Test 7

Question 1

Describe single radial immunodiffusion test

Answer 1

Antibody is premixed in a gel. Antigen is put in holes in the gel, and diffuses out. A ring of precipitate is formed around the hole.

Question 2

What is Ouchterlony’s technique?

Answer 2

Double immunodiffusion.
Result: precipitation line (qualitative)

Question 3

Describe double immunodiffusion?

Answer 3

Radial diffusion of antigen and antibody from holes forming a line of precipitation

Question 4

How are the results of double immunodiffusion interpreted?

Answer 4

reaction of identity; reaction of non-identity; reaction of partial identity

Question 5

What is protein electrophoresis used for, and what medium is used?

Answer 5

screening of serum and other fluids for protein abnormalities, agarose gel

Question 6

What are the protein fractions in protein electrophoresis?

Answer 6

Albumin
Alpha-1 globulins
Alpha-2 globulins
Beta-1 globulins
Beta-2 globulins
Gamma globulins

Question 7

What is immunofixation?

Answer 7

Technique used to identify abnormal bands in electrophoretic pattern of serum proteins.
Detect monoclonal components.

Proteins are anchored in situ after electrophoresis by forming an insoluble complex with antibody.

Question 8

What are the four stages of immunofixation?

Answer 8

1. Separation of proteins by electrophoresis in agarose gel
2. Fixation and immunoprecipitation of electrophoresed proteins
3. Non-precipitating, soluble proteins are removed by blotting and washing
4. Precipitated proteins are visualized by staining

Question 9

Name the tracks of immunofixation:

Answer 9

electrophoretic pattern, gamma antiserum, alpha antiserum, mu antiserum, kappa, lambda

Question 10

What is immunoelectrophoresis a combination of?

Answer 10

electrophoresis and gel diffusion

Question 11

How are antigens separated in immunoelectrophoresis?

Answer 11

charge, size, conformation, composition, pH of buffer

Question 12

Describe classical immunoelectrophoresis:

Answer 12

A gel is prepared with alternating wells and slots with antigen mixture placed in the wells and electrophoresis is performed to separate proteins in the sample.
Antibodies are added into the slots. Antigens and antibodies migrate towards each other and form arcs of precipitate.

Question 13

How are proteins separated in classical immunoelectrophoresis?

Answer 13

positively charged proteins move to the negative electrode, and negatively charged proteins move to positive electrode

Question 14

What is identified in classical immunoelectrophoresis?

Answer 14

serum proteins

Question 15

What type of analysis is classical immunoelectrophoresis?

Answer 15

qualitative

Question 16

Describe counter-immunoelectrophoresis

Answer 16

Antigens placed in one well, antibodies in the other. Electric current is used to drive the antigen towards the antibody.

Question 17

What is rocket immunoelectrophoresis used for?

Answer 17

• quantitative
  Determining levels of serum proteins

Question 18

Describe rocket immunoelectrophoresis

Answer 18

Antigens are loaded into wells along the edge of a gel containing incorporated antibody.
Antigens are electrophoresed into the gel where they interact with antibody forming precipitation line.

Results: height of precipitation arc is directly proportional to the concentration of the antigen, compared to known concentrations.

Question 19

What is the basic principle of enzyme immunoassays?

Answer 19

Enzyme of conjugate reacts with a colourless substrate to generate a colour reaction product

Question 20

What is a chromogenic substrate?

Answer 20

A substrate used in enzyme immunoassays which produce colour

Question 21

How are enzyme immunoassays divided?

Answer 21

Enzyme-linked immunosorbent assay (ELISA),
enzyme immunoblotting and
enzyme immunohistochemistry

Question 22

What are the enzymes used for enzyme immunoassays?

Answer 22

Alkaline phosphatase and Horse Radish peroxidase

Question 23

What system is used to increase sensitivity of enzyme-immunoassays?

Answer 23

Biotin-avidin systems.
Avidin can non-covalently bind up to four biotin molecules - used in amplification step.
Used in case of: low level of antigen or need for rapid detection

Question 24

Steps of ELISA:

Answer 24

1. binding of known component to solid surface
2. blocking of the surface to prevent further non-specific binding
3. incubation of the test sample with adsorbed antigen or antibody
4. detection of antigen-antibody complexes with enzyme-linked antibody which produce a colour product from substrate to be detected

Question 25

What groups are ELISA techniques divided into?

Answer 25

Detection of antibodies - indirect, blocking
Detection of antigens - sandwich, competitive

Question 26

Describe sandwich ELISA

Answer 26

1. primary antibody coats the place
2. antigen is added and binds to antibody
3. secondary enzyme-linked antibody recognizes Ag-Ab complex and catalyses substrate to produce specific colour

Question 27

In competitive ELISA

Answer 27

antigen and enzyme-labelled antigens competitively bind to antibody

Question 28

Describe blocking ELISA

Answer 28

1. binding of antigen to solid phase
2. incubation with test sample
3. incubation with enzyme labelled antibody
4. enzyme catalyzes substrate to produce specific colour

Question 29

What are typical blocking solutions?

Answer 29

phosphate buffered saline
sodium carbonate containing bovine serum
albumin
casein
gelatine

Question 30

Describe how lateral-flow immunoassays work

Answer 30

1. antigen solution flows through a porous strip
2. solution passes through zone with soluble polyclonal or monoclonal antibodies
3. soluble immune complexes are formed
4. fluid flows through test zone where immune complexes are bound to immobilised antibody against target antigen
5. in control zone, unbound conjugates bind to immobilised antiglobulins