Immunology- Basic Techniques

Question 1

What is an immunological assay?

Answer 1

A biochemical test that measures the presence or concentration of a specific molecule using of an antibody

Question 2

What is serology?

Answer 2

measurement of antigen-antibody reactions for diagnostic purposes

Question 3

What is a primary binding test?

Answer 3

Directly measuring the binding of an antigen to an antibody (e.g ELISA testing)

Question 4

What is a secondary bhinding test?

Answer 4

Measures the results of antigenn-antibody reactions in vitro (e.g precipitation assays, haemoagglutination inhibition, complement fixation

Question 5

What is an in-vivo test?

Answer 5

A test where you are testing on whole living organisms or cells
Measures the actual protective effects of antibodies in animals

Question 6

What is the most common source of antibodies?

Answer 6

serum obtained from clotted blood

Question 7

How can you deplete serum of complement activity?

Answer 7

Heating it to 56 degrees for 30 minutes

Question 8

What is the difference between serum and plasma?

Answer 8

Serum = plasma clotting factors
Plasma = whole blood, blood cells

Question 9

What is an antiglobulin?

Answer 9

made after immunoglobulins are injected of an animal of a different species (specifically against other antibodies) (secondary antibodies)

Question 10

What are polyclonal antibodies?

Answer 10

Mixed populations of antibodies
bind to different areas of the target antigen

Question 11

What are the pros and cons of polyclonal antibodies?

Answer 11

• Pros- Cheap to produce
• Cons- Non-specific
• May bind to other antigens (cross
  reactive
• Different bleeds may yield different
  quality of antibody

Question 12

How can you produce polyclonal antibodies?

Answer 12

1. Inject a target antigen into an animal
2. Bleed the animal and isolate the serum
3. Purify the antibody from the serum

Question 13

What are monoclonal antibodies?

Answer 13

Single antibodies produced by a single B cell clone
Binds to a single specific site on the target antigen
* Derived from hybridomas
* pure and specific

Question 14

What are the pros of monoclonal antibodies?

Answer 14

• • No batch-to-batch differences
• less likely to cross react with other antigens
• can be obtained in almost unlimited
  amounts

Question 15

What are the cons of monoclonal antibodies?

Answer 15

Expensive to produce

Question 16

How can you produce monoclonal antibodies?

Answer 16

1. Inject a target antigen into animal
2. Isolate B cells (able to produce specific antibody) in the
   laboratory
3. Fuse the B cells with immortalized tumour cells
   (myeloma cells)

Question 17

What are three ways you can modify monoclonal antibodies?

Answer 17

Linking with-
* Enzymes
* Fluorescent markers
* Biotin

Question 18

What does ELISA stand for?

Answer 18

Enzyme-Linked immunosorbent assay
ELISAs may be used to detect and measure (quantify) either antibody or antigen (peptides, proteins, and hormones)

Question 19

What are the four different types of ELISA?

Answer 19

• Direct
• Indirect
• Sandwich
• Competitive

Question 20

What are the available enzyme systems for ELISA assays?

Answer 20

• Alkaline phosphatase (AP)
• Horseradish peroxidase (HRP)

Question 21

What is a Chromogenic substance?

Answer 21

Gives colour

Question 22

What is a chemiluminogenic substance?

Answer 22

emits light

Question 23

What is a flurogenic substance?

Answer 23

Emits light

Question 24

What is biotin?

Answer 24

(water-soluble B complex vitamin) that binds to antibody Fc region

Question 25

What is Streptavidin?

Answer 25

(bacterial tetrameric protein that binds to biotin with HIGH affinity) attached to enzyme molecule

Question 26

How do immunoprecipitation based techniques work?

Answer 26

If a solution of soluble antigen is mixed with a strong antiserum * The mixture becomes cloudy within a few minutes, * then flocculent * finally, a precipitate settles to the bottom of the tube within an hour. ## Footnote precipitate contains antigen/ antibody complexes

Question 27

When are immunoprecipitation based techniques used?

Answer 27

* Formation of such large immune complexes that they fall out of solution (precipitate) * Only occurs when Ag/Ab concentrations are roughly equal―leading to formation of large complexes *Can be used to purify antigen molecules or to remove antigens from a solution*

Question 28

What is the Ouchterlony method?

Answer 28

Ag placed in center well, serum samples placed in outer wells. Visible “precipitin line” forms where large immune complexes form.

Question 29

What can haemoagglutination reactions detect?

Answer 29

detect Ag conjugated to or present naturally on the surface of red blood cells.

Question 30

What are immunofluorescence techniques?

Answer 30

Immunofluorescence technique uses specialized microscopy which detects antibodies conjugated with fluorescent dyes

Question 31

What is flow cytometry?

Answer 31

Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells.

Question 32

How does flow cytometry work?

Answer 32

a) Cells are stained with fluorescently conjugated mAbs b)A sample containing labelled cells is suspended in a fluid and injected into the flow cytometer instrument.