Immunoassay Formats

Question 1

1D Western blotting

Answer 1

Heat proteins to denature disulfide bonds then treat them with SDS to provide all proteins with the same charge to mass ratio, then load them onto the gel. Smaller proteins will migrate quicker to the bottom.

Question 2

2D western blotting

Answer 2

Separate according to charge first using pH 4-10. Flip it over and separate by weight using current. Get spots

Question 3

nitrocellulose or PVDF transfer membrane

Answer 3

take the polyacrylamide gel and place it next to the transfer membrame, surrounded by filter paper and support pads. Run a current through and the proteins come onto the transfer membrane.

Question 4

Ouchtelory immunodiffusion

Answer 4

in the middle, well with antibody. on the outside, wells with antigen. allow the antigens to diffuse towards the antibody. antibody-antigen equivalent forms a precipitin =immune complex formation

Question 5

scedosporium prolificans

Answer 5

pot plants. affect CNS. has dark colour. mAb CA4. all have tetrahydroxynaphthalene reductase enzyme (virulence factor)

Question 6

Immunofluorescence

Answer 6

Use FITC or TR. UV light - different absorbance and emission spectra. Direct or indirect antibodyies.

Question 7

Immuno-electron gold microscopy

Answer 7

ab tagged with gold. powerful visualisation of ag-ab complexes (at nm scale)

Question 8

PTA-ELISA

Answer 8

plate trapped antigen - elisa (immobilized ag

Question 9

DAS-ELISA

Answer 9

double antibody sandwich - elisa (2 Ab)

Question 10

Rhizoctonia saloni

Answer 10

soilborne plant pathogen. develop 2 mAb that bind to the glycoprotein (which is only produced by live cells). EH2 binds to the protein, EE1 binds to the carbohydrate.

Question 11

Baiting module of rhizoctonia solani

Answer 11

put soil in agar plate and cover with nylon microfilament cloth. overlay with quinoa and incubate. pathogen grows over –> test with ELISA.

Question 12

DAS-ELISA of rhizoctonia solani

Answer 12

Coat plates with mAb (IgM) EH2 - 16h 4C.
Bait incubation (antigen) - 2h, 23.
IgM EE1 mAb - 1h 23C.
Anti IgM (u-specific) - 1h 23C.
Substrate (30m 23C)

Question 13

Global amphibian decline

Answer 13

Btarachochyntium dendrobatidis

Question 14

Batrochochytrium dendrobatidis

Answer 14

chytridiomycota - aquoatic and motile. colonises keratinised skin of frogs causing electrolyte unbalance leading to cardiac arrest.

Question 15

Diagnosis of chytridiomycosis

Answer 15

qPCR or histology - not practical so developed mAb. using hybridomas. 5C4

Question 16

frog in a pot

Answer 16

place frog in a pot of solution, antigen shredding. lateral flow device.

Question 17

lateral flow assay

Answer 17

sample with antigen is put on the assay.

antigen binds with gold conjugated 5C4 Ab which binds to the immobilised 5C4 Ab. Gives a line of detection.