Immunology- Basic Techniques Flashcards
What is an immunological assay?
A biochemical test that measures the presence or concentration of a specific molecule using of an antibody
What is serology?
measurement of antigen-antibody reactions for diagnostic purposes
What is a primary binding test?
Directly measuring the binding of an antigen to an antibody (e.g ELISA testing)
What is a secondary bhinding test?
Measures the results of antigenn-antibody reactions in vitro (e.g precipitation assays, haemoagglutination inhibition, complement fixation
What is an in-vivo test?
A test where you are testing on whole living organisms or cells
Measures the actual protective effects of antibodies in animals
What is the most common source of antibodies?
serum obtained from clotted blood
How can you deplete serum of complement activity?
Heating it to 56 degrees for 30 minutes
What is the difference between serum and plasma?
Serum = plasma clotting factors
Plasma = whole blood, blood cells
What is an antiglobulin?
made after immunoglobulins are injected of an animal of a different species (specifically against other antibodies) (secondary antibodies)
What are polyclonal antibodies?
Mixed populations of antibodies
bind to different areas of the target antigen
What are the pros and cons of polyclonal antibodies?
- Pros- Cheap to produce
- Cons- Non-specific
- May bind to other antigens (cross
reactive - Different bleeds may yield different
quality of antibody
How can you produce polyclonal antibodies?
- Inject a target antigen into an animal
- Bleed the animal and isolate the serum
- Purify the antibody from the serum
What are monoclonal antibodies?
Single antibodies produced by a single B cell clone
Binds to a single specific site on the target antigen
* Derived from hybridomas
* pure and specific
What are the pros of monoclonal antibodies?
- No batch-to-batch differences
- less likely to cross react with other antigens
- can be obtained in almost unlimited
amounts
What are the cons of monoclonal antibodies?
Expensive to produce
How can you produce monoclonal antibodies?
- Inject a target antigen into animal
- Isolate B cells (able to produce specific antibody) in the
laboratory - Fuse the B cells with immortalized tumour cells
(myeloma cells)
What are three ways you can modify monoclonal antibodies?
Linking with-
* Enzymes
* Fluorescent markers
* Biotin
What does ELISA stand for?
Enzyme-Linked immunosorbent assay
ELISAs may be used to detect and measure (quantify) either antibody or antigen (peptides, proteins, and hormones)
What are the four different types of ELISA?
- Direct
- Indirect
- Sandwich
- Competitive
What are the available enzyme systems for ELISA assays?
- Alkaline phosphatase (AP)
- Horseradish peroxidase (HRP)
What is a Chromogenic substance?
Gives colour
What is a chemiluminogenic substance?
emits light
What is a flurogenic substance?
Emits light
What is biotin?
(water-soluble B complex vitamin) that binds to antibody Fc region
What is Streptavidin?
(bacterial tetrameric protein that binds to biotin with
HIGH affinity) attached to enzyme molecule
How do immunoprecipitation based techniques work?
If a solution of soluble antigen is mixed with a strong antiserum
* The mixture becomes cloudy within a few minutes,
* then flocculent
* finally, a precipitate settles to the bottom of the tube within an hour.
precipitate contains antigen/ antibody complexes
When are immunoprecipitation based techniques used?
- Formation of such large immune complexes that
they fall out of solution (precipitate) - Only occurs when Ag/Ab concentrations are
roughly equal―leading to formation of large
complexes
Can be used to purify antigen molecules or to
remove antigens from a solution
What is the Ouchterlony method?
Ag placed in center well, serum
samples placed in outer wells.
Visible “precipitin line” forms
where large immune complexes
form.
What can haemoagglutination reactions detect?
detect Ag conjugated to or present
naturally on the surface of red
blood cells.
What are immunofluorescence techniques?
Immunofluorescence technique uses specialized
microscopy which detects antibodies conjugated with
fluorescent dyes
What is flow cytometry?
Flow cytometry is a technique used to detect and measure
physical and chemical characteristics of a population of cells.
How does flow cytometry work?
a) Cells are stained with fluorescently conjugated mAbs
b)A sample containing labelled cells is suspended in a fluid and
injected into the flow cytometer instrument.
