Practice #3

Question 1

ELISA

Answer 1

enzyme linked immunosorbent assay

Question 2

What are the steps of direct ELISA?

Answer 2

96 well plate
Antigen
wash
bovine albumin
wash
Ab from pt sera with chromagen
colored reaction

To detect the presence of Ab in the patient sera

Question 3

What are the steps of indirect ELISA?

Answer 3

96 well plate
Antigen
wash
bovine albumin
wash
Ab from pt sera
wash
Ab against the pt antibodies with chromagen
colored reaction

To detect the presence of Ab in the patient sera

Question 4

What are the steps of sandwich ELISA?

Answer 4

96 well plate
Antbodies 
wash
bovine albumin
wash
Ag from pt sera
wash
Ab against the Ag with chromagen
colored reaction

To detect the presnce of an Ag in the patient sera

Question 5

Indications for ELISA

Answer 5

Autoimmune diseases
Infections diagnosis
Tumor diagnosis
Endocrinology

Question 6

Biotin-avidin Biotin-streptavidin system

Answer 6

One of the strongest non covalent bonds in nature
Used in ELISA in order to enhance the signal of the colored reaction
We tag Fc region of the Ab with biotin molecules that can bind streptavidin with 2 ormore chromagens (ex HRP)

Question 7

QuantiFERON

Answer 7

ELISA based method in which we detect the levels of IFN gamma in the plasma fraction.
sandwich ELISA is used in this case
The indication is TB detection

Question 8

IRMA

Answer 8

Immunoradiometric assay

sandwich ELISA to detect Ag

Question 9

RIA

Answer 9

Radioimmunoassay
competitive ELISA to detect Ag
in competitive ELISA we add radioactive labled Ag that competes with the serum Ag on the Ab binding site
Stronger colored reaction means lower Ag in the serum

Question 10

Western blot

Answer 10

Purified proteins by gel electrophoresis can be transferred to a nitrocellulose membrane

1. positive electrode
2. sponge
3. filter paper
4. membrane
5. gel
6. filter paper
7. sponge
8. negative electrode

On the nitrocellulose membrane we can detect specific antigens by primary Ab against the Ag and secondary labled Ab against the primary Ab

Question 11

Lateral flow test

Answer 11

contains from right to left
1. latex bead with specific Ab against the Ag we want to detect
2. Test Ab specific for th Ag as well
3. Control Ab specific for the Ab on the bead
Positive reaction: 2 colored lines
Negative reaction: only control line colores