Antibodies as Clinical Tools

Question 1

Define titer

Answer 1

measure of the concentration of specific antibody in a sample

Question 2

How is a titer expressed?

Answer 2

• ratio – dilution series – the higher the second number, the more antibodies are present
• last dilution determines the value

Question 3

How are antibodies produced commercially?

Answer 3

animals are injected with antigen –> animals will produce antibodies in response –> sample the blood over several weeks –> when ready, sacrifice animal and harvest monoclonal or polyclonal antibodies

Question 4

Define monoclonal antibodies

Answer 4

recognize a single epitope

Question 5

Define polyclonal antibodies

Answer 5

• recognize multiple epitopes

- many antibodies are secreted from various activated B cells during an infection

Question 6

What are common modifications of antibodies?

Answer 6

• addition of fluorophore with flourescence

- enzymes catalyze colorimetric reactions in assays

Question 7

What is a primary antibody?

Answer 7

antibody that recognizes a specific target antigen

Question 8

What is a secondary antibody?

Answer 8

antibody that recognizes a specific primary antibody

Question 9

What is agglutination?

Answer 9

clumping together of particles, usually by antibodies binding to antigens on adjacent particles

Question 10

What is hemagglutination?

Answer 10

clumping of RBCs when antibodies bind cell surface antigens

Question 11

What is the Coombs test?

Answer 11

• antiglobulin test used to test the presence of Rh in pregnant women and fetuses
• develop anti-human Ig antibodies

Question 12

Why are we only worried about Rh antibodies and not ABO antibodies?

Answer 12

ABO antigens can’t pass through the placenta

IgM is much larger than IgG

Question 13

Direct vs Indirect Coombs test

Answer 13

Direct:
-use FETAL RBCs
-detects anti-Rh antibody bound to fetal RBCs
-wash RBCs to get rid of free Abs
-anti-human Ig added and agglutinates antibody-coated cells
Indirect:
-use mother’s serum
-detects anti-Rh in mother’s serum
-incubate with Rh+ RBCs
-unbound antibodies washed away
-add anti-human Ig and agglutinates antibody-coated cells

Question 14

Describe the ELISA test

Answer 14

coat wells with antigen
add patient serum
add secondary antibody with fluorescence
add substrate
measure absorbance with an spectrometer

Question 15

Define immunohistochemistry

Answer 15

• tissue sample removed from patient

- a modified antibody is added to identify if and how much of the target protein there is

Question 16

Define immunocytochemistry

Answer 16

• cells are collected from patient

- modified anitbody added to detect if and how much of the target protein is present

Question 17

What is a flow cytometer?

Answer 17

• detects and counts individual cells as they pass through a laser beam (1 at a time)
• detected and stained with antibodies for the target antigen (primary or secondary antibody have fluorescent molecule)

Question 18

What is a Flourescence-Activated Cell Sorter (FACS)?

Answer 18

-flow cytometer that can sort the identified cells into pure populations of cells with specific characteristics

Question 19

Define dip-stick tests

Answer 19

• dip-stick has the antibodies for a target antigen
• reaction site: free antibody + dye enzyme
• test site: fixed antibody + dye substrate
• control site: fixed antibody that recognizes a different epitope + dye substrate