Lecture 15: Immune Diagnosis

Question 1

What is a precipitin curve?

Answer 1

• Kinetics of antigen-antibody interaction is driven by concentration
• When antibody is kept constant and concentration of antigen increases then the zone of equivalence is reached
• Driven by ‘bivalency’ of antibodies
• Equivalence: antibody cross-linking of antigen, immune complex precipitates
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Question 2

What was the historical use of the precipitin reaction?

Answer 2

Radical immunodiffusion: width proportional to concentration of antigen

Question 3

Turbidimetry

Answer 3

Ability of light to go through, antigen-antibody complex

Question 4

Nephelometry

Answer 4

Ability of light to deflect off, antigen-antibody complex at 90 degree angle

Question 5

What are some examples of clinical tests based on antibody precipitation?

Answer 5

• Anti-SLO antibody test
• Anti-ds-DNA antibody test

Question 7

Aggulitination definition

Answer 7

When antigen is displayed on the surface of a large particle, antibodies can cause bacteria to clump. This clumping is “agglutination”

Question 9

What is a polyclonal antibody?

Answer 9

A Polyclonal Antibody represents a collection of antibodies from different B cells that recognize multiple epitopes on the same antigen

Question 10

What are the characteristics of Conventional Polyclonal Antisera?

Answer 10

• Multiple immunisations (2-3)
• Recognise multiple epitopes
• Range of affinities
• Required specificity is small fraction of total antibodies
• Need extensive purification
• Batch-to-batch variation

Question 11

How are monoclonal antibodies produced?

Answer 11

Spleen cells and myeloma cells are mixed and fused forming hybrid myelomas. Separate clones.

Question 12

What are the properties of monoclonal antibodies?

Answer 12

• Homogeneous with respect to specificity and antibody class
• Recognize single determinant on the antigen of interest
• Produced with little or no contamination by irrelevant antibodies
• Invariant reagent that is available in infinite supply
• Ready to use as standardized reagents

Question 13

Examples of assays and tests that utilize monoclonal antibodies

Answer 13

Radioactive atoms
Enzymes
Fluorochromes

Question 14

ELISA: general information

Answer 14

Enzyme-linked immunosorbant assay
HRP = horse radish peroxidase
Catalyses the oxidation of substrates that are chromogenic

can now use lumnex bead based immunoassays - can use different beads at the same time

Question 15

What is a mixed lymphocyte reaction?

Answer 15

Usually looked at for immune rejection. Inactive one bodies WBCs culture for 4-5 days, add H-3 (thymidine). Proliferation = rejection, no proliferation = ok

Question 16

Cytotoxic Assays: Chromium Release Assays

Answer 16

Get target cells to take up 51-Cr, spin and wash. Plate out with effector cells, and look at release of chromium (radioactivity), measure 51-Cr in supernatant