ELISA
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62 Terms
What does ELISA stand for?
Enzyme-Linked Immunosorbent Assay.
What is immunology?
The study of the immune system.
What is an antigen?
A foreign substance that can trigger an immune response and be recognized by antibodies.
What is an antibody?
A protein/immunoglobulin produced by B cells that specifically recognizes an antigen.
What is an epitope?
The specific part of an antigen recognized by an antibody.
What do B cells do?
Produce antibodies.
What do T cells do?
Stimulate B cells and kill infected cells.
What is humoral immunity?
Immune response involving antibodies circulating in blood and lymph.
What is cell-mediated immunity?
Immune response involving T lymphocytes that bind and destroy infected cells.
What is innate immunity?
Defenses present from birth that are not highly specific to particular pathogens.
What is adaptive/acquired immunity?
A specific immune response that develops after exposure to an antigen.
What is a primary immune response?
The first response to an antigen; initially dominated by IgM, followed by IgG.
What is a secondary immune response?
A faster and stronger response after a second exposure, with much more IgG production.
What are immunoglobulins?
Another name for antibodies.
What are the five classes of immunoglobulins?
IgG, IgM, IgA, IgE, and IgD.
Which immunoglobulin is most abundant in internal body fluids?
IgG.
What is IgG?
The most abundant immunoglobulin in internal body fluids; about 15% of total adult serum protein.
What is IgM?
The immunoglobulin responsible for the primary immune response.
What is IgA?
An antibody found in external secretions such as tears, saliva, milk, and mucosal secretions.
What is IgE?
An immunoglobulin that is a primary component in allergic reactions.
What is IgD?
An immunoglobulin that may be involved in regulating the immune response.
What are polyclonal antibodies?
Antibodies produced by many different B-cell clones that recognize multiple epitopes.
What is an advantage of polyclonal antibodies?
They are simple and inexpensive to produce.
What is a disadvantage of polyclonal antibodies?
Different batches are not exactly the same.
What are monoclonal antibodies?
One antibody type produced from a single B-cell clone.
What is a hybridoma?
A hybrid cell made by fusing an antibody-producing B cell with an immortal tumor-like cell.
Why are hybridomas useful?
They can produce monoclonal antibodies indefinitely with little batch-to-batch variability.
What is direct detection?
The antibody specific for the antigen is directly labeled to produce the detectable signal.
What is indirect detection?
An unlabeled primary antibody binds the antigen and an enzyme-linked secondary antibody binds the primary antibody.
What does the primary antibody bind?
The target antigen.
What does the secondary antibody bind?
The primary antibody.
Why is indirect detection useful?
One enzyme-linked secondary antibody can detect many primary antibodies from the same animal species.
Why does indirect detection amplify the signal?
Multiple secondary antibodies can bind to one primary antibody, allowing more labels to accumulate.
What is the basic principle of ELISA?
Specific antigen-antibody binding is detected using an enzyme-linked antibody that produces a color signal.
What is attached to the secondary antibody in this ELISA?
HRP enzyme.
What does HRP do?
It changes the enzyme substrate from colorless to blue.
What does a blue well indicate?
The target antigen was detected.
What does a colorless well indicate?
The target antigen was not detected.
Why are washing steps important in ELISA?
They remove unbound material so the signal comes from specific binding.
How much body-fluid sample is transferred during sharing?
750 µL.
How many other students do you share your sample with?
Three students.
What must you record during sample sharing?
The names of the three sharing partners in order.
How are the 12 ELISA wells labeled?
First 3 = positive controls, next 3 = negative controls, remaining wells = student initials.
How much positive control is added to each positive well?
50 µL.
How much negative control is added to each negative well?
50 µL.
How much student sample is added to each sample well?
50 µL.
Why use a fresh pipette tip for each sample?
To prevent contamination or carryover between samples.
How long do the samples bind to the plastic wells?
5 minutes.
What happens after the sample incubation?
Wash the wells to remove unbound sample.
How many initial washes are performed?
Two washes.
What is used to wash the wells?
Wash buffer.
How much primary antibody is added?
50 µL to all 12 wells.
How long does the primary antibody bind?
5 minutes.
How many washes occur after the primary antibody?
Two washes.
How much secondary antibody is added?
50 µL to all 12 wells.
How long does the secondary antibody bind?
5 minutes.
How many washes occur after the secondary antibody?
Three washes.
What enzyme is attached to the secondary antibody?
HRP.
How much enzyme substrate is added?
50 µL to all 12 wells.
What happens after adding the enzyme substrate?
Wait 5 minutes, then observe and record the results.
What is the basic ELISA sequence?
Sample → Wash → Primary antibody → Wash → Secondary antibody → Wash → Substrate → Color.
What is the most important reason for the wash steps?
Remove unbound material and reduce nonspecific signal.