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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

22
Q
A
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
Q

What are the steps required to carry out western blotting?

A

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
Q

What types of epitope does ELISA work for?

A

Linear and non-linear

27
Q

How sensitive is ELISA?

A

Very

28
Q

What is ELISA used to detect?

A

or detecting the presence of a specific
biological material in a wide range of complex sample

29
Q

How do we use ELISA for diagnostics?

A

Can be used to quantitate the amount of AG present

30
Q

What are the three types of ELISA?

A

direct, indirect and sandwich

31
Q

What occurs in direct ELISA?

A

biological sample containing AG added to well, add labelled Ab for Ag & substrate for enzyme label.

Measure substrate conversion by observing the colour

32
Q

What occurs in indirect ELISA?

A

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
Q

What occurs in sandwich ELISA

A

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
Q

What is flow cytometry

A

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
Q

What does flow cytometry do?

A

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
Q

What is FACS

A

Fowrward Scatter results - cell size
Side scatter results - granularity

37
Q

How can different cell types be identified

A

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
Q

How can immune cells be identified using FACS

A

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
Q

What sllows further definition of the cells in FACS

A

Incubating the cells with a fluorescently (FITC)-conjugated
antibody before analysis allows further definition of the the cells

40
Q

will different fluorochromes have emission “cross-over”
when detected

A

yes

41
Q

how is the detector cross-over removed

A

compensation

42
Q

What is RPE

A

large molecule used for fluorescence-based detection

43
Q

What is FITC

A

is a green fluorescent dye that’s used in flow cytometry and other applications to label proteins, antibodies, and other molecules

44
Q

Only once compensation has been been
performed, can we be confident of the results
seen with multi-colour staining. true or false

A

true

45
Q
A

CMB2004 (12 decks)

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