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Immunology > Lecture 4 Slides effector responses > Flashcards

Lecture 4 Slides effector responses Flashcards

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0
Q

Purpose of virus and toxin neutralization

A

Prevents pathogen-host binding

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1
Q

Four categories of antibody effector function

A

Virus and toxin neutralization
Opsonization
Complement fixation and formation of the membrane attack complex
Antibody-dependent cell-mediated cytotoxicity (ADCC)

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2
Q

Purpose of opsonization

A

Phagocytosis of bacteria

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3
Q

Purpose of complement fixation and formation of membrane attack complex

A

Phagocytosis or lysis

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4
Q

ADCC purpose

A

NK induced apoptosis

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5
Q

How neutralization works

A

Antibody binds to site on pathogen or toxin with host proteins, masking them, and inhibiting entry of that pathogen or toxin into the host, antibody-pathogen complexes are then eliminated, often after phagocytosis

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6
Q

How opsonization works

A

Antibody binds to pathogen and is then bound by Fc receptors on phagocytosic cells. The antibody-antigen binding to FcRs induces internalization and destruction by the phagocytic cell

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7
Q

How complement fixation works

A

Antibody-antigen complex becomes bound by complement components in serum and is either phagocytosed via cells expressing C3 receptors or lyses as a result of pore formation by the complement components C7,8,9

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8
Q

How does ADCC work

A

Antibody antigen complexes are bound by Fc receptors on NK cells and granulocytes, thus directing the cytotoxicity of these cells toward the antigen targeted by the antibody and inducing apoptosis of the target cell

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9
Q

Functions of Fc receptors

A 
De granulation (FccR and IgE)
Opsonization of bacteria and phagocytosis 
Maintaining serum levels of antibodies
ADCC
Trams cytosine into secretions
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10
Q

On what four types of noncovalent forces so antigen antibody reactions depend? Are reactions reversible?

A

Hydrogen bonds
Ionic bonds
Hydrophobic
Van der Waals

Reactions are reversible

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11
Q

What do weak noncovalent interactions depend on to happen

A

Close Complementarity of the shapes of the antibody and antigen

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12
Q

Antibody-combining site

A

Cleft formed by heavy and light antibody chains where antigen molecule nestles

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13
Q

Where does antigen make contact with antibody

A

Amino acids in hyper variable regions of borh heavy and light chains

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14
Q

Antibody affinity

A

Combined forces that helps antibody bind antigen. Defined as a constant.

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15
Q

What is affinity the result of

A

A balance between the attractive and repulsive forces. High affinity implies good fit and vice versa. (Kb)

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16
Q

Antibody avidity

A

Affinity plus valence.

How strong plus how many arms to bind

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17
Q

Sensitivity

A

How little or how much antigen can be detected

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18
Q

Six antigen-antibody based assays

A
  1. Precipitation
  2. Agglutination
  3. Radioimmunoassay
  4. Enzyme linked immunosorbent assay (most widely used)
  5. Immunofluorescence/immunocytochemistry
  6. Flow cytometry
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19
Q

Precipitation reaction

A

Antibody and soluble antigen (both soluble alone) interact in aqueous solution and form a precipitate

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20
Q

Precipitin

A

Antibody that can precipitate out of solution once bound to antigen

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21
Q

In precipitation reaction, antibody must be

A

Bivalent

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22
Q

In precipitation reaction, antigen can be

A

Bivalent or multivalent

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23
Q

Disadvantages of precipitation reaction

A

Slow, taking day or two
Requires large quantity of Ag or Ab
Replaced by modern assays

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24
Radial and double immunodiffusion
``` A precipitation reaction Immune precipitate in gel Ag and Ab diffuse towards each other Visible line of precipitation occurs at the line of equilibrium Only antigen diffuse in radial Both Ag and Ab diffuse in double ```
25
Radial immunodiffusion (Mancini)
``` In gel Circle Antibody incorporated in agar Antigen in center and diffuses Precipitate forms a ring ```
26
Double (Ouchterlony) immunodiffusion
Antibody and antigen samples next to each other and can mix in middle, where thy form a precipitate. Both Ab and Ag diffuse.
27
Why can't univalent antigens precipitate out of solution
They bind to antibody and nothing else. Can't form lattice.
28
Levels of precipitation reaction
Antibody excess zone Equivalence zone Antigen excess zone
29
Why does excess antigen lead to a reduction in the amount of antibody precipitated
Fall is due to solubilization of antigen-antibody complexes by excess antigen.
30
Immuno-double-diffusion line of identity
Single constant line between Ab and two antigen samples tested
31
Line of nonidentity
No constant line, but lines that cross form
32
Line of partial identity
A continuous line exists, but with a spur, indicating that complex is related but not identical
33
Coggin's test
Example od Immuno-double-diffusion
34
Immunoelectrophoresis
Combines electrophoresis and double immunodiffusion Antigen mixture is first electrophorised to separate its component by charge Specific antibody and antigen will diffuse and produce line precipitation Qualitative study (rocket immunoelectrophoresis) Reduces time and forces reaction.
35
Immunoelectrophoresis steps
1.separate antigens in an agar gel by placing electric charge across it. The gel's pH is chosen so that positively charged proteins move to negative electrode and negative to the positive. 2. A trough is then cut between the walls and filled with the antibody, which is left to diffuse 3. The antigens and antibody form precipitin arcs
36
Immunoprecipitation
Collect immunoprecipitates using magnetic beads coupled to secondary antibody
37
Immunoprecipitation steps
1. Treat cell extract with antigen by placing antibody so complex forms 2. Add magnetic beads to which second antibody is linked. This binds to complex. 3. Place magnet against side of tube to collect antigen-antibody complexes 4. Rinse 5. Dissociate to study antigen
38
Agglutination
Visible clumping formed by the interaction between Ab and a particulate Ag
39
Agglutinin
Antibody
40
Solubility in agglutination
One is soluble. One is insoluble. Nothing is precipitating out of solution. We are looking for a complex.
41
Uses of agglutination
Widely used Used to detect blood type RBCs mixed with anti sera to A or B antigen Bacterial agglutination Passive agglutination (coating RBC antigen and add serum)
42
Agglutination inhibition
Absence of agglutination is diagnostic of antigen
43
Blood type A has which antigens and antibodies
Antigens A | Ab anti-B
44
Blood type B has which antigens and Ab
b and anti-A
45
AB has which antigens and antibodies
A and B. Neither
46
O has which antigens and antibodies
Neither antigen. Anti-A and anti-B
47
Hemagglutination assay
Used for certain viruses | Hemagglutination is positive
48
Radioimmunoassay (RIA)
Immunoassay for antibody Most sensitive technique for detecting Ag or Ab A labeled and unlabeled Ag compete for the Ab binding site Ag is labeled with radioisotope, added to Ab and the test Ag is added Displaced radioactive isotope is measured
49
For what is radioimmunoassay used
Detecting hormones
50
Radioimmunoassay steps
1. Labeled Ag and unlabeled Ag are mixed with Ab (infected because of unlabeled) 2. Make uninfected batch, only labeled Ag 3. Allow for incubation period 4. Remove supernatant and measure radioactivity of antigen-antibody complexes 5. If sample is infected, the amount of label bound will be less than in controls with uninfected serum
51
Obtaining standard radioimmunoassay curve
Add increasing concentrations of unlabeled antigen to a fixed quantity of labeled Ag and antibody. Notice that amount of labeled Ag decreases as amount of unlabeled AG increases.
52
Determine amount of unlabeled antigen with this curve
Concentration of unlabeled antigen can be determined by using linear part of course. How? Well, the amount of labeled displaced, wchi can be measured because of radioactivity, is equal to amount of unlabeled put in.
53
Elisa
Similar to RIA with enzyme rather than radioactive labeling | An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product
54
How to solve false positives with Elisa
Confirm positive with western blotting. Western blotting is highly specific. Elisa highly sensitive.
55
Elisa stands for
Enzyme-linked immunosorbent assay
56
Is Elisa quantitative or qualitative
Both
57
ELISA steps
1. Sensitize plate with antigen 2. Wash 3. Add test antibody 4. Wash 5. Add ligand 6. Wash 7. Add Chromogen 8. Develop plate
58
ELISA ligand
A molecule that can detect the antibody and is covalently coupled to an enzyme such as peroxidase. This binds test antibody. After free ligand is washed away, bound ligand is visualized by addition of chromogen
59
Chromogen
A colorless substrate which is acted on by the enzyme portion of the ligand to produce a colored end product.
60
ELISA: how is amount of test antibody measures
By assessing the amount of colored end product by optical density scanning of the plate
61
Indirect ELISA
``` Antibody determined Steps -antigen coated well -wash -add specific antibody to be measured -wash -add enzyme-conjugated secondary antibody -wash -add substrate and measure color ```
62
Sandwich ELISA
``` Antigen is determined Steps -antibody coated well -wash -add antigen to be measured -wash -add enzyme conjugated secondary antibody -wash -add substrate and measure color ```
63
Competitive ELISA
inhibition type assay Concentration of antigen is inversely proportional to the color produced STEPS -incubate antibody with antigen to be measures -add Ag-Ab mixture to antigen-coated well -wash -add enzyme conjugated secondary antibody -wash -add substrate and measure color
64
Immunofluorescence
Fluorescence molecules absorb light of one wavelength and emit light with another wavelength The emitted light can be viewed with fluorescence microscope
65
Immunofluorescence types
1. Direct 2. Indirect with fluorochrome-labeled anti-isotype antibody 3. Indirect method with fluorochrome-labeled protein A
66
Direct immunofluorescence
Primary Antibody to mAg is labeled with fluorochrome | Primary antibody binds directly to antigen
67
Indirect method immunofluorescence
Primary antibody bonds to antigen Fluorochrome is used to label secondary anti-isotype antibody, which binds to primary antibody OR Fluorochrome is used to label protein that binds to antibody Fc portio
68
Two techniques used to localize viral antigens in tissues
Immunofluorescence | Immunohistochemistry
69
Flow cytometry
Designed to automate analysis and separation of cells stained with fluorescent dye Uses laser beam and light detector to count intact cells in suspension Quantitative whereas immunofluorescence is qualitative only

Immunology (5 decks)

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