Immunological testing

Question 1

What is ELISA? What does it measure?

Answer 1

Enzyme linked immunosorbent assay

Measures Ag or Ab, infection or hypersensitivity

Question 2

What are the advantages of ELISA tests?

Answer 2

Quantifiable
Quick
Cheap
Reproducible

Question 3

ELISA can detect specific antibodies in animal plasma/ serum. How does this work?

Answer 3

Coat plate wells with antibody 
Add serum samples 
Add enzyme tagged antibodies
Add substrate
(Wash between each stage to remove unbound reagents)
Colour change = more antibody

Question 4

Immune complex formations can be large or small. What does this depend on?

Answer 4

Relative amounts of antigen/antibody
Number of binding sites on antigen
Class of antibody

Question 5

What is the method to test for agglutination for antibodies?

Answer 5

RBCs coated with antigen
Mixed with test serum
Positive sera = agglutination of RBCs

Question 6

How do immunoflouresence tests work?

Answer 6

Specific antibodies conjugated to fluorscent dyes
Antibodies bind directly (primary) or indirectly (secondary) to the antigens
Put under short wavelength high energy light - immune complexes glow fluorescently

Question 7

What is the most common serology test in small animal practice?

Answer 7

Snap test

Fast, done in practice

Question 8

How does double diffusion work?

Answer 8

Wells cut in agar plate - one filled with antigen, one filled with antibody
Line of precipitation forms if two reactants meet

Question 9

Double diffusion causes precipitation if the immune complex is produced at equivalence. What does this mean?

Answer 9

Equal amount of antibody to antigen

Question 10

What does the concentration of each reactant depend on in double diffusion?

Answer 10

Concentration
Molecular size
Ability to diffuse

Question 11

What are the 3 basic precipitation factors of double diffusion?

Answer 11

Lines of identity - identical antigens
Lines of non-identity - two lines that cross, serum contains 2 antigens
Lines of partial identity - serum contains an antigen with 2 different epitopes

Question 12

What is single radial diffusion?

Answer 12

Antiserum containing known antibodies incorporated into agar
Wells cut out and antigen placed in wells
Immune complexes form radial diffusion ring - larger diameter = higher antigen concentration

Question 13

What is immunoelectrophoresis used to detect?

Answer 13

B lymphocyte lymphoma (type of leukaemia)

Question 14

What change in serum levels of antibodies does B-lymphocyte lymphoma result in

Answer 14

Increased antibodies

Due to clonal expansion of B cells

Question 15

What is the difference between electrophoresis and immunoelectrophoresis?

Answer 15

Electrophoresis - identification of any increased antibodies

Immune - identifies specific pathological changes

Question 16

How does electrophoresis work?

Answer 16

Patient serum (contains antigens) put into well in agar gel
Electric current is applied to cause antigens to migrate to either positive/negative ends of agar gel
Antiserum (containing specific antibodies) added to trough below agar plates
If immune complexes form - precipitation
Normal serum compared to patient serum

Question 17

What is the snap assay?

Answer 17

Type of ELISA
Enzyme labeled conjugate mixed with serum/blood and added to sample well
Sample-conjugate mix flows through the matrix
The device is then activated by ‘snapping’ which releases wash buffer
Coloured precipitation = positive result

Question 18

What is affinity?

Answer 18

Strength of reaction between single antigen and binding site (sum of attractive and repulsive forces)

Question 19

What is avidity?

Answer 19

Overal strength of binding between an antigen with many determinants and multivalent antibodies

Question 20

Complex size depends on relative amount of Ag/Ab, affinity and class of antibody. How can class of antibody affect complex size?

Answer 20

Different number of binding sites

E.g. IgG has 2 antigen binding sites, IgM has 10

Question 21

What are useful properties of immune complex formation that helps with immunological testing?

Answer 21

Large complexes form (at optimal ratio of Ag:Ab)

Multidimensional lattice means visible precipitate can form

Question 22

What holds the light chain of an antibody to the heavy chain?

Answer 22

Disulfide bond

Question 23

What is an epitope?

Answer 23

The part of an antigen that interacts with the antigen specific receptor/antibody

Question 24

What is immunoelectrophoresis mainly used to identify?

Answer 24

Myelomas (mainly)
Deficiencies
Acute bacterial infections