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Generation of Antibody Diversity. The big question:how do you get all those different antibodies? Two other big questions- getting different classes with the same variable regions, and getting secreted and membrane-bound forms of the same class

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generation of antibody diversity
Generation of Antibody Diversity
  • The big question:how do you get all those different antibodies?
  • Two other big questions- getting different classes with the same variable regions, and getting secreted and membrane-bound forms of the same class
  • WARNING: This is hard stuff- and I'll cover more than you need to know- partly to impress you on the capabilities of what's happening to you daily.
overall picture
Overall picture
  • Overall picture- diversity produced by combining a number of variable regions with at least one joining region, plus the constant regions. Diversity is increased by the total number of possible rearrangements. The regions in between are deleted.
  • Both light and heavy chains variable regions involved. We have multiples of multiples resulting in millions of combinations from ~245 gene segments.
slide4

The story in mice

3 possibilities

Gene segments

Chromosome 16

85X 4 possible combinations=340

Chromosome 6

134X 13X 4 possible combinations =6968

Chr. 12

Total = 343X6968= 2,390,024- a lot, but not gazillions.

slide5

How we know this-

Southern blot, using J region as a probe. In a germ line culture, the probe only finds the J region. In a B cell line, it finds the rearranged V region as well. Further cloning and sequencing verified this result.

Fig. 22-9 for more information

slide7

http://www.bio.davidson.edu/COURSES/Immunology/Flash/somaticrecomb.htmlhttp://www.bio.davidson.edu/COURSES/Immunology/Flash/somaticrecomb.html

http://www.uni-greifswald.de/~immuteach/basics/somrec/somrec.html

slide17

Joining flexibility adds more diversity, and also contributes to nonproductive rearrangements

These are the ends of the J and V regions after any P-nucleotide addition has taken place, and also N-nucleotide addition (in H chain)

slide18

Allelic exclusion: successful rearrangement of 1 allele excludes rearrangement of the other allele.

The cell “knows” when it’s done an unproductive rearrangement!

slide19

+

Error!

more diversity somatic hypermutation
MORE Diversity- Somatic Hypermutation!!
  • This occurs AFTER B cell formation, and in response to antigen in the germinal centers.
  • Rate of mutation ~ 10-3 per bp per cell generation- 105 X higher than normal, and just in the CDR 1,2 &3 regions.
  • The process is random, and there’s selection for those that are better at binding antigen.
slide21

Immunize mice w/ antigen (a hapten)

Periodically remove cells and make monoclonals- after primary immunization, 2o, 3o immunizations

Sequence H and V chain mRNA from these monoclonals

Compare sequences and antibody affinity

slide22

Quiz on Wed:

  • Ab classes
  • Somatic recombination: sample calculation
  • Definitions:
  • “sloppy”- DJ v-DJ, or VJ joining produces a variety of outcomes
  • Non-productive rearrangement
  • Allelic exclusion
  • Somatic hypermutation
class switching no not what happens between 9 50 and 10 00
Class switching (no- not what happens between 9:50 and 10:00!)
  • Antibodies come in classes
  • In the maturation process, there is class switching- initially IgM is made, this switches to IgG or A or E due to cytokine influences- e.g. IL-4 induces switch to IgG and sometimes also to IgE.
  • The switching come from deleting previous heavy classes.
  • ?consequences?
slide24

May be sequential, may be Su recombines directly with SE. IL-4 induced

Switch recombinase, signaled by cytokine

Consequences? Can’t go backwards!

These have been found

membrane vs secreted
Membrane vs secreted
  • Apparently all antibody classes can be membrane-bound or secreted, but this mainly gets involved in the initial change of a B cell w/ mIgM and mIgD to a plasma cell that’s secreting IgM.
  • The switch involves alternative splicing.
slide28

In eukaryotes, we make a primary transcript, that can then be cut- where it is cut is where we add a “polyA tail”

Mu really consists of six exons

Delta really conists of 5 exons

antibodies for health and wealth
Antibodies for health and wealth
  • Monoclonals are quite useful, but typically of mouse origin.
  • For use in humans, we want to minimize the antibody response to the mouse-specific Ag determinants.
  • There are a variety of ways to put mouse variable regions onto human antibodies, but we’re not going to talk about them, just the results.
things to know from this chapter lots of stories
Things to know from this chapter- lots of stories!!
  • Be able to tell the generation of Ab diversity story- with some numbers and estimates.
  • Be able to define the processes that add to diversity: p and n-nucleotide addition, joining flexibility, somatic hypermutation.
  • RSS’s, RAGs, nonproductive rearrangement, allelic exclusion, class switching.
  • Be able to tell the story of how class switching and the switch from membrane to secreted Ig occurs.