MB - Immunoglobulins II

Question 1

What is the structure of the first Ig domain in both light and heavy chains?

Answer 1

First Ig domain in both light and heavy chain is variable in amino acid sequence and structure (VL and VH)

Question 2

What is the antigen recognition site made up of?

Answer 2

Antigen recognition site
formed by VL and VH

Question 3

What regions are present in the VH domain?

Answer 3

Multiple hypervariable regions

• Hypervariable regions are found in loops

Question 4

What are some features of H and L chains? (3)

Answer 4

• VH and VL both have highly variable sequence/structure
• Antigen binding site a combination of both
• 2 possible L chains per H chain

Question 5

What is the genetic basis for the variability? (2)

Answer 5

Not enough genes in genome for one gene/Ab

1) Combining of genes during maturation of B cells
2) Mutation of genes during activation of B cells

Question 6

What occurs during Gene recombination - Light chain (kappa)?

Answer 6

3 sub-genes combined to make final version

• V(ariable) [40 copies]
• J(oining) [5 copies]
• C(onstant) [1 copy]

V + J = VL domain (110aa)
(1-97) (98-110)

Any of the 40 V can be combined with any of the 5 J. Joining point can vary → Many different VL

Question 7

What occurs during Gene recombination - Light chain (lambda)?

Answer 7

3 sub-genes combined to make final version

• V [30 copies]
• J [4 copies]
• C[4 copies]

Question 8

What occurs during gene recombination - heavy chain?

Answer 8

4 genes combined to make final version

• V [51 copies]
• D(iversity) [27 copies]
• J [6 copies]
• C [Cμ,Cδ etc]

V + D + J = VH domain

Additional nucleotides added between V and D gene

Question 9

What is class switching in Igs?

Answer 9

• Naïve B cells express IgM and IgD (1º response)
• Activated B cells express IgG, IgE or IgA (2º response)

Switch in the C gene used switches function of Ab, but antigen recognition is the same

Question 10

What are 6 useful properties of antibodies as laboratory tools?

Answer 10

1. Resistant to denaturation
2. Highly specific
3. Very strong binding
4. Easily generated and collected in polyclonal form (from blood)
5. Easily purified (because of 1, 2 and 3)
6. Easily labelled

Question 11

How can antibodies be collected and purified? (5)

Answer 11

Affinity purification from diluted serum

• Coat chromatography bead with antigen
• Make affinity column
• Pass antibodies through the column and collect attatched antibodies
• (Pass acid through) Ab denatures and elutes from beads
• Ab collected in basic solution and refolds

Question 12

How can labelling be used to collect Abs?

Answer 12

Purified Ab can be modified

Enzyme, radioactive or fluorescent tag attached

(ELISA, Immunohistochemistry, Immunofluorescence, Immunoblots)

Key tool: Ab against Ab

Ab can be raised to Fc region of other species’ Ab = 2º Ab

Question 13

What are some drawbacks of antibodies as laboratory tools?

Answer 13

1. Large, can’t enter cells
2. Can only investigate interior of isolated, dead, fixed cells/tissue slices
3. Hard/expensive to produce in large quantities as monoclonals
4. Can’t make in bacteria

• Assembly X
• Glycosylation X

Question 14

What are Camelid and Shark antibodies? (6)

Answer 14

Single domain for antigen recognition

• Can be expressed in E.coli
• “Nanobodies”
• Can enter living cells and bind cytoplasmic proteins
• Even more stable (can be boiled)
• Still very expensive to produce

Heavy chain only