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analytical techniques > immunological techniques > Flashcards

immunological techniques Flashcards

1
Q

what is an immunoassay

A

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

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2
Q

what does sensitivity and specificity mean

A

sensitivity - can detect very small concentrations

specificity - measuring only 1 substance

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3
Q

how does an antibody bind to an antigen

A

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

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4
Q

what is a hapten

A

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

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5
Q

what parts of an antibody reacts with what?

A

Fc interacts with other elements

Fab interacts with ag

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6
Q

what are the classes of antibodies

A

(Ig) M A D G E

Ig is immunoglobin

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7
Q

what are the types of antibodies that B cells produce

A

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

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8
Q

what is an immune response

A

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

this is regulated by cytokine production

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9
Q

describe immune complexes

A

antibodies bind to antigen = immune complex

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

measured quantitatively

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10
Q

describe Ag-Ab reactions

A

Ab + Ag = AgAb
weak = strong

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

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11
Q

what is affinity and avidity

A

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

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

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12
Q

what are the noncellular immunoprecipitation assays

A

single radial immunodiffusion
rocket immunoelectropheresis
double diffusion
haemagluttination

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13
Q

describe single radial immunodiffusion

A

SRID

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

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14
Q

describe rocket immunoelectropheresis

A

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

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15
Q

describe double diffusion

A

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

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16
Q

describe agglutination

A

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

17
Q

how does cell lysis happen

A

antigen on cell + antibody + c’ = cell lysis

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

18
Q

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

A

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

19
Q

how does a viral neutralisation test work

A

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)

20
Q

what is a ligand and its immunoassays

A

something that binds to something else eg antibody to HIV envelope

radioimmunoassay (RIA)
ELISA

21
Q

describe radioimmunoassay

A

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

tells you if unlabelled antigens present

22
Q

describe ELISA

A

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

23
Q

what are the uses of cellular assays

A

identify cells and their functions
organ transplants
disease diagnosis

24
Q

what is a ficoll hypaque density gradient

A

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

25
Q

what is flow cytometry

A

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

26
Q

what are the cell function assays

A 
lymphocyte transformation
mixed lymphocyte culture
cytoxicity assays
cell movement
phagocytosis
27
Q

describe lymphocyte transformation

A

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

28
Q

describe mixed lymphocyte culture

A

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

29
Q

describe cytoxicity assays

A

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

30
Q

describe cell movement

A

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

31
Q

describe phagocytosis

A

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

done via spectrophotometer

32
Q

what is immunohistochemical staining

A

observe variety of antigens undetected by cytochemical staining eg hormones

analytical techniques (6 decks)

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