L12 Flashcards
What secrete AB?
BCR (plasma cells)
What do AB bind to?
Native AG
Why can binding be well characterised?
They are monoclonal
What is the plasma
fluid phase of blood
What is serum
plasma - blood cot
What is antiserum
serum from immunised people/animal
What does antiserum not contain
cells or clotting proteins
What does antiserum contain?
Ab, soluble blood components (like growth factors, other proteins, etc)
Why can antiserum contain diff AB binding to the same Ag?
Different ab may bind to different epitopes
What are the methods employed to purify Ab from other serum proteins?
gel filtration chromatography - seperates based on MW
affinity chromatography
What are the limitations of using antiserum
also once the antiserum has been used, then another
individual will need immunizing and the antibodies
generated will never be exactly the same
(remember BCRs; hence antibodies in blood
are generated randomly)
What are myelomas?
Plasma cell tumour
How were monocolonal Ab generated
Immunise mouse with Ag, harvest spleen, fuse spleen cells and myeloma cells with PEG = immortal hybridomas
What can myelomas do?
Produce
large amounts of homogenous antibodies
What make hybridomas?
Mouse myeloma cells and spleen Ab producing cells
Do hybridomas grow forver?
Yes, in a culture
Do labels affects Ab/Ag binding?
No
Why are labels used?
Easily detected & allow presence of Ag to be confirmed.
What does the detection of a lebelled or flurochrome AB show?
Presence of Ag of interest
What are secondary antibodies used for?
Detecting primary (which is binding to its Ag)
What does the use of secondary Ab result in?
Increase in sensitivity
Why are Ab used in research and diagnostics?
Excellent purifying tools - isolating/identifying biological molecules of interest
What are the uses of Ab in research?
affinity chromatography, immunofluorescence microscopy, western blotting, ELISA,
What are the steps required to carry out western blotting?
Lyse the virus, seperate viral proteins based on MW, mAB binds to target protein, visualised by secondary binding - identifies that known protein was present in virus
What types of epitope does ELISA work for?
Linear and non-linear
How sensitive is ELISA?
Very
What is ELISA used to detect?
or detecting the presence of a specific
biological material in a wide range of complex sample
How do we use ELISA for diagnostics?
Can be used to quantitate the amount of AG present
What are the three types of ELISA?
direct, indirect and sandwich
What occurs in direct ELISA?
biological sample containing AG added to well, add labelled Ab for Ag & substrate for enzyme label.
Measure substrate conversion by observing the colour
What occurs in indirect ELISA?
iological sample containing Ag. added to well in plastic plate
* add unlabelled primary antibody specific for Ag
* measure substrate conversion (colour)
Ag
* add labelled secondary and then substrate for enzyme label
What occurs in sandwich ELISA
unlabelled antibody specific for Ag added to plate (capture antibody)
* add sample
* measure substrate conversion (colour)
* add a different Ag specific labelled secondary and then substrate
What is flow cytometry
a technique used for characterisation of cells based on light scattering properties - can be natural or induced by the pre-incubation of cells with antibodies labelled with dyes that fluoresce.
What does flow cytometry do?
measures how single cells in a population affect (scatter)
a laser beam as they pass through it
power of flow cytometry is that it can measure the light scatter
from individual cells in a mixed population very rapidly
(up to 2000 cells/sec)
in addition, multiple parameters from each individual cell can be
measured simultaneously
CMB2004 L12
What is FACS
Fowrward Scatter results - cell size
Side scatter results - granularity
How can different cell types be identified
can be identified by how they effect the flow cytometer’s laser
(as they will have different FSC and SSC values)
software allows us to focus in on distinct
populations (P) by drawing a “gate”
How can immune cells be identified using FACS
can be identified by their expression of
various molecules
e.g. CD3, CD4, CD8, CD19,
antibodies specific for these molecules can be labelled
(conjugated) with fluorescent dyes, and used to identify (and
count) the number of antibody “positive (+)” cells in a mixed
population as the dye attached to the cell surface will emit
light when stimulated by the the flow cytometer’s laser
What sllows further definition of the cells in FACS
Incubating the cells with a fluorescently (FITC)-conjugated
antibody before analysis allows further definition of the the cells
will different fluorochromes have emission “cross-over”
when detected
yes
how is the detector cross-over removed
compensation
What is RPE
large molecule used for fluorescence-based detection
What is FITC
is a green fluorescent dye that’s used in flow cytometry and other applications to label proteins, antibodies, and other molecules
Only once compensation has been been
performed, can we be confident of the results
seen with multi-colour staining. true or false
true
