Lecture 16; Immune System In health Diagnosis

Question 1

What is precipitation?

Answer 1

It is the formation of Ag:Ab complexes

Question 2

What allows immune complexes to form?

Answer 2

Multi-valency of Ag and AB. (bivalency)

The size of immune complexes is determined by the concentration of Ag and number of epitopes.

Question 3

Describe the precipitation curve;

Answer 3

Protein precipitated vs Antigen added

• Antibody excess is to the left
• Equivalence
• Ag excess

Therefore the curve has kinetics

Question 4

What drives the kinetics of the precipitation curve?

Answer 4

The kinetics are driven by the concentrations

Question 5

How is the zone of equivilence reached in the precipitation curve?

Answer 5

AB must remain constant and Ag increased.

Question 6

What is Nephelometry?

Answer 6

Measures light at 90 degrees to incidence (i.e refracted) of the test tube containing Ag:AB complexes

Quantifies the amount of Ab

Question 7

What is Turbidity?

Answer 7

Measures light passing straight through the test tube containing Ag:AB complexes

Quantifies the amount of Ab

The solution of AgAB goes turbid (insoluable precipitate)

Question 8

What does nephelometry and turbidity rely on?

Answer 8

Ag:AB complexes forming an insoluble precipitate

Question 9

Whats an example where the nephelometry and turbidity measurements may be used?

Answer 9

Quantitate the antibodies against DNA in SLE patients

Question 10

How does agglutination work?

Answer 10

AB are polyvalent 2+ binding sites for the same antigen.

Large particles i.e RBC, bacteria etc have many antigens / epitopes on the surfaces. They can therefore be bound by many AB

= Agglutination

Question 11

What specifically is agglutination?

Answer 11

Mixture of antigens and antibodies that form an interlacing network. This stops the particles from moving around freely (agglutination)

Haemagglutination when RBC are used

Question 12

Antibodies that agglutinate RBC are also known as;

Answer 12

Agglutinins (IgM more effective than IgG because 10 v 2 binding sites)

Question 13

What are some variations of agglutination?

Answer 13

Antigens can be artificially coated onto RBC in a process known as tanning

Alternatively this can also be done to microscopic latex beads

i.e Latex beads coated in IgG for RF assay

Question 14

How are polyclonal antibodies produced?

Answer 14

Antisera is produced by immunising a mouse against a particular antigen a number of times. The responding B cells will proliferate into plasma cells and produce an array of antibodies against the epitopes of the antigen.

Thus the antiserum collected will contain an array of antibodies with varying affinities for the set of antigen determinants for the antigen (polyclonal antibodies)

Question 15

What is one method of producing monoclonal antibodies?

Answer 15

Via a hybridoma

Question 16

How are monoclonal antibodies produced in a hybridoma?

Answer 16

• Activated B cells are taken from the spleen of an immunised mouse and fusing them with myeloma cells in culture to produce hybridomas

Question 17

Describe some features of the produced hybridomas;

Answer 17

• Only specific for the b cell that it was fused with so therefore will only produce that single antibody
• Produces pure clones of cells each secreting antibodies of a single specificity

Question 18

What is another method of producing monoclonal antibodies?

Answer 18

Process called; Antibody Phage Display

Question 19

Describe antibody phage display;

Answer 19

• Molecular Technique
• Gene segments encoding the variable domains of antibodies are fused to genes encoding the coat protein of bacteriophage.
• This enables a library of recombinant bacteriophage to be engineered that display variable domains on their surface
• This library can be screened (biopanning) to identify clones for a specific antigen
• This clones contain the coding sequence to enable production of monoclonal antibodies

Question 20

What are the properties of monoclonal antibodies?

Answer 20

• Homogenous with respect to specificity and antibody class
• Recognises a single determinant on the antigen of interest
• Produced with little or no contamination by irrelevant antibodies
• Reagents easily available
• Ready use as standardised reagents

Question 21

What are monoclonal antibodies usually labelled with?

Answer 21

• Radioactively
• Enzyme
• Flurochrome

Question 22

Describe the steps in an elisa assay;

Answer 22

ELISA steps;

• Coat antigen onto well base
• Add AB
• Wash
• Add HRP substrate, catalyses reaction = colour
• Colour proportional to AB
• Stop reaction
• Plate reader

Many Ways ELSIA can be done

Question 23

What is ELISA?

Answer 23

Enzyme-linked immunosorbent assay

• Direct binding assay for an antibody
• Commonly used to measure antibodies in blood (level)

Question 24

What are some variations of ELISA?

Answer 24

• Standard/Direct ELISA
• Capture or Sandwich assay (Antigen specific antibodies are bound to plate, and antigen is introduced to be quantified using secondary ab)

Question 25

Describe ELISA using beads;

Answer 25

Multiplex bead based ELISA - Quantification of multiple antigens in a single sample. - Small microspheres labelled with flourescent dye are coated with antibody. - Can be detected simultaneously with machine luminex

Question 26

What is essential to the immunoflourescence technique?

Answer 26

Flurochromes can be chemically coupled to antibody molecules

Question 27

Describe direct IF;

Answer 27

- Staining tissue section with flurochrome tagged antibodies specific for an antigen - The amount of flourescence is proportional to the amount of antibody

Question 28

Describe two colour IF;

Answer 28

Two antibodies (each coupled with to a different flurochrome), are used to examine two different antigens to see how their distribution relates to each other.

Question 29

Describe indirect IF;

Answer 29

A primary antibody that detects the antigen of interest is added to the tissue. Bound antibody is detected with flourescently labelled secondary (anti-Ig)

Question 30

Whats the struggle with flourescent imaging?

Answer 30

Flourescent images at high magnification are hard to examine because of the glare from slightly out-of-focus planes above and below the area of interest. (blurred) Confocal Microscope solves this

Question 31

How does a confocal microscope work?

Answer 31

A confocal microscope focus' a sharp laser beam on a fine plane within the tissue. Photomultiplier tubes scan the area and collect emitted light only from that exact plane. This information is analysed digitally. = High definition image of a thin cross section of tissue. Therefore protein interactions can be observed

Question 32

What does the solution contain in flow cytometry?

Answer 32

- Cells in suspension are incubated with flurochrome tagged antibodies The tagged cells then individually pass through the cytometer in droplets of buffer in a single cell stream (different cell types (antigens) can be labelled differently)

Question 33

What does flow cytometry measure?

Answer 33

Size = Foward scatter Granularity = side scatter - Can detect up to 14 different antigens on a cell i. e CD4 to CD8 ratios - Immunotyping of leukemias

Question 34

What are the methods of HLA tissue typing?

Answer 34

Microlymphcytotoxicity PCR Sequence Base Typing Mixed Lymphocyte reaction

Question 35

What is Microlymphcytotoxicity?

Answer 35

Peripheral blood lymphocytes are incubated with a panel of antibodies directed against all the main HLA antigens. Complement is added. The cells are lysed if they have the antigen of interest.

Question 36

What is PCR?

Answer 36

Requires the DNA of HLA molecules to match the primers. Only the DNA containing the correct sequence will be amplified indicating whether or not the cell has the HLA molecule

Question 37

What is sequence base typing?

Answer 37

HLA typing by directly DNA sequencing the HLA genes.

Question 38

What is mixed lymphocyte reactions?

Answer 38

If lymphocytes of patients with two different HLA types are mixed together , they recognise the other HLA antigens as foreign and begin to proliferate One set of cells are irradiated so they cant respond but still have HLA molecules

Question 39

What are cytotoxic assays?

Answer 39

- Incubate target cells with radioactive chromate, spin and wash. - Incubate chromate-labelled target cells with cytotoxic effectors. Cyotoxic cells lyse targets and radioactive material released. - Measure the amount of radioactive released into the supernate

Question 40

What are cytotoxic assays used for?

Answer 40

Functional assays used to determine killing by; - Antigen specific CD8 cytotoxic lymphocytes - NK cells - ADCC