MCB 4211 Sept 15 2020 Lecture: - PowerPoint Presentation

Slide1

MCB 4211

Sept 15 2020

Lecture:

What are the structural characteristics of antibodies?

Reading: Immunobiology textbook Chapter 4: “Antigen recognition by B cell and T cell receptors”A. Immunoglobulin structure1. subunit structure2. heavy and light chains3. Immunoglobulin fragments and their uses: Fc, Fab, and F(ab’)2B. Antigenic determinants of immunoglobulins1. isotypes and subclasses 2. idiotypes3. allotypes

Quiz #1: 10 questions

will focus on the material presented to date

Terminology

Antigen (Ag) recognized by

TcR or BcRAntibody (Ab) on surface of a B cell is BcR(note: B cells come from Bursal of Fabricius in birds, or Bursal equivalent; bone marrow in mammals)(note: T cells come from the Thymus)Antibodies were found in the gamma fraction of electrophoretic separation, and were noted to be globular proteins, so then termed ”gamma globulins”. When specifically functioning as antibodies, they were also called immunoglobulins (Ig)

Y

Basic structure of an antibody/immunoglobulin

Light Chain is 25

KDa

Heavy Chain is 50 Kda2H2L is 150KDabilaterally symmetricalInterchain bondsH and L chains held together by disulfide bridgesH chains held together by disulfide bridgesIntrachain disulfide bondsThere are disulfide bridges that define the ends of immunoglobulin domainsTypical structure of antibody

Immunoglobulin domains

The H and L chains are composed of individual domain structures that are marked by cysteines at the end (the contribute the SH groups for SS bonds)

These domains are found in many other proteins as well (examples include TcR, CD4, CD8, MHC class I and MHC class II

These are all members of the

Immunogloblulin

supergene family of genes

Possible source of amplified genetic material that produces deletions and duplications

Results of these gene duplications and deletions:

Too much duplication or deletion can be lethal configuration

But, where the deletions/duplications are smaller, they are survivable, and offer the opportunity to mutate the second copy to a new functionWe see this ABBA type structure of genes in, for example, the MHC, which may be an example of this mechanism

Fragmenting antibody with Enzymes

Papain (from Papaya) cuts above the hinge region and produces two different fragments: Fab (monovalent fragment antigen binding) and Fc (Fragment crystallizable)

Fab is monovalentFc region mediates FcR binding Pepsin cuts below the hinge and also digests the portion to smaller peptides; the main fragment is a bivalent F(ab’)2

Predict the effect of adding Fab fragments to a system of (antibody plus antigen) where the Ab + Ag can produce immune complexes

Predict the effect of adding F(ab’)

2 fragments to a system of (antibody plus antigen) where the Ab + Ag can produce immune complexes

Kabat and Wu; sequencing Bence Jones Proteins

Bence Jones proteins are monoclonal light chains that appear in urine of B cell tumor (neoplastic plasma cells)

Kabat and Wu sequenced the VL domains of a number of different cancer patients and assembled a variability plot from the data

They identified three

Hypervariable regions amongst “framework residues” that are more consistent between individual antibody sequences on both the H and L chains

Five different classes or isotypes of antibodies in mammals

https://

www.prosci-inc.com/resources/antibody-development-guide/antibody-structure-and-properties/J chainyesnoyesnono

Immunoglobulin Heavy chains

IgM

IgGIgAIgDIgEHeavy chain classm, mug,gammaa, alphad, deltae, epsilonImmunoglobulin light chains

Light chain class

l

, lambda

k

,kappa

Beyond isotypes, there are other antigens associated with antibody structure

Idiotype: the antigenic character of the antigen binding site

Allotype: allelic shapes encoded by alleles of the Ig H and L genes that are inherited

Note: you can make anti-idiotypic antibodies,

etc

NOTE: you can make antibodies to the antigens characteristic of antibodies

The T cell receptor (

TcR

) also has variable regions that interact with antigenBUT the interactions are not with soluble antigen

Now for the quiz…

Next slides for next lecture

5 forces the contribute to Ab binding to Ag

Individually weak, but many of them combine to make high affinity interactions

Affinity of antibody is measured as affinity constant[Ab] [Ag][AbAg]KA=[Ab] [Ag]

[

AbAg

]

K

A

is association constant or affinity

This is a calculation for a monovalent Fab. Bivalent antibodies have much higher apparent association with antigen called AVIDITY

How to measure affinity: Equilibrium dialysis

Other measures: Surface plasmon resonance