Imipramine (base) by LLE.
Record a scheme in the lab journal for the number of tubes for the extractions for both SPE, LLE,
acidic, basic and neutral.
What is the function of reverse phase column.
Lecture 2 Flach
How to work safely with blood
When a risk is voluntary chosen then you have another perception of this risk than when it is
imposed upon you by someone else. If risk are avoidable they are badly taken by people. Facts and
figures do not always work. There also can be advantages of risks.
Risks in the lab: fire and explosion, radiation, toxic substances, biological hazards, repetitive strain
injury, broken glassware, needle stick accidents, etc.
Because there are risks there also are regulations on how to work in the laboratory.
There are guidelines with the best practices. Other people have described what are appropriate
measures and you can adapt them to yourself. So all regulations are transformed to the guidelines of
best practices.
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,85% of the lab accidents happen by human errors. This is by thoughtlessness, inexperience,
carelessness, ignorance, blind spot or a chain thereof.
A needle stick accident is every situation where there is blood-blood contact. As long as the skin is
intact there is no real exposure. Intact skin is an excellent protection because it will not be internal. If
you want to know if your skin is intact you can put ethanol on it. If it stings, the skin is not intact and
blood on the skin can go through and come in contact with your blood.
Risks are hepatitis B and C, HIV, blood-borne diseases and others.
When working with fixated material there is not a risk. This is e.g. in our practical. HepB can survive
for days in dry blood on a surface. As soon as it is fixated there is no risk.
Hepatitis B is a liver disease with an incubation time or 2-6 months. When people are infected
people are 2-6 months more or less sick with liver inflammation and fatigue. 10% have it chronic and
become a carrier high risk on liver failure and cirrhosis. 10% of these chronic patients develop liver
cancer which is often lethal. So there is a mortality rate of 1%. Carriers are also at risk of transmitting
the virus to a new person. For hepatitis B we have vaccination. Gay men, immigrants and intravenous
drug users are more at risk.
Hepatitis C is often unnoticed for a long time. If it is symptomatic it has symptoms like hepatitis B.
after 5-20 years usually chronic hepatitis develops with a few symptoms. So it goes unnoticed but not
without risks. 20% of these develop liver cirrhosis and are at risk of liver cancer. 60-85% remains a
carrier after the original infection. For hepatitis C there is no vaccination. But if there is a risk of
infection this has to be under survey and detected as early as possible then treatment is very
effective. People with tattoos, immigrants, drug users and people who have had a transfusion or
transplant are more at risk.
HIV is an immune disease. In the acute phase it is flu like and goes away within a couple weeks. Then
later people get fever, fatigue, diarrhoea, weight loss and night sweats. In specific forms cancer and
pneumonia can occur. Nowadays the life expectancy is becoming better, but it has to be treated
consistently and in time and life-long. HIV is post exposure prophylaxis. Medicines must be
administered as soon as possible after the risk of infection. Gay men and drug users are more at risk.
Much more prevalent in Africa.
Infection risk = prevalence * transmission * number of incidents
Transmission is the possibility of transmission in case of contact with a virus. This is different for all
kinds of viruses and bacteria.
Prevalence: Transmission risk:
Hepatitis B: 2,1%
Hepatitis C: 0,1%
HIV: 0,3%
The more accidents you have had in your life, you are more at risk. This was a reason to start a
vaccination program under hospital workers.
If you have such accident:
- Let the wound blead, disinfect.
- Do your risk assessment (is the source of my accident someone with a high prevalence).
- Was it a lot of blood? (for HepB even a little bit can cause infection, but for HIV is much less
infectious).
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, - What do you know about your material/patient? Check this because when you react
immediately you have the patient still in the hospital so that a test can be done.
- What is your vaccination state?
- Consult with a doctor as soon as possible after the accident.
Lecture 3 Bischoff
Chapter 1&2
Bioanalysis is the analysis of pharmaceuticals and their metabolites as well as biomarkers and
therapeutic proteins at low concentrations in complex biological samples. Low concentration is
becoming more important because drugs are given at lower dosages because of higher effectiveness.
So they circulate at lower concentrations and also need to be measured at lower concentrations. This
requires more sensitive techniques.
To do so, it is necessary to use advanced analytical techniques with high sensitivity and selectivity
and to optimize the methodology based on a profound knowledge of the physico-chemical properties
of the analyte as well as a good understanding about possibly interfering matrix components.
You need a workflow you start with the samples which need to be prepared (enrichment or
removing of molecules). Then you need to separate the molecules to avoid interference and to gain
the necessary selectivity. Then you need to detect the molecules with a sensitive method that relies
on how good the sample preparation and separation is. Last you get raw data which you will analyse
and process and interprete.
Applications of bioanalysis: doping analysis, environmental and occupational safety analysis (are
there toxins/antibiotics in e.g. ground water), clinical and forensic toxicology, drug metabolism and
pharmacokinetics, laboratory medicine (diagnosis/prognosis), therapeutic drug monitoring and
analysis of therapeutic proteins.
Plasma is a derivative of human blood. The challenge in plasma is that you have a huge dynamic
range. There are many different kinds of proteins with varying concentrations. The very low
concentrations are hard to measure. Most often the molecules of interest have a low concentration,
this will determine which methods can be used.
Example of why bioanalysis is so challenging: you need to measure a tumour biomarker in blood to
follow the efficacy of a given therapy or to assess the risk to develop cancer. Blood contains millions
of other proteins. Some of these are present at much higher concentrations than a putative cancer
biomarker. So you need an enormous selectivity to measure the tumour biomarker. A realistic
concentration of biomarker to measure is 0.6 ng/ml. Blood has an overall protein concentration of 60
mg/ml meaning that for each biomarker molecules there will be 10 8 molecules of other proteins in
the blood (assuming the same MW as the biomarker).
Qualitative bioanalysis identify the substance and determine if the substance is present or
absent. Also to make sure that you are measuring the right substance which is not sure if there are so
many other substances around.
Quantitative bioanalysis measure the concentration or amount of the substance. Again you need
to make sure that you measure the right compound. Then you need to quantify it.
Matrix biological material in which a substance must be determined. This is often blood derived
(plasma or serum), urine, cerebrospinal fluid, bronchoalveolar lavage, hair and tissue.
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, Blank matrix biological material without the analyte. So this is the matrix without the compound
that you want to measure. With this you can make a calibration curve.
Calibrator blank matrix containing a known concentration of the analyte. So you add a known
amount of the analyte to the blank matrix to create known concentrations.
Calibration line relates the measures signal to the analyte concentration. This usually is straight.
In case of e.g. ligand binding essays you do not observe a straight line.
Internal standard substance that resembles the analyte as much as possible. This should correct
for variability in the method. It should reduce the variability. This is because in every step of analysis
you are affecting the sample, so you change the analyte concentration. The analyte may absorb a bit
on the plastic or glass or bind to proteins leading to precipitation. So there is loss of analyte in the
process. To correct for this you add an internal standard that behaves in the same way as your
analyte, but still can be discriminated from the analyte using the bioanalytic method.
When a calibration curve goes through 0, indicates that there is nothing in your blank matrix. If this is
not the case this is not a big problems because you can correct for this, but it is important to realise.
Very complex molecules are not synthesized chemically but they are made in cell cultures. The
consequences are that you do not have one homogeneous molecule but you have a family of
molecules. This family has to be consistent but you can never get a unique homogeneous protein.
This just does not exist. Also not our own proteins. This is very difficult for the FDA to accept since it
is not homogeneous. Now this is not a problem anymore. Purifying would cost an endless amount of
money.
Sample preparation:
- Concentration adjustment you have to get your sample to contain a measurable
concentration. This can be via enrichment or dilution. Usually it is enrichment because you
often want to measure low concentrations which cannot be measured without enrichment.
- Removal of interfering compounds phospholipids, amino acids, carbohydrates,
metabolites, etc. You need to prepare your sample into a form that allows you to remeasure
your target analyte.
- Stabilization of analysis many analytes especially biological molecules are not particularly
stable. So when you do all kinds of manipulations of your sample for preparation you need to
make sure that your analyte is not modified and is not lost or converted to something else.
You often have to add things during preparation like protease inhibitors or antioxidants that
will prevent modifications of the analyte.
- Adjustment of analyte properties for example to make a substance volatile so that you
can use gas chromatography or mass spectrometry. For this there are very standardize
methods.
Separation:
- You need to separate your analyte from molecules that give the same or a similar response.
For example if you use a not so sensitive method like light absorption you have plenty of
molecules that will absorb an even amount of light.
- You can increase the depth of analysis separate proteins out and thereby allow certain
proteins to be measured more specifically and sensitive.
- Improve sensitivity
- Improve selectivity
- Make a sample compatible with detection systems e.g. transfer into the gaseous phase. Or
remove molecules which are absolutely not volatile and will contaminate your system.
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