Lecture 16; Immune System In health Diagnosis Flashcards
What is precipitation?
It is the formation of Ag:Ab complexes
What allows immune complexes to form?
Multi-valency of Ag and AB. (bivalency)
The size of immune complexes is determined by the concentration of Ag and number of epitopes.
Describe the precipitation curve;
Protein precipitated vs Antigen added
- Antibody excess is to the left
- Equivalence
- Ag excess
Therefore the curve has kinetics
What drives the kinetics of the precipitation curve?
The kinetics are driven by the concentrations
How is the zone of equivilence reached in the precipitation curve?
AB must remain constant and Ag increased.
What is Nephelometry?
Measures light at 90 degrees to incidence (i.e refracted) of the test tube containing Ag:AB complexes
Quantifies the amount of Ab
What is Turbidity?
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)
What does nephelometry and turbidity rely on?
Ag:AB complexes forming an insoluble precipitate
Whats an example where the nephelometry and turbidity measurements may be used?
Quantitate the antibodies against DNA in SLE patients
How does agglutination work?
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
What specifically is agglutination?
Mixture of antigens and antibodies that form an interlacing network. This stops the particles from moving around freely (agglutination)
Haemagglutination when RBC are used
Antibodies that agglutinate RBC are also known as;
Agglutinins (IgM more effective than IgG because 10 v 2 binding sites)
What are some variations of agglutination?
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
How are polyclonal antibodies produced?
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)
What is one method of producing monoclonal antibodies?
Via a hybridoma
How are monoclonal antibodies produced in a hybridoma?
- Activated B cells are taken from the spleen of an immunised mouse and fusing them with myeloma cells in culture to produce hybridomas
Describe some features of the produced hybridomas;
- 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
What is another method of producing monoclonal antibodies?
Process called; Antibody Phage Display
Describe antibody phage display;
- 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
What are the properties of monoclonal antibodies?
- 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
What are monoclonal antibodies usually labelled with?
- Radioactively
- Enzyme
- Flurochrome
Describe the steps in an elisa assay;
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
What is ELISA?
Enzyme-linked immunosorbent assay
- Direct binding assay for an antibody
- Commonly used to measure antibodies in blood (level)
What are some variations of ELISA?
- Standard/Direct ELISA
- Capture or Sandwich assay (Antigen specific antibodies are bound to plate, and antigen is introduced to be quantified using secondary ab)
Describe ELISA using beads;
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
What is essential to the immunoflourescence technique?
Flurochromes can be chemically coupled to antibody molecules
Describe direct IF;
- Staining tissue section with flurochrome tagged antibodies specific for an antigen
- The amount of flourescence is proportional to the amount of antibody
Describe two colour IF;
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.
Describe indirect IF;
A primary antibody that detects the antigen of interest is added to the tissue. Bound antibody is detected with flourescently labelled secondary (anti-Ig)
Whats the struggle with flourescent imaging?
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
How does a confocal microscope work?
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
What does the solution contain in flow cytometry?
- 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)
What does flow cytometry measure?
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
What are the methods of HLA tissue typing?
Microlymphcytotoxicity
PCR
Sequence Base Typing
Mixed Lymphocyte reaction
What is Microlymphcytotoxicity?
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.
What is PCR?
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
What is sequence base typing?
HLA typing by directly DNA sequencing the HLA genes.
What is mixed lymphocyte reactions?
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
What are cytotoxic assays?
- 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
What are cytotoxic assays used for?
Functional assays used to determine killing by;
- Antigen specific CD8 cytotoxic lymphocytes
- NK cells
- ADCC
