Immunology Flashcards
What is ELISA?
Enzyme Linked ImmunoSorbent Assay
What is an ELISA?
An enzyme linked ImmunoSorbent assay is an immunological assay used to measure antibodies, antigens, proteins and glycoproteins in biological samples.
Why do they use ELISA’s?
The samples used are measured to diagnose HIV, pregnancy, measuring of cytokines, supernatant, soluble receptors and supernatants
How is an ELISA done?
They are done on a 96 well plate, allowing multiple samples to be measures. The plates need to be special absorbent plates to ensure antibodies and antigens stick to the surface. Each Elisa measure a specific antigen and multiple kits are available for different antigens.
What is the method to do a sandwich ELISA?
1- two sets of antibodies are used to detect secreted products. You first coat the plate with capture antibodies and any excess are washed away.
2- next you add the sample, any antigens present bind with the capture antibodies. The sample is added in triplicate to ensure detection and analysis. Excess is washed away again.
3- next a detection antibody is added. This antibody is labelled with an enzyme. This antibody binds to the target antigen bound in the plate. Finally a substrate is added and a chromogenic reaction occurs, converting the substrate into a coloured product which can be measured
How do you determine an antigen concentration?
Determining the antigen concentration in a sample requires a production of a standard curve using antigens of a known concentration.
What are Antibodies and antigens
Antibodies are Y shaped proteins that recognise and bind antigens- they’re made by B cells.
An antigen is anything that can cause an immune response. It can be a pathogen component, or components of proteins, carbohydrates, lipids, drugs, toxins etc
What is analytical sensitivity?
The ability of a test to detect small amounts of Ab and Ag
What is clinical sensitivity?
Ability of a test to give positive results if patients have the disease (no false negatives)
What is a false negative?
This is where the result is negative as the concentration of the Ab or Ag is too low to detect. In reality, the patient has the disease.
What is analytical specificity?
The ability of a test to detect a substance without interference from cross-reacting substances
What is clinical specificity?
The absolute of test to give a negative result if the patient doesn’t have the disease (no false positive)
What is a false positive?
The test result is positive however the test is detecting a cross reacting substance. In reality the result should be negative as the patient doesn’t have the disease
What is highly sensitive?
False negative reactions are absent or minimal
What is highly specific?
False positive reactions are absent or minimal
Why are antibodies used as research tools?
- They’re highly specific for corresponding antigens
- easy to isolate and study
- invaluable as probes of biological processes
- can be generated in large amounts
- have high affinity for their antigen
Monoclonal vs polyclonal antibodies
Polyclonal (pAb)- Heterogeneous, secreted different by B cells, same antigen but different epitope.
Monoclonal (mAb)- Homogeneous, secreted by one B cell clone, has the same antigen and same epitope
Monoclonal antibody production
1- specific antigen injected in mouse
2- spleen B cells producing desired antibodies are isolated
3- fuse the spleen cells with myeloma cancer cells which are immortal
4- produces Hybridoma- subclonal hybdrodoma cells produce antibody
Polyclonal antibody production
1- inject antigen into rabbit
2- antigen activates B cells
3- Plasma B cells produce polyclonal antibodies
4- Obtain antiserum from rabbit containing antibodies
ELISA controls
Positive control- Confirm the assay is working, can be done as part of the standards
Negative control- confirm a positive result is not due to a non-specific binding of Abs or faulty reagents
How do you determine ELISA sensitivity?
As you dilute the standard further, it will eventually reach an Absorbance thag is undetectable above the negative control.
You identify the lowest possible concentration of the primary antibody that still gives a positive result.
This is the sensitivity limit of your assay.
High limit= low sensitivity = potential for false negatives as the concentration of the measured analysts is too low for detection
