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Body Defence > Practicals > Flashcards

Practicals Flashcards

1
Q

What is an immunoassay?

A

A biochemical test that measures the presence or concentration of a small molecule in a solution - usually through antibody/antigen

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2
Q

What is the ELISA assay?

A

A common serological test to determine the presence of particular antigens or of specific antibodies

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3
Q

Give 3 things the ELISA assay is used for?

A
  • Prenatal diagnosis of the foetal rhesus type
  • Determination of tissue transplant compatibility
  • Detection of specific infections (e.g. viral and bacterial)
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4
Q

What are the three types of ELISA assay?

A
  • Direct ELISA
  • Indirect ELISA
  • Capture ELISA (variation of indirect)
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5
Q

What does the direct ELISA do?

A

Use monoclonal antibodies to detect presence of particular antigen in a sample

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6
Q

What does the direct ELISA use to measure the amount of antigen?

A

Labelled primary antibody

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7
Q

Describe the process of a direct ELISA (5)

A
  • 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
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8
Q

What type of assay plate is an ELISA performed in?

A

Multiwell microtiter plate

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9
Q

Why are ELISAs performed in multiwell microtiter plates?

A

Dilutions of serums can be easily prepared and tested

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10
Q

What does ELISA stand for?

A

Enzyme-linked immunosorbent assay

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11
Q

What is observed in a direct ELISA plate?

A

Reaction less intense as serum is diluted (2 fold dilutions) and
Titre isdevelopment

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12
Q

How can the development of colour in wells with specific antibody be observed in an ELISA plate? (2)

A
  • Naked eye

- Quantified with electronic plate reader

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13
Q

How does the serum appear across the direct ELISA plate?

A

Diluted (2 fold)

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14
Q

How does the serum appear diluted across a

direct ELISA plate? (2)

A
  • Reaction less intense

- Amount of antibody captured in the wells decreases

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15
Q

What titre is taken from a direct ELISA plate?

A

Highest dilution with definite colour

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16
Q

What antibodies does an indirect ELISA involve?

A

At least two: primary and secondary

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17
Q

What is the primary antibody in an indirect ELISA?

A

Specific to antigen of interest but not linked to enzyme

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18
Q

What is the secondary antibody in an indirect ELISA? (3)

A
  • Conjugated to an enzyme
  • Specific to primary antibody
  • Quantifies how much primary antibody bound to antigen
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19
Q

Describe the process of an indirect ELISA (7)

A
  • 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
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20
Q

What is the main difference between a direct and indirect ELISA?

A
  • Direct = only a labelled primary antibody is used

- Indirect = antigen bound by primary antibody detected by a labelled secondary antibody

21
Q

What is a variation of the indirect ELISA known as?

A

Capture/sandwich ELISA

22
Q

What does a capture/sandwich ELISA allow? (2)

A
  • Complex antigen samples e.g serum to be assayed for specific antigen presence
  • Presence of antibody against specific infection
23
Q

Give an example of when a capture/sandwich ELISA is used

A

To detect HIV virus in a serum sample

24
Q

What is the correlation between colour change and antibody concentration in an ELISA plate?

A

The higher the concentration of antibody present in the serum, the stronger the colour change.

25
Q

How is an ELISA plate - quantified with electronic plate reader?

A

Detector reports absorbance of light as a numerical (optical density) OD405 reading

26
Q

What wavelength of light is used to read ELISA plates?

A

405nm

27
Q

What is the purpose of flow cytometery?

A 
Count and analyse:
- Cell size 
- Cell shape 
- Cell properties
Within heterogenous cell population
28
Q

What is flow cytometery data like?

A

Extremely quantitative

29
Q

What does flow cytometery data analysis require?

A

In depth analysis with specific programmes

30
Q

Describe how to carry out flow cytometery (9)

A
  • 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
31
Q

What flow cytometry was examined in the practical?

A

Human peripheral blood leucocyte isolated from a normal healthy individual

32
Q

What does a flow cytometer contain? (5)

A
  • Sample
  • Lasers
  • Optics gathering light
  • Detectors sense the light
  • Computer system to output data into form that can be analysed
33
Q

In flow cytometery, what is forward scatter light from each cell detected by?

A

A detector on a far side of the cell from the laser

34
Q

What is forward scatter proportional to in flow cytometery?

A

Cell size

35
Q

How is forward scatter in flow cytometery analysed?

A
  • 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
36
Q

How is side scatter in flow cytometery detected?

A

By detector perpendicular to path on laser beam

37
Q

What is side scatter proportional to in flow cytometery? (2)

A
  • Cell shape

- Cell complexity

38
Q

How is side scatter in flow cytometery analysed?

A

Flow cytometer converts detected side scatter into a voltage pulse directly proportional to side scattered light

39
Q

What can be divided and how from flow cytometery by analysing forward and side scatter data together?

A

A hetereogenous cell population into differing cell size, shape and complexity

40
Q

What is a strength of flow cytometery experimental technique?

A

Capability of analysing multiple populations in a sample

41
Q

Explain how flourescent molecules are used in flow cytometery (5)

A
  • 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
42
Q

List 3 examples of fluorescent molecules in a cell

A
  • Florescent labelled antibodies
  • Fluorescent dye
  • Fluorescent stain
43
Q

What does the use of fluorescent molecules in flow cytometery allow analysis of?

A

Individual populations of cells within a heterogeneous mix of cells in a sample

44
Q

What is cellular cytotoxicity?

A

Ability of immune system effect cell to actively lyse a target cell

45
Q

What treatment effects was cellular cytotoxicity used to investigate in the practical? (2)

A
  • Lysis of tumour cells

- DNA fragmentation of tumour cells

46
Q

What is the measurement of cellular cytotoxicity used for? (2)

A
  • Identifying compounds that might pose certain health risks in humans
  • Cytotoxicity levels of cancer cells: anti-cancer medications hinder proliferation of target cells
47
Q

What 3 ways is cellular cytotoxicity measured? (3)

A
  • Vital (formazan) dyes
  • Protease biomarkers
  • Measuring ATP content
48
Q

How are vital (formazan dyes) relevant to cellular cytotoxicity?

A

They are formed by the reduction of salts released at cell death

Body Defence (11 decks)

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