Immunoglobulins

Question 1

The 3 most abundant plasma proteins

Answer 1

Question 2

Antigen

Answer 2

Any foreign molecule bound selectively by antibodies

Question 3

Immunogen

Answer 3

Molecule (protein, carbohydrate, nucleic acid) that induces antibody production

Question 4

Antigenic determinant/ epitope

Answer 4

Small region of a larger molecule (like a protein) that elicits the production of a specific antibody

Question 5

There are __ peptide chains in an antibody

___ holds the 2 heavy chains of an antibody together and a light chain to the heavy chain.

Within the ___ chain there are 3 constant domains and 1 variable domain.

Answer 5

4 peptide chains

Disulfide bonds.

The heavy chain has the 3 constant domains (C_H1-C_H3) and 1 variable domain (V_H); defines the class of immunoglobulin

Question 6

The light chain can be one of two types

Answer 6

lambda or kappa

Both have constant (C_L) and variable (V_L) regions

Question 7

The variable domains from both the heavy and light chains all face to one end of the molecule and serve as

Answer 7

antigen binding sites to the specific epitopes

Question 8

The five immunoglobulin classes GADEM all have different ___ chains.

Match the chain with teh class.

Question 9

Which Igs contain multiples of the 2heavychain,2lightchain tetramer?

Answer 9

IgA is a dimer

IgM is a pentamer

Question 10

The individual constant and variable domains are supersecondary structures.

Describe the Immunoglobulin fold in constant domains vs variable domains

Answer 10

Each domain is composed of B-strands that form anti-parallel B-sheets.

• Constant domains* - 7 strands held together in a 4-3 arrangement by hydrogen bonds or hydrophobic interactions between the R groups.
• Variable domains* - 9 strands held together in a 5-4 arrangement

Question 11

Complementarity Determining Regions

What are they and where are they?

Answer 11

Area of hypervariable amino sequence that is complementary to the epitope; serve as antigen-binding sites.

In the variable region of antibody light and heavy chains; they’re in the loops that connect the antiparallel B strands (3 loops from light chain, 3 loops from heavy chain)

Question 12

The combine dvariability of the 12 CDR sites from all 4 peptide chains provides the diversity for

Answer 12

antibody:epitope interactions

Question 13

The regions between CDRs are called

Answer 13

Framework regions

Question 14

Steps of an ELISA immunoassay

Answer 14

ELISA shows the quantitation of any protein in a sample

The primary antibody is specific for the protein/antigen of interest.

Question 15

ChIP

Purpose & steps

Answer 15

• Purpose
  
  • Demosntrates whether specific proteins are associated with specific regions on the genome
  • Determines specific locations in the genome where epigenetic modifications occur
• Steps
  
  • Crosslink protein to DNA
  • Shear DNA into short pieces
  • Use antibodies to immunoprecipitate proteins from the mixture
  • Sequence the immunoprecipitated DNA

Question 16

Polyclonal vs Monoclonal antibodies

Answer 16

Polyclonal antibodies are produced by B lymphocytes in response to antigens; it’s a range of different antibodies that recognize all the different epitopes on the target antigen.

• Occurs in your body all the time

Monoclonal antibodies: synthesized by a population of identical B lymphocytes in a cell culture all producing antibodies to only one epitope; it’s a homogenous antibody population that all recognize tehs ame epitope.

• Used for therapy against one epitope

Question 17

Myelomas

Answer 17

• Cancer of plasma cells (B-lymphocytes that secrete Igs)
• Elevated Ca2+, renal failure, anemia, bone lesions (CRAB)
• Markers
  
  • High serum [monoclonal antibody/Mprotein]
  • Urinate Bence Jones protein (monoclonal light chains)

Question 18

Producing monoclonal antibodies: how can cells making antibodies against one specific epitope be isolated from the rest of the B cells in the polyclonal population and also be made immortal (myeloma cells)?

Answer 18

1. Inject an antigen into a mouse to produce a polyclonal population of antibodies against the antigen
2. Remove and fuse the spleen cells with myeloma cells, which are engineered to be HGPRT negative (won’t grow in a HAT medium)
3. Spleen cells that can’t fuse with myeloma cells will die because they are mortal; myeloma cells will die since they can’t grow in the HAT medium
4. Only the hybridomas (antibody-producing spleen cells + immortal myeloma cells) can grow in the HAT medium
5. Titrate the survivors so no more than 1 cell in a single well and let them grow.
6. Assay the supernatant for an antibody against the antigen of interest and then clone –> monoclonal populations

Question 19

Our monoclonal antibody may be very specific for the target epitope, but humans would hae an immune response to a mouse Ig.

how did we address this?

Answer 19

Chimeric monoclonal antibody: variable domain from mouse, but the rest is from humans

mouse hypervariable CDRs cloned in-frame to the human framework for both light and heavy chains

Question 20

If something ends in -mab, what is it?

Answer 20

Monoclonal antibody .

A drug ending in -mab is an antibody-based therapeutic agent

Question 21

Western blots / Immunoblots

Answer 21

1. Separate proteins on SDS gels
2. Transfer to membrane
3. Block membrane with nonspecific protein
4. Incubate with primary antibody
5. Incubate with antibody-enzyme that binds to primary antibody
6. Add substrate
7. Colored product precipitates on membrane where the specific protein is located

Question 22

Most abundant plasma protein

Answer 22

albumin

Question 23

Most abundant immunoglobulin in plasma

Answer 23

IgG

Question 24

The antigen binds at the __ terminal of the antibody

Answer 24

N

Question 25

Where in this molecule is a protease most likely to attach? Also recognize the diff regions

Answer 25

Hinge region