Mim - Lecture 6

Front 1

6.1

bonds in Ag-Ab interactions:

Back 1

- no covalent
- H bonds
- Van der Waals
- hydrophobic interactions
- reversible

Front 2

6.2

combining site of an antibody:

Back 2

- in the Fab portion
- constructed from the hypervariable regions of HC and LC

Front 3

6.3

Antibody affinity:

Back 3

- strength of the rxn b/w a single antigenic determinant and a single combingins ite on the antibody
- sum of attractive and repulsive forces b/w Ag and Ab's variable site

Front 4

6.4

Avidity

Back 4

- measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies
- influenced by both the valence of the antibody and the valence of the antigen
- more than the sum of the individual affinities

Front 5

6.5

affinity and avidity

Back 5

- affinity strength of binding between a single antigenic determinant and an individual antibody combining site, whereas avidity refers to the overall strength of binding between multivalent antigens and antibodies

Front 6

6.6

antibodies can distinguish differences in:

Back 6

1) the primary structure of an antigen
2) isomeric forms of an antigen
3) secondary and tertiary structure of an antigen

Front 7

6.7

cross reactivity

Back 7

- cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant, or the ability of a population of antibody molecules to react with more than one antigen

Front 8

6.8

factors affecting measurement -> physical form of the antigen:

Back 8

- particulate antigens will cause agglutination or clumping after binding to antibodies
- soluble antigens -> precipitate after binding to antibodies

Front 9

6.9

IgM and agglutination:

Back 9

- b/c of its high valence, its especially a good agglutinin

Front 10

6.10

O blood type

Back 10

- universal donor
- no blood group antigens on RBC

Front 11

6.11

AB blood

Back 11

- universal recipient
- no anti A or anti B antibodies in serum

Front 12

6.12

titer

Back 12

max dilution that gives visible agglutination

Front 13

6.13

prozone effect

Back 13

- excess antibodies and not enough antigens -> smaller clumps or complexes

Front 14

6.14

passive hemagglutination:

Back 14

- coat erythrocytes w/ a soluble antigen -> use that in an agglutination test for antibody to the soluble antigen

Front 15

6.15

complement fixation tests:

Back 15

- Ag/Ab complexes will 'consume' complement if its present
- free Ag or Ab cannot
- quantitate Ag/Ab rxns
- IgG and IgM

Front 16

6.16

direct comb's test

Back 16

- used to detect nor-agglutinating antibodies on RBC
- add a second antibody that binds specifically to the immunoglubulin coating the RBC
- second Ab can cross link and cause agglutination

Front 17

6.17

incomplete antibodies

Back 17

- bind to erythrocytes, but do not cause agglutination of RBC

Front 18

6.18

comb's test/antiglobulin test:

Back 18

- use anti-immunoglobulin antibodies to detect antibodies for ABO blood screening and Rh compatibility b/w mother and fetus

Front 19

6.19

indirect comb's test

Back 19

- 2 steps
- used when serum has antibody for a particular RBC
- incubate RBC in serum w/ Ab, wash out unbound
- 2nd anti-immunoglobulin Ab is added to cause agglutination

Front 20

6.20

hemagglutination inhibition

Back 20

- measure soluble antigens' ability to inhibit the agglutination of Ag-coated RBC
- soluble Ag bind to Ab preventing binding on Ag on RBC and cause agglutination

Front 21

6.21

radial immunodiffusion (Manuni)

Back 21

- precipitation text
- quantitative
- Ab in agar gel
- different dilutions of Ag placed in holes
- equivalence pt = precipitation
- used to determine immunoglobulin levels

Front 22

6.22

immunoelectrophoresis

Back 22

- qualitative
- analyze components in a serum
- evaluate purity of isolated serum proteins
- electric field separate Ag based on change
- Ab added -> diffuse across gel
- Ag-Ab complex precipitate to form a series of visible lines based on their change and reactivity

Front 23

6.23

countercurrent electrophoresis:

Back 23

- Ab, Ag opposite charge
- qualitative
- fast

Front 24

6.24

radioimmunoassay (RIA)

Back 24

- based on the measurement of radioactivity associated with immune complexes
- label may be on either the antigen or the antibody (for detection of hormones thyroxine, insulin)

Front 25

6.25

enzyme linked immunosorbent assays (ELISA)

Back 25

- based on the measurement of an enzyme reaction associated with immune complexes.
- enzyme may be linked to either the antigen or the antibody

Front 26

6.26

competitive RIA/ELISA for Ag detection

Back 26

- use a standard curve made from known amounts of an antigen and the cooresponding enzyme activities/radioactivity as reference to determine the amount of antigen in an unknown sample
- separate immune complexes from the remainder of the components

Front 27

6.27

noncompetitive RIA/ELISA for Ag or Ab

Back 27

- RAST (radioallergo sorbent test)
- use a solid phase w/ immobolized antigen
- add Ab sample and a labeled anti-immunoglobulin
- test for IgE due to allergies
- anti-Ig can be labeled w/ radioactive or enzyme (horseradish peroxidase or alkaline phosphatase)

Front 28

6.28

flow cytomery

Back 28

- used to identify and enumerate cells containing a certain antigen

Front 29

6.29

fluorescence-activated cell sorting (FACS)

Back 29

- cells in suspension are labeled with a florescent tag by either direct or indirect immunofluorescence. The cells are then analyzed on the flow cytometer
- laser hit the cell, scatter the laser light
- detector senses the light scattering
- measure # of cells
- size
- Ag expression

Front 30

6.30

One parameter FACS analysis

Back 30

- X = amount of fluorescence
- Y = # of cells exhibiting the fluorescence

Front 31

6.31

two parameter FACS analysis

Back 31

- x = 1 fluorescence
- y = another fluorescence
- # of cell = contour and intensity