MCB 4211 SEPT 15 2020 Lecture What are

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 structure 1. subunit structure 2. heavy and light chains 3. Immunoglobulin fragments and their uses: Fc, Fab, and F(ab’)2 B. Antigenic determinants of immunoglobulins 1. isotypes and subclasses 2. idiotypes 3. allotypes Quiz #1: 10 questions will focus on the material presented to date

Terminology Antigen (Ag) recognized by Tc. R or Bc. R Antibody (Ab) on surface of a B cell is Bc. R (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)

Basic structure of an antibody/immunoglobulin Y

Typical structure of antibody Light Chain is 25 KDa Heavy Chain is 50 Kda 2 H 2 L is 150 KDa • bilaterally symmetrical • Interchain bonds • H and L chains held together by disulfide bridges • H chains held together by disulfide bridges • Intrachain disulfide bonds • There are disulfide bridges that define the ends of immunoglobulin domains

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 Tc. R, CD 4, CD 8, MHC class I and MHC c 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 function We 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 monovalent Fc region mediates Fc. R 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 J chain yes no no https: //www. prosci-inc. com/resources/antibody-development-guide/antibody-structure-and-properties/

Immunoglobulin Heavy chains Ig. M Heavy chain class m, mu Ig. G Ig. A Ig. D Ig. E g, gamma a, alpha d, delta e, epsilon Immunoglobulin light chains Light chain class l, lambda k, kappa NOTE: you can make antibodies to the antigens characteristic of antibodies 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

The T cell receptor (Tc. R) also has variable regions that interact with antigen BUT 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] [Ab. Ag] KA is association constant or [Ab] [Ag] affinity This is a calculation for a monovalent Fab. Bivalent antibodies have much higher apparent association with antigen called AVIDITY KA=

How to measure affinity: Equilibrium dialysis Other measures: Surface plasmon resonance