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CMB2004 > L12 > Flashcards

L12 Flashcards

1
Q

What secrete AB?

A

BCR (plasma cells)

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2
Q

What do AB bind to?

A

Native AG

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3
Q

Why can binding be well characterised?

A

They are monoclonal

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4
Q

What is the plasma

A

fluid phase of blood

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5
Q

What is serum

A

plasma - blood cot

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6
Q

What is antiserum

A

serum from immunised people/animal

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7
Q

What does antiserum not contain

A

cells or clotting proteins

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8
Q

What does antiserum contain?

A

Ab, soluble blood components (like growth factors, other proteins, etc)

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9
Q

Why can antiserum contain diff AB binding to the same Ag?

A

Different ab may bind to different epitopes

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10
Q

What are the methods employed to purify Ab from other serum proteins?

A

gel filtration chromatography - seperates based on MW
affinity chromatography

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11
Q

What are the limitations of using antiserum

A

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)

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12
Q

What are myelomas?

A

Plasma cell tumour

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13
Q

How were monocolonal Ab generated

A

Immunise mouse with Ag, harvest spleen, fuse spleen cells and myeloma cells with PEG = immortal hybridomas

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14
Q

What can myelomas do?

A

Produce
large amounts of homogenous antibodies

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15
Q

What make hybridomas?

A

Mouse myeloma cells and spleen Ab producing cells

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16
Q

Do hybridomas grow forver?

A

Yes, in a culture

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17
Q

Do labels affects Ab/Ag binding?

A

No

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18
Q

Why are labels used?

A

Easily detected & allow presence of Ag to be confirmed.

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19
Q

What does the detection of a lebelled or flurochrome AB show?

A

Presence of Ag of interest

20
Q

What are secondary antibodies used for?

A

Detecting primary (which is binding to its Ag)

21
Q

What does the use of secondary Ab result in?

A

Increase in sensitivity

23
Q

Why are Ab used in research and diagnostics?

A

Excellent purifying tools - isolating/identifying biological molecules of interest

24
Q

What are the uses of Ab in research?

A

affinity chromatography, immunofluorescence microscopy, western blotting, ELISA,

25
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
26
What types of epitope does ELISA work for?
Linear and non-linear
27
How sensitive is ELISA?
Very
28
What is ELISA used to detect?
or detecting the presence of a specific biological material in a wide range of complex sample
29
How do we use ELISA for diagnostics?
Can be used to quantitate the amount of AG present
30
What are the three types of ELISA?
direct, indirect and sandwich
31
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
32
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
33
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
34
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.
35
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
36
What is FACS
Fowrward Scatter results - cell size Side scatter results - granularity
37
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”
38
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
39
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
40
will different fluorochromes have emission “cross-over” when detected
yes
41
how is the detector cross-over removed
compensation
42
What is RPE
large molecule used for fluorescence-based detection
43
What is FITC
is a green fluorescent dye that's used in flow cytometry and other applications to label proteins, antibodies, and other molecules
44
Only once compensation has been been performed, can we be confident of the results seen with multi-colour staining. true or false
true
45

CMB2004 (12 decks)

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