Practicals

Question 1

What is an immunoassay?

Answer 1

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

Question 2

What is the ELISA assay?

Answer 2

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

Question 3

Give 3 things the ELISA assay is used for?

Answer 3

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

Question 4

What are the three types of ELISA assay?

Answer 4

• Direct ELISA
• Indirect ELISA
• Capture ELISA (variation of indirect)

Question 5

What does the direct ELISA do?

Answer 5

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

Question 6

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

Answer 6

Labelled primary antibody

Question 7

Describe the process of a direct ELISA (5)

Answer 7

• 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

Question 8

What type of assay plate is an ELISA performed in?

Answer 8

Multiwell microtiter plate

Question 9

Why are ELISAs performed in multiwell microtiter plates?

Answer 9

Dilutions of serums can be easily prepared and tested

Question 10

What does ELISA stand for?

Answer 10

Enzyme-linked immunosorbent assay

Question 11

What is observed in a direct ELISA plate?

Answer 11

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

Question 12

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

Answer 12

• Naked eye

- Quantified with electronic plate reader

Question 13

How does the serum appear across the direct ELISA plate?

Answer 13

Diluted (2 fold)

Question 14

How does the serum appear diluted across a

direct ELISA plate? (2)

Answer 14

• Reaction less intense

- Amount of antibody captured in the wells decreases

Question 15

What titre is taken from a direct ELISA plate?

Answer 15

Highest dilution with definite colour

Question 16

What antibodies does an indirect ELISA involve?

Answer 16

At least two: primary and secondary

Question 17

What is the primary antibody in an indirect ELISA?

Answer 17

Specific to antigen of interest but not linked to enzyme

Question 18

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

Answer 18

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

Question 19

Describe the process of an indirect ELISA (7)

Answer 19

• 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

Question 20

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

Answer 20

• Direct = only a labelled primary antibody is used

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

Question 21

What is a variation of the indirect ELISA known as?

Answer 21

Capture/sandwich ELISA

Question 22

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

Answer 22

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

Question 23

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

Answer 23

To detect HIV virus in a serum sample

Question 24

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

Answer 24

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

Question 25

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

Answer 25

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

Question 26

What wavelength of light is used to read ELISA plates?

Answer 26

405nm

Question 27

What is the purpose of flow cytometery?

Answer 27

``` Count and analyse: - Cell size - Cell shape - Cell properties Within heterogenous cell population ```

Question 28

What is flow cytometery data like?

Answer 28

Extremely quantitative

Question 29

What does flow cytometery data analysis require?

Answer 29

In depth analysis with specific programmes

Question 30

Describe how to carry out flow cytometery (9)

Answer 30

- 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

Question 31

What flow cytometry was examined in the practical?

Answer 31

Human peripheral blood leucocyte isolated from a normal healthy individual

Question 32

What does a flow cytometer contain? (5)

Answer 32

- Sample - Lasers - Optics gathering light - Detectors sense the light - Computer system to output data into form that can be analysed

Question 33

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

Answer 33

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

Question 34

What is forward scatter proportional to in flow cytometery?

Answer 34

Cell size

Question 35

How is forward scatter in flow cytometery analysed?

Answer 35

- 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

Question 36

How is side scatter in flow cytometery detected?

Answer 36

By detector perpendicular to path on laser beam

Question 37

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

Answer 37

- Cell shape | - Cell complexity

Question 38

How is side scatter in flow cytometery analysed?

Answer 38

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

Question 39

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

Answer 39

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

Question 40

What is a strength of flow cytometery experimental technique?

Answer 40

Capability of analysing multiple populations in a sample

Question 41

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

Answer 41

- 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

Question 42

List 3 examples of fluorescent molecules in a cell

Answer 42

- Florescent labelled antibodies - Fluorescent dye - Fluorescent stain

Question 43

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

Answer 43

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

Question 44

What is cellular cytotoxicity?

Answer 44

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

Question 45

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

Answer 45

- Lysis of tumour cells | - DNA fragmentation of tumour cells

Question 46

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

Answer 46

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

Question 47

What 3 ways is cellular cytotoxicity measured? (3)

Answer 47

- Vital (formazan) dyes - Protease biomarkers - Measuring ATP content

Question 48

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

Answer 48

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