immunological techniques

Question 1

what is an immunoassay

Answer 1

a biological test that uses 1 or more immunologic products or reagents

Question 2

what does sensitivity and specificity mean

Answer 2

sensitivity - can detect very small concentrations

specificity - measuring only 1 substance

Question 3

how does an antibody bind to an antigen

Answer 3

an antibody binds to one molecule on the antigen - the epitope.

once the antigen is eliminated the immune response stops

an antigen with 4 epitopes will need 4 antibodies - each specific to the epitopes

Question 4

what is a hapten

Answer 4

a small molecule requiring coupling to another molecule to produce an immune response. it needs a carrier molecule to bind to an antibody

Question 5

what parts of an antibody reacts with what?

Answer 5

Fc interacts with other elements

Fab interacts with ag

Question 6

what are the classes of antibodies

Answer 6

(Ig) M A D G E

Ig is immunoglobin

Question 7

what are the types of antibodies that B cells produce

Answer 7

Polyclonal antibody - broad spectrum of antibodies - targets the different epitopes but could have an Ab you dont want

Monoclonal antibody - only recognise the same epitopes - less affinity - used for immunotherapy and in vitro diagnosis

Question 8

what is an immune response

Answer 8

the body recognising foreign substances with phagoctyes, T cells kill the invaders, B cell produce antibodies

this is regulated by cytokine production

Question 9

describe immune complexes

Answer 9

antibodies bind to antigen = immune complex

primary - complex forms
secondary - interaction between complex - agglutination
tertiary - complex interacts with the immune cell

measured quantitatively

Question 10

describe Ag-Ab reactions

Answer 10

Ab + Ag = AgAb
weak = strong

hydrophobic and electrostatic interactions
stable if complementary shape of Ag and binding site of Ab
reversible

Question 11

what is affinity and avidity

Answer 11

affinity - strength of binding - defined by eqbm constant
avidity - sum of binding strength

K= [AgAb]/[Ag][Ab]

Question 12

what are the noncellular immunoprecipitation assays

Answer 12

single radial immunodiffusion
rocket immunoelectropheresis
double diffusion
haemagluttination

Question 13

describe single radial immunodiffusion

Answer 13

SRID

antibody within gel
antigen added to wells in gel
incubate to diffuse
diameter can be graphed against antigen conc

Question 14

describe rocket immunoelectropheresis

Answer 14

antibody throughout gel
antigen in wells
move antigen by electropheresis
rocket height can be graphed against antigen conc

Question 15

describe double diffusion

Answer 15

antibody in a well
antigen in other wells
both diffuse - precipitates form line/arc

if antigens are identical - arc forms
if antigens are partly identical - spur forms
if antigens not identical - cross forms

Question 16

describe agglutination

Answer 16

best results with IgM as it is pentomeric - 5 antibodies
RBC coated in antigen

button - negative reaction
carpet - positive reaction

used for blood grouping, antibody detection

Question 17

how does cell lysis happen

Answer 17

antigen on cell + antibody + c’ = cell lysis

c’ is an enzyme cascade with 20 serum proteins - ruptures cell membrane

Question 18

how to find if antibody is in serum via complement fixation assay

Answer 18

add inactive c’ to antigen and c’
complexes will form if antibody in serum is specific for antigen
check for free c’

if c’ fixes - no lysis - positive reaction
if c’ doesnt fix - no antibody - lysis - negative reaction

Question 19

how does a viral neutralisation test work

Answer 19

serum + virus + cell culture

if there is no change - antibody present - no cytopathic effect
if there is a cytopathic effect - no antibody present

(virus causes cytopathic effect, antibody coats virus so virus cannot enter cell)

Question 20

what is a ligand and its immunoassays

Answer 20

something that binds to something else eg antibody to HIV envelope

radioimmunoassay (RIA)
ELISA

Question 21

describe radioimmunoassay

Answer 21

antigens and sample compete for antibody binding site
B/F
bound ag/free ag

tells you if unlabelled antigens present

Question 22

describe ELISA

Answer 22

enzyme linked immunosorbent assay

antigen attaches to antibody
(possibly secondary antibody with enzyme added)
substrate added - colour change

direct - first antibody has enzyme
indirect - needs secondary antibody
sandwich - antibody binds to plate, then antigen, then secondary antibody
indirect sandwich - antibody, antigen, antibody, secondary antibody

used for antigen detection in infection or presence of antibody eg HIV

Question 23

what are the uses of cellular assays

Answer 23

identify cells and their functions
organ transplants
disease diagnosis

Question 24

what is a ficoll hypaque density gradient

Answer 24

ficoll is a polymer of sucrose
hypaque is a dense organic compound
obtains cells for assays via centrifugation eg lymphocytes from blood

Question 25

what is flow cytometry

Answer 25

uses monoclonal for cell surface antigens the Fc portion of antibody is removed label the antibody with fluorescent dye cells stream, lasers through, each cell is detected this determines cell size, granularity and surface molecules

Question 26

what are the cell function assays

Answer 26

``` lymphocyte transformation mixed lymphocyte culture cytoxicity assays cell movement phagocytosis ```

Question 27

describe lymphocyte transformation

Answer 27

measures lymphocytes ability to respond to stimulants eg mitogens lymphocytes synthesises dna add radiolabel 3H thymidine - takes up more if stimmed to divide ficoll hypaque lymphocytes - culture with mitogen - add thymidine - isolate - count

Question 28

describe mixed lymphocyte culture

Answer 28

measures ability of histoincompatible lymphocyte to stim lymphocyte from someone else - 1 way - 1 group irradiated - no response - 2 way - both cell groups respond used to select kidney donor

Question 29

describe cytoxicity assays

Answer 29

detects antibody-antigen interaction on cell surface end point is cell death lymphocyte + c' + eosin dye = cell death the c' contains antibodies, binds to antigen on lymphocyte surface , ruptures cell membrane, gets dyed red

Question 30

describe cell movement

Answer 30

capillary tube movement (neutrophils) - migrate out chemoattractant diffuses towards tube and stims the cells, making the cells diffuse further micropore filter technique cells diffuse through filter into filter meshwork, endpoint where 2 cells travel the same distance

Question 31

describe phagocytosis

Answer 31

determines visible particles that can be counted in the cell identify ingestible substance that can be extracted and measured done via spectrophotometer

Question 32

what is immunohistochemical staining

Answer 32

observe variety of antigens undetected by cytochemical staining eg hormones