Genscript interview preparation

Question 1

What is the purpose of blocking solutions with antibodies

Answer 1

To prevent primary and secondary antibodies from binding non-specifically to other exposed surfaces and proteins, resulting in high background

Question 2

What is the purpose for primary antibody

Answer 2

To ensure specificity

Question 3

What is the function of secondary antibodies

Answer 3

To ensure sensitivity

Question 4

What does the secondary antibody have that the primary antibody doesn’t?

Answer 4

Secondary antibodies have enzyme products, which forms colour development when substrate is added

Usually conjugated with alkaline phosphatase or horseradish peroxidase

Question 5

What are some important principles of an assay?

Answer 5

Specificity
Sensitivity
Working range
Repeatability and reproducibility
Interfering substances –> False negatives?
Contaminating substances –> False positives?

Question 6

What is the consequence of an insufficient amount of blocking buffer in ELISA?

Answer 6

Allows probe antibodies to bind directly to well surface, leading to high background

Causes non-specific binding of primary or secondary antibodies to the blocking agent

Question 7

What is the purpose of washing in ELISA

Answer 7

To remove unbounded antibodies, reagents and other contaminants in the sample, to minimise non-specific probe binding

Question 8

What is the prozone effect?

Answer 8

Immunologic phenomenon whereby the effectiveness of antibodies to form immune complexes is sometimes impaired when concentrations of antibody or antigen are very high