L20: Immunological techniques/use of antibodies

Question 1

Antibodies

Answer 1

Glycoproteins secreted by B cells, recognise a particular antigen through Fab region. Generation of Diversity results in huge number of antibodies with unique antigen-recognition region. One antibody will recognise a single epitope of an antigen but multiple antibodies (from different B cell clones) might recognise the same antigen

Question 2

Polyclonal antibodies

Answer 2

1. Animal immunised (antigen + adjuvant) followed by boosters
2. Serum (containing complex mixture of antibodies) is collected
3. Antibody can be affinity purified
4. Antibody is heterogeneous, limited supply, batch specific

Question 3

Monoclonal antibodies

Answer 3

1. Animal immunised (antigen + adjuvant) followed by boosters
2. Spleen cells harvested and fused with myeloma cells
3. Single cell clones screened for reactivity against antigen
4. Unlimited supply of antibody with defined specificity

Question 4

Antibody use

Answer 4

1. Cell isolation and identification: magnetic bead isolation, flow cytometry, panning
2. Cell biology: ELISA, immunoblotting, immunoprecipitation, immunocytochemistry/immunofluorescence
3. In vivo studies: antibody-blocking experiments, immunohistochemistry/immunofluorescence
4. Diagnostics

Question 5

Common research questions

Answer 5

1. What is the function of a specific cell type in an immune response? depletion and adoptive transfer of immune cells in vivo, isolation and functional studies of a particular cell type in vitro
2. What is the function of the protein/gene that I’m interested in? blockade of action by antibody/gene knock-out, transgenic over-expression and administration of recombinant protein

Question 6

Functional studies of a specific cell type in an immune response (2)

Answer 6

1. Depletion and adoptive transfer of immune cells in vivo: focus on immune cell development and function and mechanistic understanding in an in vivo setting
2. Isolation and functional studies of a particular cell type in vitro: physiological relevance and mechanistic studying

Question 7

Depletion and adoptive transfer of immune cells in vivo

Answer 7

Can deplete animals of specific cellular compartment, then perform adoptive transfer

1. Haematopoeitic cells: radiation kill, donor cell study
2. Lymphoid cells: radiation, donor cell and immune functions studied
3. T cells: thymectomy, bone marrow reconstituted
4. B cells: lineage-specific Abs
5. Mutant/gene targeted mice: knock-out mutant mice

Question 8

Isolation and functional studies of a particular cell type in vitro

Answer 8

Cellular isolation through:
1. Panning: plate coated with antibody against a particular cell surface protein
2. Magnetic beads: antibody against a particular cell surface protein linked to magnetic beads - cells selected by either positive or negative selection
3. Flow cytometry: antibody linked to fluorescent tag. Cells detected by flow cytometry and sorted into desired populations
Functional studies - proliferation:
1. Antigen specific
2. Polyclonal
3. Method: 3H-thymidine assay and CFSE assay
Cytokine production: sandwich ELISA or ELISPOT assays
Cytotoxic activity: CTLs mixed with target cells loaded with peptide, killing of target monitored through chromium release assay

Question 9

Blocking gene/protein function: antibody or gene-knockout

Answer 9

Blockage of action by antibody: administer antibody or isoptype control in vivo or in vitro and assess biological response
Blockage of action by gene-knockout: use embryonic stem cells and homologous recombination and compare WT vs KO responses

Question 10

Enhancing gene/protein function: transgenic over-expression or recombinant protein administration

Answer 10

Transgenic over-expression: DNA construct injected by pronuclear injection into fertilised egg, implanted into uterus and offspring backcrossed onto stable genetic background
Administration of recombinant protein: test in vivo or in vitro for response