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QIHC maryjw79 @ gmail email 4 access > GENERAL IMMUNOLOGY > Flashcards

GENERAL IMMUNOLOGY Flashcards

1
Q

Innate immunity

A

leukocytes, dendritic cells, natural killer cells, and plasma proteins as front-line defenders against pathogens.

mounts a general response to any antigen.

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2
Q

List the two types of lymphocytes

A

T-cells
B- cells

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3
Q

T-cells provide what kind of immunity?

A

Cell mediated (cellular)

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4
Q

What organs creates T-cells?

A

Thymus - humans
Bursa - chickens

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5
Q

What is immunohistochemistry?

A

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.

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6
Q

What is an Antigen (Ag)?

A

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

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7
Q

What is an Antibody? (Ab)

A

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

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8
Q

List the two main types of antibodies

A

Monoclonal and Polyclonal.

Either be primary or secondary.

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9
Q

What is a monoclonal Antibody?

A

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

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10
Q

B-cells provide what type of immunity?

A

Humoral immunity via the adaptive immune system.

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11
Q

What is a polyclonal Antibody?

A

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.

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12
Q

List the antibody classes

A

IgG, IgA, IgM, IgE, and IgD

Ig standing for immunoglobulin.

GAMED

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13
Q

Describe an antibody structure

A

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).

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14
Q

Immune response

A

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)

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15
Q

Acquired immunity

A

Specific to an antigen

Carried out by white blood cells called lymphocytes

Two types: antibody responses and cell-mediated immune response

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16
Q

Humoral immunity

A

Immunity by antibody molecules that are secreted by plasma cells.

Named so because it involves substances found in the humors, or body fluids.

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17
Q

What organ creates B-cells?

A

Bone marrow

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18
Q

Peptide

A

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

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19
Q

Hapten

A

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

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20
Q

Immunogen

A

Any substance that generates B-cell and/or T-cell adaptive immune responses upon exposure to a host organism

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21
Q

Epitope

A

The part of an antigen that is recognized by the immune system,
a.ka. antigenic determinant,

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22
Q

Affinity

A

The strength of the individual bond between the paratope and epitope contact

Capacity to form insoluble immune complexes

a.k.a. binding affinity

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23
Q

Avidity

A

The overall strength of the antibody-antigen interaction

a.k.a. functional affinity

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24
Q

Which part of the antibody determines its class?

A

Heavy chain

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25
Q

Which part of the antibody determines its specificity to the antigen?

A

The variable region

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26
Q

Which region of the antigen does the antibody bind to?

A

Epitope

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27
Q

List the two light chains

A

kappa (κ)
lambda (λ)

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28
Q

Two antibodies used widely in IHC

A

IgG
IgM

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29
Q

Describe antigen-antibody interaction

A

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

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30
Q

Paratope

A

The part of an antibody that binds to the epitope is called a paratope

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31
Q

What creates the Y-chain shape of an antibody?

A

Two heavy chains and two light chains joined

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32
Q

What are the variable regions of an antibody?

A

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

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33
Q

Define Fab

A

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

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34
Q

What are the constant regions of an antibody?

A

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

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35
Q

Define Fc

A

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

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36
Q

Which portion of an antibody structure governs its ability to recognize and bind with a specific antigen?

A

Primary structure

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37
Q

What do the terms light and heavy refer to?

A

The molecular weight of the amino acids within each respective chain

38
Q

A “hybridoma” is formed when an immunoglobulin-producing spleen cell is fused with what non-immunoglobulin-producing cell?

A

Myeloma

Are immortal, malignant plasma cells

39
Q

An antibody’s ability to recognize and bind with a specific protein antigen is due to its

A

Amino acid sequences.

40
Q

I’m what two ways do heavy chains differ?

A

Antigenic
Structural

41
Q

Heavy chains determine what two properties?

A

Class
Subclass

42
Q

What type of bonds join Light chains to Heavy chains?

A

Covalent

Covalent bonds create the tertiary structure providing stability

43
Q

What region contributes to the subclasses specificity of IgG?

A

The hinge region

44
Q

In a newly immunized species, what is the first detectable humoral antibody?

A

IgM

45
Q

Define Latent Period

A

The time from the first introducing the antigen until the appearance of newly formed IgM

~ 1 week

46
Q

What is the life span of IgG antibodies?

A

Three weeks

47
Q

What is the life span of IgM antibodies?

A

Four to six days

48
Q

What carrier proteins are used when peptides are used as antigens?

A

KLH - keyhole limpet hemocyanin
BSA - bovine serum albumin
OVA - ovalbumin

49
Q

Why do antibodies directed against linear conformation perform better?

A

The conformation of proteins in a tissue is affected by formal fixation and antigen retrieval

50
Q

What two features are important when determining if an antigen will elicit a good immune response?

A

Amino acid sequence
Structure and spatial position of the epitope

51
Q

Why are polyclonal antibodies made in rabbits?

A

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

52
Q

What organ is used when creating B-cell immotal cell lines from mice?

A

Spleen

53
Q

What advantages does a rabbit monoclonal antibody have over a mouse monoclonal?

A

More diverse epitope recognition

Better immune response to small sized epitopes

Higher affinity and overall avidity

Enhanced binding properties due to heavy glycosylation

54
Q

What advantages does a mouse monoclonal antibody have over a rabbit monoclonal?

A

Generate higher yield of antibodies

More stable in culture

55
Q

What factors affect/weaken antigen-antibody complexes?

A

High salt concentration

High temperature

Low pH

56
Q

Define cross-reactivity

A

An antigen reacts with several antibodies

ex: CEA antigen reacts with antibodies against CEA, blood group antigens and normal tissue proteins

57
Q

Why does cross-reactivity occur?

A

Due to sharing of at least one common epitope between several antigens

Can also be caused by antigen retrieval changing epitopes

58
Q

What issue does gross overfixation cause?

A

Makes antibody penetration more difficult

59
Q

What are the two important factors when storing antibodies?

A

Type of storage container

Temperature

60
Q

What type of containers should be used to store antibodies?

A

Polypropylene

Polycarbonate

Borosilicate glass

61
Q

Before being stored, what can be added to antibodies with low concentration of protein?

A

0.1 - 1.0% BSA

Reduces loss of polymerization and adsorption onto the container

62
Q

What are the advantages of using peptides as antigens?

A

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

63
Q

What are the disadvantages of using peptides as antigens?

A

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

64
Q

Concentrated antibodies can be mixed with what substance to prevent ice crystal formation?

A

Glycerine

65
Q

Why should frost free −20°C freezers should be avoided for the storage of antibodies?

A

Due to the damage caused by
the freeze-thaw cycles that these types of freezer perform

66
Q

Define antibody specificity

A

The ability of an antibody to bind
selectively to a single epitope on an antigen

67
Q

List three forms in which antibodies are supplied

A
  1. Concentrated
  2. Diluted
  3. Ready To Use (RTU)
68
Q

What are the two most common isotypes of primary antibodies?

A

IgG
IgM

69
Q

Define lectin

A

Class of proteins from plant origins which bind specifically to certain sugars

Cause agglutination of particular cell types

70
Q

List three differences outside normal staining methods that must be considered when using lectins

A
  1. It is important to protect the sugar-binding sites and prevent lectin from self-agglutinating
  2. Must use the correct type of lectin
  3. Stock solution should contain 1.0mg of protein and kept at 4C
71
Q

Define conjugated antibody

A

Chemically linked antibody to a specific tag/label

72
Q

Describe the conjugation process when created a conjugated antibody

A

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.

73
Q

Define antibody titer

A

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

74
Q

How does anitbody affinity affect antibody titers?

A

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

75
Q

Define antibody dilution

A

Optimal concentration in which binding with the antigen is optimized while non-specific interactions with surrounding proteins or surfaces are minimized

76
Q

How are dilutions normally expressed?

A

As a ratio of the parts of solute to the total number of parts

77
Q

Explain protein concentration

A

The amount of protein in solution

78
Q

Define optimal antibody dilution

A

Optimal antibody dilution is when a good signal-to-noise ratio is achieved.

79
Q

Explain signal vs noise

A

Signal is true detection binding while noise is non-specific binding

80
Q

Define titration study

A

Diluting antibodies to determine the optimal concentration of antibody that gives the best stain index

81
Q

Explain checkerboard titration

A

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

82
Q

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?

A

1 part antibody
49 parts diluent

83
Q

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?

A

20ul of antibody

980 ul of diluent

84
Q

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.

A

Serial dilutions are best:
1:100
1:200
1:400

85
Q

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
86
Q

A solution is made using 10 uL concentrate to 3990 ul diluent. What dilution was made?

A
87
Q

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?

A
88
Q

What is meant by using IHC as a theranostic device?

A

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

89
Q

What are some examples of theraostic IHCs?

A

HER2/neu analysis
c-kit testing for GISTS
CD20 in B cell lymphomas

90
Q

What is meant by using IHC as a genomic device

A

Locating specific genomic aberrations in the patient’s tissues by identifying (or not identifying) the presence or absence of specific aberration or gene signature

91
Q

What are some examples of genomic IHCs?

A

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

QIHC maryjw79 @ gmail email 4 access (16 decks)

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