Week 7 Lec: Antibodies Flashcards
True or false: The term immunoglobulin is synonymous with antibody.
True
What protein is found in the serum portion of the blood?
Glycoprotein
Percentage of polypeptides (protein) in immunoglobulins?
82-96%
Percentage of carbohydrates in immunoglobulins?
2-14%
What method is used to determine the structure of immunoglobulins?
serum electrophoresis
Materials that can be used for serum electrophoresis?
- Cellulose acetate
- Citrate agar
pH of cellulose acetate?
alkaline @ 8.6
pH of citrate agar?
acidic @ 6-6.3
Immunoglobulins appear primarily where?
Gamma Regions
Immunoglobulins are part of the ___________ branch of the immune response.
humoral branch
Immunoglobulins play an essential role during?
Antigen recognition
Immunoglobulins play an essential role in biological activities related to immune response such as?
opsonization and complement activation
Abbreviation for immunoglobulin?
Ig
Proteins in Alpha 1 Globulin?
- alpha-1 antitrypsin
- alpha-fetoprotein
Proteins in Alpha 2 Globulin?
- ceruloplasmin
- haptoglobin
- alpha-2 macroglobulin
Proteins in Beta Globulin?
- transferrin
- hemopexin (pero hemopoietin sa notes ko? ewan)
- complement system
- fibrinogen
- lipoproteins (LDL, HDL, VLDL)
Beta-gamma bridging is associated with what health condition?
liver cirrhosis
A toll-like spike in the gamma regions indicates what health condition?
multiple myeloma
Patients with multiple myeloma have an elevated?
plasma protein
The urine of patients with multiple myeloma contains what type of proteins?
Bence Jones proteins
Bence Jones proteins are what type of chains?
L chains
Bence Jones proteins are being secreted by?
malignant plasma cells
What happens to Bence Jones proteins heated to 60°C?
precipitate
What happens to Bence Jones proteins heated to 80°C?
dissolve
All immunoglobulin molecules are made up of a basic?
four-chain polypeptide unit
The four-chain polypeptide unit of immunoglobulins consists of?
- 2 large chains, or heavy (H) chains
- 2 smaller chains, or light (L) chains
The chains of immunoglobulins are held together by noncovalent forces and?
disulfide linkage/bond or interchain disulfide bridges
The structure of immunoglobulins was first described by two scientists, namely?
Gerald Edelman and Rodney Porter
Gerald Edelman and Rodney Porter won the Nobel Prize in Physiology and Medicine for describing the structure of immunoglobulins in what year?
1972
Edelman’s work centered on using the _______________ to separate out immunoglobulins on the basis of molecular weight.
analytical ultracentrifuge
Edelman’s work centered on using the analytical ultracentrifuge to separate out immunoglobulins on the basis of?
molecular weight
Edelman found that intact IgG molecules had a sedimentation coefficient of?
7S (Svedberg unit)
This indicates the sedimentation rate in an analytical ultracentrifuge.
Svedberg unit
On obtaining a purified preparation of IgG, Edelman used ______ to unfold the molecule.
7 M urea
Once unfolded, the exposed sulfhydryl bonds could be cleaved by a reducing agent, such as?
2-Mercapthoethanol, or simply Mercaptoethanol
After such treatment, the material was subjected again to ultracentrifugation, and two separate fractions were obtained. What are those fractions?
3.5 S and 2.2 S
The molecular weight of the 3.5 S fraction?
50,000
The 3.5 S fraction was designated as what chain?
H chain
The molecular weight of 2.2 S fraction?
22,000
The 2.2 S fraction was designated as what chain?
L chain
Porter’s work was based on the use of what proteolytic enzyme?
papain
Papain, which was used to cleave IgG into three pieces of about equal size, had a sedimentation coefficient of _____, and represented a molecular weight of approximately __________.
3.5 S; 45,000 to 50,000 d
Porter then subjected the immunoglobulin to?
Carboxymethyl cellulose ion exchange chromatography
After the carboxymethyl cellulose ion exchange chromatography process, the material was separated into 2 types of fragments. The one spontaneously crystallized at 4°C is called?
Fc fragment (for “fragment crystallizable”)
The remaining two identical fragments were found to have antigen-binding capacity and were named?
Fab fragments (fragment antigen binding)
How many fragments are there in papain digestion?
3 fragments
What are the 3 fragments in papain digestion?
2 Fab, 1 Fc
The Fab fragment in papain digestion consists of what chains?
1 light chain, 1/2 heavy chain held by disulfide bonding
The Fc fragment in papain digestion consists of what chains?
2 carboxy-terminal halves of a heavy chain
True or false: Fc has an antigen-binding ability.
False. Only Fab has an antigen-binding ability.
The Fc fragment is important in effector functions of immunoglobulin molecules, which include?
opsonization and complement fixation
He used pepsin to obtain additional evidence for the structure of immunoglobulins.
Alfred Nisonoff
This proteolytic enzyme was found to cleave IgG at the carboxy-terminal side of the interchain disulfide bonds.
pepsin
Pepsin was found to cleave IgG at the __________ side of the interchain disulfide bonds.
carboxy-terminal
Cleave IgG yielded?
- one single fragment, molecular weight of 100,000 d, have antigen-binding ability - F(ab’)2
- An additional fragment called FC’, which was similar to FC except that it disintegrated into several smaller pieces
How many fragments are there in pepsin digestion?
2 fragments
What are the 2 fragments in pepsin digestion?
1 F(ab)2, 1 Fc
The F(ab)2 fragment in pepsin digestion consists of what chains?
2 light chains, 2 halves of a heavy chain
The Fc fragment in pepsin digestion consists of what chains?
same with the Fc fragment in papain digestion (2 halves of a heavy chain) except that it disintegrated into several smaller pieces
Regions where the antigen binds to the immunoglobulin?
Variable and Constant regions
The region where the amino-terminal end is located?
Variable region
The region where the carboxyl terminal is located?
Constant region
Fab consists of what region?
Variable region
Fc consists of what region?
Constant region
Which immunoglobulins have 2 domains of L chain and 4 domains of H chain?
IgG, IgA, IgD
What are the 2 domains of L chain and 4 domains of H chain in IgG, IgA, and IgD?
- 2 domains of L chain: Variable light chain and Constant light chain
- 4 domains of H chain: Variable heavy chain and Constant heavy chains 1, 2, and 3
Which immunoglobulins have 2 domains of L chains and 5 domains of H chains?
IgE, IgM
What are the 2 domains of L chain and 5 domains of H chain in IgE and IgM?
- 2 domains of L chain: Variable light chain and Constant light chain
- 4 domains of H chain: Variable heavy chain and Constant heavy chains 1, 2, 3, and 4
Analysis of several Bence Jones proteins revealed that there were two main types of L chains, which were designated as?
kappa (κ) chains and lambda (λ) chains
Kappa chain is coded on chromosome?
chromosome 2
How many amino acids does a kappa chain have?
200
Lambda chain is coded on chromosome?
chromosome 22
How many amino acids does a lambda chain have?
220
The region from position number 111 (the amino terminus is position number 1) is called?
Constant region
The first approximately 110 amino acids at the aminoterminal end constitute which region?
Variable region
True or false: The remaining amino acids after position 110 can typically be divided into three or more constant regions with very similar sequences.
True
True or false: Constant regions of the H chain are unique to each class and give each immunoglobulin type its name.
True
IgG has a/an?
Gamma chain “ɣ”
IgM has a/an?
Mu chain “µ”
IgE has a/an?
Epsilon chain “ɛ”
IgD has a/an?
Delta chain “ẟ”
IgA has a/an?
Alpha chain “α”
A unique amino acid sequence in the H chain that is common to all immunoglobulin molecules of a given class in a given species is referred to as?
Isotype
Genetic variations in the constant regions are referred to as?
Allotype
Minor variations of amino acid sequences that are present in some individuals but not others are known as?
Allotype
Where are allotypes seen?
- In 4 IgG subclasses (IgG1, IgG2, IgG3, and IgG4)
- In 2 IgA subclasses (IgA1 and IgA2)
- Kappa chain of light chain
Variations in variable regions that give individual antibody molecules specificity are referred to as?
Idiotype
The segment of H chain located between the CH1 and CH2 regions is known as the?
Hinge Region
The hinge region is mainly composed of an amino acid called?
Proline
The high proline content of the hinge region allows for?
flexibility
The ability of a hinge region to bend allows?
the two antigen-binding sites operate or work independently
Hinge regions are also part or segment of?
heavy chains
Immunoglobulins that have a hinge region?
IgG, IgA, IgD
Immunoglobulins that DO NOT have a hinge region?
IgE, IgM
The ______________ will determine the number of antigen-binding sites.
valence of immunoglobulin
True or false: The more valence in antibodies, the more antigen-binding sites there are.
True
3 basic immunoglobulin structures?
- Monomer
- Dimer
- Polymer
Immunoglobulin structure that has 2 antigen binding sites?
Monomer
Immunoglobulin/s having a monomer structure?
- IgG
- IgA1/serum IgA
- IgD
- IgE
- IgM (specifically only on the surface of B cells)
Immunoglobulin structure that has 4 antigen binding sites?
Dimer
Immunoglobulin/s having a dimer structure?
IgA2/secetory IgA
Immunoglobulin structure that has more than 4 antigen binding sites?
Polymer or Pentameric
Immunoglobulin/s having a polymer structure?
IgM because it has a valence of 10
True or false: Attempts to explain the specificity of antibody for a particular antigen began long before the actual structure of immunoglobulins was discovered.
True
The 4 theories of antibody diversity?
- Ehrlich Side-Chain Theory
- Clonal Selection Theory
- Template Theory
- Selective Theory
The theory included as part of Erlich Side-Chain Theory?
Selective Theory
Theory involved in the lock and key model of antigen and antibody.
Erlich Side-Chain Theory
He postulated that certain cells had specific surface receptors for antigen that were present before contact with antigen occurred.
Ehrlich
Determine what theory is being described: Once antigen was introduced, it would select the cell with the proper receptors, combination would take place, and then receptors would break off and enter the circulation as antibody molecules.
Erlich Side-Chain Theory
In the 1950s, they independently supported the idea of a clonal selection process for antibody formation.
Niels Jerne and Macfarlane Burnet
When did Niels Jerne and Macfarlane Burnet independently supported the idea of a clonal selection process for antibody formation?
1950s
Most acceptable theory in antibody diversity.
Clonal Selection Theory
Determine what theory is being described: The key premise is that individual lymphocytes are genetically preprogrammed to produce one type of immunoglobulin and that a specific antigen finds or selects those particular cells capable of responding to it, causing them to proliferate.
Clonal Selection Theory
Who created the Template Theory?
Felix Haurowitz
Second major theory of antibody diversity in 1930’s?
Template Theory
Determine what theory is being described: Antibody-producing cells are capable of synthesizing a generalized type of antibody, and when contact with an antigen occurs, the antigen serves as a mold or template and alters protein synthesis so the antibody with a specific fit is made. This specific antibody produced enters the circulation, while antigen remains behind to direct further synthesis.
Template Theory
They discovered a technique to produce antibody arising from a single B cell.
George Kohler and Cesar Milstein
George Kohler and Cesar Milstein discovered a technique to produce antibody arising from a single B cell, which is called?
Monoclonal Antibody
In result, the discovery of monoclonal antibody revolutionized?
serological testing
Kohler and Milstein’s technique fuses an __________ with a __________ that can be grown indefinitely in the laboratory.
activated B cell, myeloma cell
Myeloma cells lacks an enzyme called?
HGPRT or Hypoxanthine-guanine phosphoribosyltransferase
It is an important enzyme to synthesize nucleotides from hypoxanthine and thymidine.
HGPRT or Hypoxanthine-guanine phosphoribosyltransferase
They are cancerous plasma cells.
Myeloma cells
Method used to produce monoclonal antibodies?
Hybridoma Production or Hybridoma Technique
It is a high-end type of antibody.
Monoclonal Antibody
True or false: The knowledge that B cells are genetically preprogrammed to synthesize very specific antibody has been used in developing monoclonal antibodies for diagnostic testing.
True
Hybridoma production: One pathway, which builds DNA from degradation of old nucleic acids, is?
blocked
Hybridoma production: The other pathway, which makes DNA from new nucleotides, is blocked by the presence of?
aminopterin
Hybridoma production: The remaining hybridoma cells are diluted out and placed in ___________, where they are allowed to grow.
microtiter wells
Hybridoma production: Each well, containing one clone, is then screened for the presence of the desired antibody by?
removing the supernatant
Hybridoma production: Once identified, a hybridoma is capable of being maintained in cell culture indefinitely, and it produces a permanent and uniform supply of monoclonal antibody that reacts with a?
single epitope
Fusing medium for plasma cells and myeloma cells?
PEG - Polyethylene glycol
True or false: During Hybridoma production, a large percentage of cells will fuse.
False. Only a small percentage of cells will actually fuse.
In Hybridoma production, the fused cells are placed in a selective culture media called?
HAT Medium (hypoxanthine-aminopterin-thymidine medium)
Why will myeloma cells die after placing the fused cells in HAT medium?
Because myeloma cells lack the enzyme HGPRT, they cannot utilize hypoxanthine and thymidine
Why will spleen cells die after placing the fused cells in HAT medium?
Because normal B cells cannot be maintained continuously in cell culture
Which are the only cells that will grow after Hybridoma production?
Hybridoma cells
