Practicals Flashcards
What is an immunoassay?
A biochemical test that measures the presence or concentration of a small molecule in a solution - usually through antibody/antigen
What is the ELISA assay?
A common serological test to determine the presence of particular antigens or of specific antibodies
Give 3 things the ELISA assay is used for?
- Prenatal diagnosis of the foetal rhesus type
- Determination of tissue transplant compatibility
- Detection of specific infections (e.g. viral and bacterial)
What are the three types of ELISA assay?
- Direct ELISA
- Indirect ELISA
- Capture ELISA (variation of indirect)
What does the direct ELISA do?
Use monoclonal antibodies to detect presence of particular antigen in a sample
What does the direct ELISA use to measure the amount of antigen?
Labelled primary antibody
Describe the process of a direct ELISA (5)
- Measure amount of antigen
- Coat/bind wells of plate with chosen antigen
- Fill wells with dilution of patient serum with primary antibodies conjugated to enzyme
- If antigen specific antibodies present against antigen, they would bind to it and fix to bottom of wells
- Wells washed out (to remove unbound antibody)
Add chromogenic enzyme substrate - Interaction of substrate with enzyme on primary antibody generates visible colour
What type of assay plate is an ELISA performed in?
Multiwell microtiter plate
Why are ELISAs performed in multiwell microtiter plates?
Dilutions of serums can be easily prepared and tested
What does ELISA stand for?
Enzyme-linked immunosorbent assay
What is observed in a direct ELISA plate?
Reaction less intense as serum is diluted (2 fold dilutions) and
Titre isdevelopment
How can the development of colour in wells with specific antibody be observed in an ELISA plate? (2)
- Naked eye
- Quantified with electronic plate reader
How does the serum appear across the direct ELISA plate?
Diluted (2 fold)
How does the serum appear diluted across a
direct ELISA plate? (2)
- Reaction less intense
- Amount of antibody captured in the wells decreases
What titre is taken from a direct ELISA plate?
Highest dilution with definite colour
What antibodies does an indirect ELISA involve?
At least two: primary and secondary
What is the primary antibody in an indirect ELISA?
Specific to antigen of interest but not linked to enzyme
What is the secondary antibody in an indirect ELISA? (3)
- Conjugated to an enzyme
- Specific to primary antibody
- Quantifies how much primary antibody bound to antigen
Describe the process of an indirect ELISA (7)
- Antigen coating
- Add patient serum sample (primary antibody)
- Wash
- Add enzyme labelled/covalently conjugated anti human immunoglobulin (secondary antibody)
- Wells washed again (removes unbound enzyme conjugated antibody)
- Add chromogenic enzyme substrate
- Interaction of substrate with enzyme on second antibody generates visible colour
What is the main difference between a direct and indirect ELISA?
- Direct = only a labelled primary antibody is used
- Indirect = antigen bound by primary antibody detected by a labelled secondary antibody
What is a variation of the indirect ELISA known as?
Capture/sandwich ELISA
What does a capture/sandwich ELISA allow? (2)
- Complex antigen samples e.g serum to be assayed for specific antigen presence
- Presence of antibody against specific infection
Give an example of when a capture/sandwich ELISA is used
To detect HIV virus in a serum sample
What is the correlation between colour change and antibody concentration in an ELISA plate?
The higher the concentration of antibody present in the serum, the stronger the colour change.
How is an ELISA plate - quantified with electronic plate reader?
Detector reports absorbance of light as a numerical (optical density) OD405 reading
What wavelength of light is used to read ELISA plates?
405nm
What is the purpose of flow cytometery?
Count and analyse: - Cell size - Cell shape - Cell properties Within heterogenous cell population
What is flow cytometery data like?
Extremely quantitative
What does flow cytometery data analysis require?
In depth analysis with specific programmes
Describe how to carry out flow cytometery (9)
- Suspension of individual cells prepared from cells/tissues/organisms
- Sample placed in flow cytometer
- Sucks up sample
- Mixes it into saline solution in cytometer
- Pushes cell suspension through narrowing channel
- Causes cell to form single file line before cells pass through laser at integration point
- Each cell passes through beam for individual analysis
- Flow cytometer detects light scattered in a forward manner= forward scatter
- Flow cytometer detects light scattered in a sideways manner = side scatter
What flow cytometry was examined in the practical?
Human peripheral blood leucocyte isolated from a normal healthy individual
What does a flow cytometer contain? (5)
- Sample
- Lasers
- Optics gathering light
- Detectors sense the light
- Computer system to output data into form that can be analysed
In flow cytometery, what is forward scatter light from each cell detected by?
A detector on a far side of the cell from the laser
What is forward scatter proportional to in flow cytometery?
Cell size
How is forward scatter in flow cytometery analysed?
- Detector converts the scattered light into a voltage pulse directly proportional to forward scatter light
- Computer converts to histogram with forward scatter light on X axis and cell count on Y axis
How is side scatter in flow cytometery detected?
By detector perpendicular to path on laser beam
What is side scatter proportional to in flow cytometery? (2)
- Cell shape
- Cell complexity
How is side scatter in flow cytometery analysed?
Flow cytometer converts detected side scatter into a voltage pulse directly proportional to side scattered light
What can be divided and how from flow cytometery by analysing forward and side scatter data together?
A hetereogenous cell population into differing cell size, shape and complexity
What is a strength of flow cytometery experimental technique?
Capability of analysing multiple populations in a sample
Explain how flourescent molecules are used in flow cytometery (5)
- Fluorescent molecules = excited by the correct wavelength of laser light when the cell passes
through the laser beam - After excitation, light emitted by fluorophore = directed along path with emission filters
- Allow for detection of multiple different flurophore/colours emitting light in a cell
- Quantifies relative amount of fluorophore within a cell
- Data presented as histogram/dot plot
List 3 examples of fluorescent molecules in a cell
- Florescent labelled antibodies
- Fluorescent dye
- Fluorescent stain
What does the use of fluorescent molecules in flow cytometery allow analysis of?
Individual populations of cells within a heterogeneous mix of cells in a sample
What is cellular cytotoxicity?
Ability of immune system effect cell to actively lyse a target cell
What treatment effects was cellular cytotoxicity used to investigate in the practical? (2)
- Lysis of tumour cells
- DNA fragmentation of tumour cells
What is the measurement of cellular cytotoxicity used for? (2)
- Identifying compounds that might pose certain health risks in humans
- Cytotoxicity levels of cancer cells: anti-cancer medications hinder proliferation of target cells
What 3 ways is cellular cytotoxicity measured? (3)
- Vital (formazan) dyes
- Protease biomarkers
- Measuring ATP content
How are vital (formazan dyes) relevant to cellular cytotoxicity?
They are formed by the reduction of salts released at cell death
