Immunology Chapter 6

Front 1

the combined strength of the noncovalent interactions b/t a single antigen-binding site on an antibody and a single epitope is the ______ of the antibody for that epitope

Back 1

affinity

Front 2

forward (association) rate divided by the reverse (dissociation) rate constant

Back 2

association constant (Ka)

Front 3

in immunology, the association constant (Ka) is called the

Back 3

affinity constant

Front 4

can be calculated from the ratio of the molar concentration of bound Ag-Ab complex to the molar concentrations of unbound antigen and antibody at equilibrium

Back 4

Ka
affinity constant

Front 5

low affinity ag-ab complexes have Ka values b/t...
high-affinity can be as high as...

Back 5

10^4 - 10^5
10^11

Front 6

old method for determining affinity constant

Back 6

equilibrium dialysis

Front 7

based on repeated equilibrium dialysis with a constant concentration of antibody and varying concentration of ligand

Back 7

Scatchard plots

Front 8

ratio of the concentration of bound ligand to total antibody concentration

Back 8

r

Front 9

concentration of free ligand

Back 9

c

Front 10

number of binding sites per antibody molecule

Back 10

n
valency

Front 11

r/c = Ka(n) - Ka(r)

Back 11

scatchard equation

Front 12

number of binding sites per antibody molecule

Back 12

valency
n

Front 13

strength of multiple interactions b/t a multivalent antibody and antigen is called

Back 13

avidity

Front 14

occurs if two different antigens share an identical or very similar epitope
often observed among polysaccharide antigens that contain similar oligosaccharide antigens

Back 14

cross-reactivity

Front 15

glycolipids expressed on red blood cells
individual lacking one or both of these antigens will have serum antibodies to the missing antigens -- induced not by exposure to red blood cell antigens but by exposure to cross-reacting microbial antigens present on common intestinal bacteria

Back 15

ABO blood-group antigens

Front 16

more sensitive, convenient, and rapid method of measuring antibody affinity than equilibrium dialysis
since mid-1990s

Back 16

surface plasmon resonance (SPR)

Front 17

beam of polarized light is directed through a prism onto a thin gold film
at a unique angle, some incident light is absorbed by the gold layer, its energy transformed into charge waves called surface _______
a sharp dip in the reflected light can be measured at that angle, called the _____ angle
angle depends on the optical properties of the material close to the gold layer's surface <-----important

Back 17

plasmons
resonant

Front 18

using surface plasmon resonance (SPR) in order to determine the reactivities of antibodies and the locations of the epitopes w/in the original antigen molecule can be isolated

Back 18

epitope mapping

Front 19

antibodies that aggregate soluble antigens are called

Back 19

precipitins

Front 20

these two _______ reactions are used to determine relative concentrations of antibodies or antigens, to compare antigens, or to determine the relative purity of an antigen preparation

Back 20

immunodiffusion reactions

radial immunodiffusion
double immunodiffusion

Front 21

antigen sample is placed in a well and allowed to diffuse into agar containing a suitable dilution of an antiserum
as antigen diffuses into the agar, the region of equivalence is established and a ring of precipitation, a precipitin ring, forms around the well

Back 21

radial immunodiffusion

Front 22

both antigen and antibody diffuse radially from wells toward each other, thereby establishing a concentration gradient
as equivalence is reached, a visible line of precipitation, a precipitin line, forms

Back 22

double immunodiffusion

Front 23

the interaction b/t antibody and a particulate antigen results in visible clumping called ________
antibodies that produce such reactions are called ______

Back 23

agglutination
agglutinins

Front 24

inhibition of agglutination reactions due to excess of antibody

Back 24

prozone effect

Front 25

antibodies that bind to the antigen but do not induce agglutination
often of the IgG class

Back 25

incomplete antibodies

Front 26

reciprocal of the greatest serum dilution that elicits a positive agglutination reaction

Back 26

agglutinin titer

Front 27

in this test, absence of agglutination is diagnostic of antigen
commonly used for illicit drug tests, parental rubella immunity testing

Back 27

agglutination inhibition

Front 28

principle of this test involves competitive binding of radiolabeled antigen and unlabeled antigen to a high-affinity antibody

decrease in the amount of radiolabeled antigen bound to specific antibody in the presence of the test sample is measured in order to determine the amount of antigen present in the test sample

Back 28

RIA
radioimmunoassay

Front 29

similar in principle to RIA but depends on an enzyme rather than a radioactive label
an enzyme conjugated w/ an antibody reacts w/ a colorless substrate to generate a colored reaction product

Back 29

enzyme-linked immunosorbent assay
ELISA

Front 30

substrate in ELISA is called

Back 30

chromogenic substrate

Front 31

serum or some other sample containing primary antibody is added to an antigen-coated microtiter well and allowed to react w/ the antigen coated to the well
free Ab1 is washed away, the the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary antibody that binds the primary antibody
excess Ab2 is washed away, and a substrate for the enzyme is added--> amount of colored reaction product is measured by specialized spectrophotometric plate readers
method of choice to detect presence of serum antibodies against HIV

Back 31

Indirect ELISA (enzyme-linked immunosorbent assay)

Front 32

in this technique, antibody is immobilized on a microtiter well
sample containing antigen is added and allowed to react w/ the immobilized antibody
well is then washed, and second enzyme-linked antibody specific for a different epitope of the antigen is added and allowed to react w/ the bound antigen
after any free second antibody is removed, substrate is added, and the colored reaction product is measured

Back 32

sandwich ELISA

Front 33

in this technique, antibody is first incubated in solution w/ a sample containing antigen
antigen-antibody complex is then added to an antigen-coated microtiter well
the more antigen present in the sample, the less free antibody will be able to bind to the antigen-coated well
addition of an enzyme-conjugated secondary antibody specific for the isotype of the primary antibody can be used to determine the amount of primary antibody bound to the well
the higher the concentration of antigen in the original sample, the lower the absorbance

Back 33

competitive ELISA

Front 34

light produced by chemiluminescence during certain chemical reactions is used in these techniques
a luxogenic substrate takes the place of the chromogenic substrate in conventional ELISA reactions
light produced during luxogenic reactions may be detected by its ability to expose photographic film
quantitative measurement of light emission can be made by use of a luminometer
enhanced sensitivity over chromogenic assays

Back 34

chemiluminescence variants of ELISA

Front 35

allows the quantitative determination of the number of cells in a population that are producing antibodies specific for a given antigen or an antigen for which one has a specific antibody
plates are coated w/ either the antigen or the antibody
suspension of the cell population under investigation is then added to the coated plates and incubated
secreted molecules reactive with the capture molecules are bound in the vicinity of the secreting cells, producing a ring of antigen-antibody complexes around each cell that produces the molecule of interest

Back 35

ELISPOT assay

Front 36

this technique makes it possible to identify a specific protein in a complex mixture of proteins

a protein mixture is electrophoretically separated on an SDS-polyacrylamide gel (SDS-PAGE), a slab gel infused w/ sodium dodecyl sulfate (SDS), a dissociating agent.
protein bands are then transferred to a nitrocellulose membrane by electrophoresis, and the individual protein bands are identified by flooding the membrane w/ radiolabeled or enzyme-linked polyclonal or monoclonal antibody specific for the protein of interest

Back 36

western blotting

Front 37

protein A and protein G found in bacteria have created a defense to the host antibodies by..

Back 37

binding to the Fc region of IgG molecules w/ high affinity

Front 38

this bacterial proteins binds to biotin (a vitamin essential for fat storage) with exquisite specificity

Back 38

streptavidin

Front 39

in this technique, an electron-dense label is either conjugated to the Fc portion of a specific antibody for direct staining or conjugated to an anti-immunoglobulin reagent for indirect staining
electron-dense label can be visualized w/ the electron microscope as small black dots
in immunogold lableling, different antibodies can be conjugated w/ gold particles of different sizes, allowing identification of several antigens w/in a cell by the different sizes of the electron-dense gold particles attached to the antibodies

Back 39

immunoelectron microscopy

Front 40

in this technique, an electron-dense label is either conjugated to the Fc portion of a specific antibody for direct staining or conjugated to an anti-immunoglobulin reagent for indirect staining
electron-dense label can be visualized w/ the electron microscope as small black dots
in immunogold lableling, different antibodies can be conjugated w/ gold particles of different sizes, allowing identification of several antigens w/in a cell by the different sizes of the electron-dense gold particles attached to the antibodies

Back 40

immunoelectron microscopy