Week 8

Question 1

explain the difference between polyclonal and monoclonal antibodies. How are they produced?

Answer 1

polyclonal:
- mixture of antibodies produced by different B-cells
- they all bind the same antigen but different epitopes
- production by immunization of animals with antigens, or blood sampling and purification

monoclonal:
- produced by one B cell clone -> antibodies are all identical
- exact binding epitope known
- produced in cell culture

Question 2

explain precipitation tests

Answer 2

we add antigens and antibodies in a mixture, and when they bind they form a precipitate that can be measured (turbidity)

Question 3

explain particle-enhanced turbidiometric immunoassay (PETIA)

Answer 3

antibodies are bound to latex particles to enhance the turbidity effect -> measured with a photometer

Question 4

what is the main principle of ELISA ?

Answer 4

Recognition of the analyte by the specificity of the antibody.
The enzyme is activated by a substrate -> generates a signal that can be measured

Question 5

explain direct ELISA and main +/-

Answer 5

antigens adsorb to well, excess washed away -> labeled antibodies are added -> addition of substrate

+: fast and few reagents
-: non-specific antigen absorption, each antibody must be labeled

Question 6

explain indirect ELISA and main +/-

Answer 6

antigens adsorb to well and washed away -> primary antibodies -> labeled secondary antibodies -> substrate

+: economical, better signal, different antigens can be detected simultaneously
-: cross-reactivity, slower

Question 7

explain sandwich ELISA and main +/-

Answer 7

antibodies in the well -> antigen added -> secondary antibody (labeled, or third that is labeled) -> substrate

+: high sensitivity, high specificity (two bindings)
-: only for large antigens, two different epitopes are needed!

Question 8

what is the competitive ELISA, +/-, and when can it be used?

Answer 8

antibodies in well -> add sample with antigens, some are labeled -> the more antigens are in the sample, the fewer labeled ones are bound -> low signal = more antigens

+: for all antigens, very robust
-: long incubation period

For small molecules, such as hormones for example

Question 9

how do the curves look like for the sandwich ELISA and the competitive ELISA ?

Answer 9

sandwich : starts low, and increases (high signal for more antigens)
competitive: starts high, ends low (low signal for more antigens)

Question 10

Explain the high-throughput lab assay (ECLIA)

Answer 10

one antibody is biotinylated, the other os labeled with ruthenium.
The biotin one binds to paramagetic streptavidin microparticles and to the antigen. The other binds to the antigen. The complex is separated with a magnetic field, and then the ruthenium is excited -> light emitted

Very fast!!! (18min)

Question 11

explain lateral flow tests

Answer 11

Gold labeled antibodies. The bounded ones stop at the test line, the others go to the control line.

More antibodies than antigen required, because some antibodies need to remain unbounded to reach the control line.