31. ELISA Flashcards
What does ELISA stand for?
Enzyme Linked Immunosorbent Assay
What are the main functions of the immune system? (2)
1) recognition of invading pathogens
2) destruction and removal of those pathogens
What are antigens?
Examples?
- any molecule or structure that can elicit an adaptive immune response
- Ex. Protein and polysaccharides
What are linked enzymes also called?
-reporter enzymes
How are antibodies produced in a lab setting?
-repeated injections of mammals or birds with antigen molecules and adjuvant > production of antibodies that bind the antigen
What is the function of adjuvants?
-stimulate inflammation, stimulating an infection;inflammation is necessary to provoke a strong immune response
Other names for antibodies
- immunoglobulin
- gamma globulin
Common lab antibody sources
-mice, rats, guinea pigs, rabbits, goats, chickens
How are antibodies named?
Ex.1) Rabbit with E.Coli O antigen 157
2) goat with mixture of antibody proteins from rabbits
1) rabbit anti-O157
2) goat anti-rabbit
What are the roles of primary antibodies?
-directly bind the antigen you wish to detect
What is the role of secondary antibodies?
-bind primary antibodies of the first species (antibodies can also be antigens)
What is the difference between direct and indirect ELISA?
- Direct: antigen attached to plastic dish; enzyme-linked antibody binds only if the antigen is present
- Indirect: Antigen attached to dish; primary antibody binds to antigen; multiple secondary antibodies bind to primary antibody, allowing more amplification (more sensitive test)
How can ELISA detect antigens using antibodies?
-the patient sample supplies the antigen; if it is absent, the antibodies don’t bind -can be direct or indirect ELISA
How can ELISA detect antibodies using only antigens?
-use the patient as the source of the primary antibodies in an indirect ELISA
Describe a Sandwich/Antigen capture ELISA
- use when antigen does not naturally bind to the plastic dish well
- used to eliminate antigen purification steps that are typically needed before a direct or indirect ELISA
- coat the dish with antibodies that capture the antigen
- similar to indirect ELISA, but uses 3 antibodies in total
What are polyclonal antibodies?
- antibodies that bind the same antigen at multiple epitopes
- Many B ells recognize the same antigen, but at different epitopes; this leads to many different plasma cell clones
What are monoclonal antibodies?
- antibodies that all bind exactly the same epitope
- single B cell leads to a single clone of plasma cells
How does the standard urine pregnancy test work?
- early in pregnancy, the placenta produces human chorionic gonadotropin (hCG)
- urine pregnancy tests detect hCG
- absorbent material (sponge) exposed to urine
- urine travels down sponge, passes through enzyme-linked mouse monoclonal anti- hCG antibodies
- If hCG is in urine, mouse anti-hCH antibodies bind hCG
- urine continues to move through absorbent material, drawing antibodies to second zone containing immobilized polyclonal anti hCH antibodies and substrate
- immobilized anti-hCG antibodies capture enzyme-linked mouse antibodies by also binding the hCG antigen, at different epitopes
- if captured, the substrate and the enzymes now react to produce colorful product
- mouse antibodies without hCH continue down absorbent material to 3rd (control) zone containing substrate and immobilized anti- mouse antibodies
What are the 2 statistics that determine how reliable a clinical test is?
1) sensitivity: probability that the test is reactive if the specimen is a true positive
- generally use samples that stimulate positive samples
2) specificity: probability that a test will not be reactive if a specimen is a true negative
- generally use samples that stimulate negative samples
