L12: Antibody Based Technologies

Question 1

Why are Abs a useful biological tool?

Answer 1

They bind free Ag which can be virtually anything and binding can be well characterised (as they’re monoclonal) and often of high affnity

Question 2

Why are Abs easy to purify?

Answer 2

They are secreted in high amounts from differentiated B/ plasma cells

Question 3

Name and explain an early approach of Ab use

Answer 3

Antiserum

• following immunisation Ag Abs are found in fluid phase of blood, called plasma
• serum is plasma once blood clot removed
• serum from immunised organism= antiserum

Question 4

How do antiserums work?

Answer 4

They contain Abs that bind Ag, along with other soluble blood components (growth factors, other proteins)

Question 5

Antiserum contains cells and clotting products. True or false?

Answer 5

False

Question 6

What does antiserum contain?

Answer 6

Many different Abs that have been secreted by different B cells

• may also contain different Abs that bind the same Ag, even if Ag is a single purified protein
• different Abs may bind a different epitope of same Ag

Question 7

How can different Abs be purified away from other serum properties?

Answer 7

Via gel filtration or affnity chromatography

Question 8

Name limitations of using antiserum

Answer 8

• if each individual Ab could be seperated, would be even better but hard to do so as they share MW
• once used, another individual will need immunuising and the Abs generated will never be identical

Question 9

Name one way to counter a limitation of antiserum use

Answer 9

Monoclonal Abs can be generated, producing hybridomas so Abs are now identical

Question 10

What do other methods using Abs rely on?

Answer 10

• Labels attached to Abs in order to detect them (label shouldn’t affect Ab/Ag binding)
• Secondary Abs: to detect primary Ab binding to its Ag, increasing sensitivity (signal from label doubled)

Question 11

How are secondary Abs produced?

Answer 11

1. Collect normal mouse sera
2. Sera contains many different mouse Abs
3. Mouse Abs purified and rat immunised
4. Rat makes Abs to this so we collect rat anti-mouse antiserum
5. Purify rate anti-mouse polyclonal Abs§

Question 12

Explain the uses of Abs in research

Answer 12

As Ab/Ag interactions are specific they make excellent tools for purifying, isolating and identifying biological molecules of interest

Question 13

Name and explains way of identifying biological molecules of interests using Abs

Answer 13

• Affinity chromatography: purifies biological molecules from a mixture
• Magnetic breed isolation (MACS): lymphocytes mixed with Abs coupled to beads poured over an iron wool mesh. When magnetic field applied unlabelled cells wash out, leaving coupled cell
• Isolating bio molecules from more limited supply of a mixture e.g. metabolically labelled cells via Immunoprecipitation
• Immunofluorescence microscopy: identifying protein location within a cell

Question 14

Why may Ab be required to recognise 3D structure of Ag?

Answer 14

This applies if Ab is required to recognise a protein in ‘situ’
- generally alpha monoclonal Abs that recognise 3D structure rquires Ag is in its proper folded state when used to immunise

Question 15

If Ab isn’t required to recognise Ag in its 3D format what is it best to immunise with?

Answer 15

Either denatured protein or synthesised peptide

Question 16

Name a commonly used research technique where Abs are required to recognised unfolded, denatured proteins

Answer 16

Western Blotting

Question 17

Explain how ELISA (enzyme linked immunosorbent assay) is used in reserach and diagnostic labs

Answer 17

• works well for detection of both 3D and linear epitopes
• very sensitive method: used for detecting presence of specific bio material & can be used to quantitate amount of Ag present (diagnostic)
• very reproducible, easily automated for scaling up to screen many samples simultaneously

Question 18

Name and explain the different formats of ELISA

Answer 18

All rely on the ‘capture’ of Ag onto a solid surface
1. Direct ELISA: bio sample containing Ag added to well in plate, labelled Ab specific for Ag and substrate for enzyme label added. Then substrte conversion (colour) measured

2. Indirect ELISA: more sensitive. bio sample containing Ag added to well in plate. Unlabelled primary Ab specific Ag added. Labelled secondary added and then substrate for enzyme label. Measure substrate conversion
3. Sandwich ELISA: even more specifc & uses less of sample. unlabelled Ab specific Ag added to plate (captures Ab). Add sample then different Ag specific labelled secondary and then substrate. Measure substrate conversion

Question 19

Explain the function of flow cytometry (FACS)

Answer 19

Used for characterisation of cells based on their light scattering properties

• properties are either natural or induced by pre-incubation of cell with Abs labelled with fluoro dyes
• used in research and clinical labs
• measures how single cells in a population affect a laser beam, as they pass through it
• works quickly & multiple parameter from each cell can be measured simultaneously

Question 20

Distinguish between forward scatter (FSC) and side scatter (SSC)

Answer 20

FSC results from a cell’s size, while SSC results from a cell’s granularity

Question 21

How can cell types in a mixture be identified using FACS?

Answer 21

By the different effects they have on flow cytometer’s laser

- software allows us to focus in on distinct populations by drawing a gate

Question 22

How can more info be obtained from FACS?

Answer 22

By adding Abs to cells before they pass through the laser e.g. immune cells can be identified by their expression of molecules such as CD3/4/8/19 etc
- Abs specific for these molecules can be labelled (conjugated) with fluoro dye and used to identify # of Ab +ve cells in population

Question 23

How is data generated from FACS, using blood as an example?

Answer 23

Cells with a single fluorescently-conjugated Ab incubated before analysis to allow furthr definition of cells.
1- labelled cells analysed for FSC/SSC then set a gate for cells of interest
2- fluoro level measured on cells in FSC/SSC gate only
3- second gate used to define a sub-population of lymphovytes based on fluoro

Question 24

How can further definition of cells be acheived? (blood used as example)

Answer 24

By incubating cells with 2 different Abs, each with a different fluoro dye
- # cells present in each quadrant allows quantification of # cells expressing CDX, CDY both or neither

Question 25

How are splenocytes analysed following incubation? (blood used as example)

Answer 25

1. setting voltages to measure FSC/ SSC of unstained cells
2. setting gate to define SSC/ FSC of cells where fluoro will be measured
3. setting ‘compensation’ to eliminate cross-over emission detectiom
4. analysis of CD4- FITC and CD8- RPE dual staining

Question 26

How can emission cross-over be eliminated or compensated for?

Answer 26

By analysing emission from cells stained with each fluorochrome individually on each detector
- allows data from cells to reflect accurate labelling