GENERAL IMMUNOLOGY Flashcards
Innate immunity
leukocytes, dendritic cells, natural killer cells, and plasma proteins as front-line defenders against pathogens.
mounts a general response to any antigen.
List the two types of lymphocytes
T-cells
B- cells
T-cells provide what kind of immunity?
Cell mediated (cellular)
What organs creates T-cells?
Thymus - humans
Bursa - chickens
What is immunohistochemistry?
The microscopic localization of specific Ag in tissues by staining with Ab labeled with fluorescent or pigmented material to test for disease, cancer or inflammation.
What is an Antigen (Ag)?
A substance not recognized by the immune system as being native to the host and stimulates an immune response.
Can be localized by the primary or secondary Ab for cancer diagnosis.
a.ka. Immunogen
What is an Antibody? (Ab)
Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses
Form complexes only with the antigens that stimulated their production
Antibodies conjugate to enzymes that catalyze reactions to form detectable compounds to visualize and localize specific antigens in a tissue sample
a.ka.a Immunoglobulin
List the two main types of antibodies
Monoclonal and Polyclonal.
Either be primary or secondary.
What is a monoclonal Antibody?
A homogenous population of immunoglobin
Detected against a single epitope
Derived from a single B-cell clone (that is fused to hybridoma cells giving immortality) from one animal and are thus immunochemically identical
Less likely to cross-react
Have monovalent affinity, binding only to the same epitope
Cannot form a lattice with antigen and form precipitates
B-cells provide what type of immunity?
Humoral immunity via the adaptive immune system.
What is a polyclonal Antibody?
A heterogeneous mix of antibodies.
Derived from the immune response of multiple B-cells, and each one recognizes a different epitope on the same antigen.
Due to their multiclonality, polyclonal Antibodies are more robust and more likely to survive in the tissue processor.
Higher risk of batch-to-batch variability.
Have the affinity for the same antigen but different epitopes.
List the antibody classes
IgG, IgA, IgM, IgE, and IgD
Ig standing for immunoglobulin.
GAMED
Describe an antibody structure
Two heavy and two light chains connected by disulfide bonds to form a “Y” shaped molecule.
The Y-shaped antibody is joined in the middle by a flexible hinge region.
Made up of a variable region and a constant region.
Variable Region: The “tips” of the Y that forms the fragment antigen-binding region (Fab). This region binds tightly to a specific part of an antigen called an epitope.
Constant Region: The antibody base and forms the fragment crystallizable region (Fc). This region is essential for the function of the antibody during an immune response. The type of heavy chain defines the overall class or isotype of an antibody (ex IgG, IgA, IgM, IgE, IgD).
Immune response
Reaction which occurs within an organism for the purpose of defense.
Three types is immune responses: innate, adaptive/acquired, and passive
Innate = nonspecific/general
Adaptive/acquired = specific
Passive = transferred (ex mom to baby)
Acquired immunity
Specific to an antigen
Carried out by white blood cells called lymphocytes
Two types: antibody responses and cell-mediated immune response
Humoral immunity
Immunity by antibody molecules that are secreted by plasma cells.
Named so because it involves substances found in the humors, or body fluids.
What organ creates B-cells?
Bone marrow
Peptide
Two or more amino acids linked in a chain
Are the structural components of cells and tissues, hormones, toxins, antibiotics, and enzymes
Shorter peptides have higher specificity and reduced cross-reactivity
Hapten
Small molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself
Immunogen
Any substance that generates B-cell and/or T-cell adaptive immune responses upon exposure to a host organism
Epitope
The part of an antigen that is recognized by the immune system,
a.ka. antigenic determinant,
Affinity
The strength of the individual bond between the paratope and epitope contact
Capacity to form insoluble immune complexes
a.k.a. binding affinity
Avidity
The overall strength of the antibody-antigen interaction
a.k.a. functional affinity
Which part of the antibody determines its class?
Heavy chain
Which part of the antibody determines its specificity to the antigen?
The variable region
Which region of the antigen does the antibody bind to?
Epitope
List the two light chains
kappa (κ)
lambda (λ)
Two antibodies used widely in IHC
IgG
IgM
Describe antigen-antibody interaction
Specific chemical interactions occur between antibodies produced by B cells and antigens when they encounter each other
Can be a reversible reaction
Can be washed away
Paratope
The part of an antibody that binds to the epitope is called a paratope
What creates the Y-chain shape of an antibody?
Two heavy chains and two light chains joined
What are the variable regions of an antibody?
The ends of the light and heavy chains
Protease can cleave this region, producing Fab (fragment antigen binding) that include the variable ends of an antibody
Define Fab
Fragment antigen binding sites
Region on an antibody that binds to antigens
Composed of one constant and one variable domain of each of the heavy and the light chain
Makes the antibody molecule bivalent
What are the constant regions of an antibody?
Determines the mechanism used to destroy antigen
Interacts with cell surface receptors called Fc receptors and some proteins of the complement system
Have the same amino acid sequence in all antibody molecules of the same class
Protease can cleave this region producing Fc ( fragment crystallizable) regions
Define Fc
Fragment crystallizable regions
The tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system
Not involved in combining with antigens
Which portion of an antibody structure governs its ability to recognize and bind with a specific antigen?
Primary structure
What do the terms light and heavy refer to?
The molecular weight of the amino acids within each respective chain
A “hybridoma” is formed when an immunoglobulin-producing spleen cell is fused with what non-immunoglobulin-producing cell?
Myeloma
Are immortal, malignant plasma cells
An antibody’s ability to recognize and bind with a specific protein antigen is due to its
Amino acid sequences.
I’m what two ways do heavy chains differ?
Antigenic
Structural
Heavy chains determine what two properties?
Class
Subclass
What type of bonds join Light chains to Heavy chains?
Covalent
Covalent bonds create the tertiary structure providing stability
What region contributes to the subclasses specificity of IgG?
The hinge region
In a newly immunized species, what is the first detectable humoral antibody?
IgM
Define Latent Period
The time from the first introducing the antigen until the appearance of newly formed IgM
~ 1 week
What is the life span of IgG antibodies?
Three weeks
What is the life span of IgM antibodies?
Four to six days
What carrier proteins are used when peptides are used as antigens?
KLH - keyhole limpet hemocyanin
BSA - bovine serum albumin
OVA - ovalbumin
Why do antibodies directed against linear conformation perform better?
The conformation of proteins in a tissue is affected by formal fixation and antigen retrieval
What two features are important when determining if an antigen will elicit a good immune response?
Amino acid sequence
Structure and spatial position of the epitope
Why are polyclonal antibodies made in rabbits?
Large amount of available anti-serum
Rarity of human antibodies to rabbit proteins
Rabbit antibodies precipitate human proteins over a wider range of either antigen or antibody excess
What organ is used when creating B-cell immotal cell lines from mice?
Spleen
What advantages does a rabbit monoclonal antibody have over a mouse monoclonal?
More diverse epitope recognition
Better immune response to small sized epitopes
Higher affinity and overall avidity
Enhanced binding properties due to heavy glycosylation
What advantages does a mouse monoclonal antibody have over a rabbit monoclonal?
Generate higher yield of antibodies
More stable in culture
What factors affect/weaken antigen-antibody complexes?
High salt concentration
High temperature
Low pH
Define cross-reactivity
An antigen reacts with several antibodies
ex: CEA antigen reacts with antibodies against CEA, blood group antigens and normal tissue proteins
Why does cross-reactivity occur?
Due to sharing of at least one common epitope between several antigens
Can also be caused by antigen retrieval changing epitopes
What issue does gross overfixation cause?
Makes antibody penetration more difficult
What are the two important factors when storing antibodies?
Type of storage container
Temperature
What type of containers should be used to store antibodies?
Polypropylene
Polycarbonate
Borosilicate glass
Before being stored, what can be added to antibodies with low concentration of protein?
0.1 - 1.0% BSA
Reduces loss of polymerization and adsorption onto the container
What are the advantages of using peptides as antigens?
Simple and quick to synthesize
Cross-reactivity with related proteins can be minimized
Antibodies can be raised to specific post-translational modifications such as methylation, acetylation
and phosphorylation
What are the disadvantages of using peptides as antigens?
Antibodies generated are less likely to recognize the protein’s
native structure
Epitopes lying within a region that is not accessible (due to the tertiary protein structure) is greater
Concentrated antibodies can be mixed with what substance to prevent ice crystal formation?
Glycerine
Why should frost free −20°C freezers should be avoided for the storage of antibodies?
Due to the damage caused by
the freeze-thaw cycles that these types of freezer perform
Define antibody specificity
The ability of an antibody to bind
selectively to a single epitope on an antigen
List three forms in which antibodies are supplied
- Concentrated
- Diluted
- Ready To Use (RTU)
What are the two most common isotypes of primary antibodies?
IgG
IgM
Define lectin
Class of proteins from plant origins which bind specifically to certain sugars
Cause agglutination of particular cell types
List three differences outside normal staining methods that must be considered when using lectins
- It is important to protect the sugar-binding sites and prevent lectin from self-agglutinating
- Must use the correct type of lectin
- Stock solution should contain 1.0mg of protein and kept at 4C
Define conjugated antibody
Chemically linked antibody to a specific tag/label
Describe the conjugation process when created a conjugated antibody
The fluorescent compound is attached via a covalent bond to the primary antibody - this is done in an alkaline solution.
Antibodies are composed of amino acids and the lysine side chain is commonly used to covalently attach labels to antibody molecules.
Define antibody titer
Measured amount of a specific type of antibody
In IHC: highest dilution of an antibody that results in max specific staining with the least amount of background under specific test conditions
How does anitbody affinity affect antibody titers?
High affinity causes faster reactions with tissue antigens and gives a more intense staining within the same incubation period as opposed to an antibody with low affinity
Define antibody dilution
Optimal concentration in which binding with the antigen is optimized while non-specific interactions with surrounding proteins or surfaces are minimized
How are dilutions normally expressed?
As a ratio of the parts of solute to the total number of parts
Explain protein concentration
The amount of protein in solution
Define optimal antibody dilution
Optimal antibody dilution is when a good signal-to-noise ratio is achieved.
Explain signal vs noise
Signal is true detection binding while noise is non-specific binding
Define titration study
Diluting antibodies to determine the optimal concentration of antibody that gives the best stain index
Explain checkerboard titration
Assess two variables at once: antibody concentration and sample concentration
Will show the optimal working dilution for an antibody showing the least amount of background staining while still identifying the target antigen
Calculate the following:
A vendor’s data sheet for a concentrated antibody recommends a dilution of 1:50.
How many parts antibody to parts of diluent are needed for this dilution?
1 part antibody
49 parts diluent
Calculate the following:
A vendor’s data sheet for a concentrated antibody recommends a dilution of 1:50.
How is a 1000 ul dilution made?
20ul of antibody
980 ul of diluent
Calculate the following:
A vendor’s data sheet for a concentrated antibody recommends a dilution of 1:50.
The 1:50 dilution is too strong. List three dilutions that could be tested to find the optimal dilution.
Serial dilutions are best:
1:100
1:200
1:400
Calculate the following:
A vendor’s data sheet for a concentrated antibody recommends a dilution of 1:50.
How would a 100ul of a 1;200 dilution be made from the remaining 1:50 dilution?
A solution is made using 10 uL concentrate to 3990 ul diluent. What dilution was made?
A solution is made using 10 uL concentrate to 3990 ul diluent.
Staining using this dilution is too weak, so 10 ul concentrated antibody is added to 1000 ul of the original dilution.
What is the new dilution?
What is meant by using IHC as a theranostic device?
IHC used as a diagnostic therapy for individual patients
Testing for possible reactions to a new medication or to tailor a treatment based on the test result
What are some examples of theraostic IHCs?
HER2/neu analysis
c-kit testing for GISTS
CD20 in B cell lymphomas
What is meant by using IHC as a genomic device
Locating specific genomic aberrations in the patient’s tissues by identifying (or not identifying) the presence or absence of specific aberration or gene signature
What are some examples of genomic IHCs?
Testing for microsatellite stability in colorectal CA (MLH1, MSH2, MSH6 or PMS2)
Identification of basal-like breast CA (high grade, ER/PR/HER2 (-), CK5/CK14/CK17 (+), variable EGFR expression
