Immunoassays

Question 1

6 type of immunoassay test

Answer 1

1) ouchterlony immunodiffusion
20 Western Blot
3) immunofluorescence
4) Immuno-electron gold microscopy
5) ELISA
6) Lateral flow assay

Question 2

Ouchterlony immunodiffusion- type of test

Answer 2

qualitative

Question 3

ouchterlony immunodiffusion detects

Answer 3

antibody-antigen interactions

Question 4

a positive ouchterlony immunodiffusion reaction will show

Answer 4

a precipitation line

Question 5

for a precipitation line to form

Answer 5

an immune complex must form- antibodies and antigens form a couples(the one that looks like a pentameric structure)

Question 6

Precipitin lines fuse completely showing

Answer 6

that wells A and b contain identical antigen determinants

Question 7

Precipitin lines cross without any interaction

Answer 7

wells A and B share no common antigenic determinants

Question 8

Precipitin lines fuse, but an additional spur forms towards

the well containing solution A

Answer 8

A and B share some common determinants in the solution- B has additional unique determinants picked by ant-serum solution

Question 9

Western Blot utilises

Answer 9

antibodies to detect specific proteins I a sample- specific antigens in a mixture of proteins

Question 10

Western blot process

Answer 10

1) take liquid sample and isolate proteins in a centrifuge
2) produce monoclonal antibodies speicifc for the protein 9with antigen of interest)
3) samples exposed to SDS gel electrophoresis4) sample separated by size and charge
4) monoclonal antibody is added t the polymer sheet containing separated proteins- will only bid to antigen of interest
5) another antibody created which is specific to the first monoclonal antibody (e.g. goat anti-mouse immunoglobulin) which is labelled radioactively is added
6) polymer sheet containing the antibody antigen complex and secondary radiolabelled antibody is visualised on an autoradiograph
- band will appear on X ray film

Question 11

what sort of polymer sheet is used in WB

Answer 11

nitrocellulose

Question 12

what also must be used in WB

Answer 12

a blocking reagent

Question 13

which is one of the most widely bused techniques

Answer 13

immunofluorescence

Question 14

in immunofluorescence antibodies

Answer 14

are tagged with fluoresecent reagent

Question 15

examples of fluoresecent reagent used in WB

Answer 15

Fluorescein Isothiocyanate- green

Texas red

Question 16

immunofluorescence can be used to

Answer 16

localise antibodies or antigens on the cell surface or inside a cell to identify pathogenic organism

Question 17

direct immunofluorescence

Answer 17

1) antigen treated with labelled antibody
2) unbound washed away
3) UV light added- fluoresce observed where antigen located

Question 18

Indirect immunofluoresce

Answer 18

1) antigen treated with unlabelled antibody
2) unbound washed away
4) treated with secondary labelled antibody
5) unbound washed away
3) UV light added- fluoresce observed where antigen located

Question 19

Immunoflorescence is

Answer 19

qualitative

Question 20

immuno-electorn gold microscopy is

Answer 20

qualitative

Question 21

EM gold allows

Answer 21

localisation of antigen-antibody interactions at the nm scale

Question 22

EM gold is a

Answer 22

very powerful method of visualising intracellular antigens or organelles

Question 23

which method allows very powerful visualising

Answer 23

EM-Gold

Question 24

which tests can both be used to visualise the extracellular and intracellular location of antigens or antibodies

Answer 24

• immunofluorescence

- Immuno-electron gold microscopy

Question 25

give an example of an immunoassay which is quantitative

Answer 25

ELISA

Question 26

two types of ELISa

Answer 26

PTA-ELISA

DAS-ELISA

Question 27

PTA-ELISA

Answer 27

Plate-trapped antigen enzyme-linked immunosorbent assay

Question 28

DAS-ELISA

Answer 28

Double antibody sandwich- enzyme-linked immunosorbent assay

Question 29

enzymes used to reveal presence of antibodies are either couple

Answer 29

directly or indirectly

Question 30

direct elisa

Answer 30

antibody is tagged with enzyme and complexes with antigen adsorbed to the well
- substrate added- colour change if present

Question 31

sandwich elisa

Answer 31

antibody absorbed to well

• antigen added
• secondary antibody added
• third antibody added = tagged with enzyme

Question 32

indirect elisa

Answer 32

• primary antibody is untagged

- secondary antibody (goat-anti-mouse immunoglobulin) which has been tagged with an enzyme is added- colour change

Question 33

describe a positive lateral flow test

Answer 33

1) sample added
2) moves up paper via capillary action
3) antigen of interest binds to antibody labelled with colloidal gold- red line
4) colloidal gold antibodies linked to enzyme will bind to immobilised antibodies specific to antigen- sandwich
5) unbound antibodies will bind to control antibodies- the 2nd line

Question 34

which element is used to label lateral flow antibodies

Answer 34

colloidal gold

Question 35

Lateral flow assay is a

Answer 35

semi-quantitative test

Question 36

point of care test

Answer 36

LFA

Question 37

when is LFA used

Answer 37

IPA and chytridiomycosis