Immuno Assay Flashcards
Each cell involved in the adaptive immunity has specialised receptors that can bind to clusters of molecules on the surface of foreign cells – known as
epitopes
One group of adaptive immune cells are known as B Lymphocytes or B cells. They secrete their receptors in large amounts – and the secreted receptors are called
Antibodies
The foreign invader is known as an
antigen
Immunoassay deals with what part of the immune system
Adaptive immune system
Facts about immunoassay
Scientists working in many fields are interested in this process. The human body has a way of specifically identifying and attacking anything foreign to the body. This can be exploited by biochemists/biomedical scientists/HCS/pharmacologists/pharmaceutical Scientists for many reasons e.g ;
vaccinations - one of the ways the immune system is exploited
Define immunoassay
immuno = immune system e.g. antibodies and antigens assay = determine how much of something is present
Therefore an immunoassay exploits the specific binding affinity an antibody has for a specific antigen to help to quantify how much of either the antibody or antigen is present.
State two major requirements for an immunoassay
An antibody
An detection system
Structure of an antibody
has two heavy and two light chains variable region gives antibody it specificity
5 types of constant region
Antibodies are immune system-related proteins called immunoglobulins. Each antibody consists of four polypeptides– two heavy chains and two light chains joined to form a “Y” shaped molecule.
The a.acid sequence in the tips of the “Y” varies among different antibodies. This region (110-130 a.acids) gives the antibody specificity.
The constant region of the antibody determines-
The constant region determines the mechanism used to destroy antigen. Antibodies are divided into 5 major classes, IgM, IgG, Iga, IgD and IgE, based on their constant region structure and immune function.
How is diversity created on antibodies variable sites?
(a) By having multiple gene sequences
(b) Inaccurate and multiple joining – random component
(c) Different heavy and light chain combinations
(d) Somatic mutation – occurs 1 million times more often in variable regions of antibodies compared to other amino acid chains.
State how to obtain a polyclonal antibody
- Immunise animals with an antigen
- reimmunize the animal to produce a secondary response
- take a blood sample and test to see if it has made the antibody
- can continue to take blood sample from the animal during its life - a continuous source of polyclonal antibody
animal that stays alive
big animal used eg rabbit
State how to obtain a polyclonal antibody
- Immunise animals with an antigen
- reimmunize the animal to produce a secondary response
- take a blood sample and test to see if it has made the antibody
- can continue to take blood sample from the animal during its life - a continuous source of polyclonal antibody
animal that stays alive
big animal used eg rabbit
State how to obtain a monoclonal antibody
a mouse is used
- innoculate animal
- reinnoculate to get a secondary response
- sacrifice animal to remove the spleen
- isolate lymphocyte and grow on a cell culture to see if antibody is produced
What happens to the removed lymphocytes in the process of obtaining a monoclonal antibody
Remove B-cells from the spleen of an animal that has been challenged with the antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer).
The fused hybrid cells (called hybridomas) will multiply rapidly and indefinitely (since they are cancer cells) and will produce large amounts of antibodies.
List how monoclonal antibody is different from polyclonal antibody
Restricted to mouse Can develop and grow cell culture (quick and cheap) Life long culture Constant antibody source Little purification needed Most often used in immunoassays
how polyclonal antibody is different from monoclonal
Many animals can be use (as long as big enough).
Need animal houses – strict regulations
Finite source – as animal will eventually die of old age and the source changes each time a new animal is used.
Must purify antibody’s from all other cell components
Best for large antigens who’s epitopes may change e.g. bacteria
What animal should you try and induce antibody production in?
For monoclonal antibodies you are restricted to mice
For polyclonal antibodies you need a large animal e.g a rabbit, goat, sheep
In choosing the animal you must consider;
FCG
Foreignness – Antibody will only be produced if the antigen is foreign to the animal (i.e. not similar to some natural substance in the body).
Convenience – need animals easy to buy and look after – that don’t take up too much space
Genome – even animals of the same species have slightly different genomes. These may respond differently to an antigen – so you may have to use the same strain of animal.
What antigen should be used to inoculate the animal?
It is not easy to make an antibody for everything. One must consider the following properties of the antigen
(a) Molecular size – Bigger the better
(b) Composition – Proteins with only a few different amino acids
(c) Heterogeneity – a difficulty is posed if a particular antigen has a number of different forms
For (c) proteins with sugars attached are a good example. The protein is the same but is modified later by sugars, but can have various amounts of sugar added and still act the same way. In this case could cause problems.
(d) Haptens – small non-protein molecules have Antigenicity (can be recognised by the immune system and bind to antibodies) but lack immunogenicity (the ability to initiate an immune response). You can trick the immune system to create antibodies by binding it to a protein – known as a carrier
(e) Solubility of the antigen – Large insoluble antigens are better at causing antibody
