Lab 31: ELISA Flashcards
what is an antigen?
an Ag is a molecule that stimulates an immune response
what is an antibody?
an Ab is a Y-shaped protein that binds to and tags the Ag for destruction
what are primary Abs?
Ab’s that directly bind to the Ag
what are secondary Abs?
Ab’s that bind to the primary Ab
what is the FAB region?
the two Y’s where the Antigens bind
where is the Fc/constant region?
the stem
how many Ag-binding sites are there on each Ab?
there are 2 Ag-binding sites on each Ab, and they are unique to each Ab
what is ELISA?
Enzyme-linked ImmunoSorbent Assay
what do we do with ELISA?
We use Abs tagged with an enzyme that catalyzes a color change reaction.
what happens if there is a match with ELISA?
If there is a match, Abs attach and we see a color after the addition of a substrate
what are the types of ELISA?
Direct: looking for the Ag
Indirect: looking for the Abs
what is the ELISA sandwich method?
you add the sample
add the labeled Ab to the virus
Add another Ab to the virus (test line)
Add the Abs to the Abs (control line)
what is the direct ELISA sandwich method?
- Add Abs to the known Ag to the well.
- Add the specimen (Ag) where the Abs catch this Ag.
- Add enzyme-labeled Abs of known specificity, where they bind to the antigen and remain there.
- Wash any off that do not bind.
- Add the substrate that changes color when acted upon by the enzyme.
- Color development indicated a positive results
what is the positive result of direct ELISA?
Color, in our case it was blue
what is the indirect ELISA method?
- Attach the known Ag to the well.
- Add the patient’s Ab serum and wash off any that do not bind.
- These Abs bind to the Ag.
- Add the enzyme-labeled anti-human IgG, and wash off any Abs that do not bind.
- Add the substrate that changes color when acted upon by the enzyme.
- Color development indicates a positive result.
what is our lab goal?
to detect the Ag in our patient’s sample
what are the 4 steps of our lab?
- Add Ag
- An unlabeled primary Ab
- Add HRP-labeled secondary Ab
- Add TMB substrate
- Observe color if positive result
how do we ensure we detect the right Ag?
Because we use Abs that are very specific to the Ag. (we use the primary Ab, which is why ELISA is very specific)
why don’t we just use a labeled primary Ab? Why do we need to use a secondary Ab?
Because the secondary Abs provide more signal, which makes ELISA sensitive and amplifies the signal.
what are two important characteristics of ELISA?
Sensitive and Speicifc
what are the sequence of steps used to detect a specific Ag?
- Patient sample: may or may not contain the specific Ag.
- Primary Ab specific to the Ag you want to detect.
- Enzyme-labeled secondary Ab specific to the primary Ab.
- Substrate
This is direct.
what are two advantages of ELISA?
- Specific: due to the specificity of Ab-Ab reactions.
- Sensitive: can detect very small amounts of Ag (0.01 ng/ml), as each primary Ab can bind to multiple labeled secondary Abs and amplify the signal (which is the color).
what is ELISA used to detect?
Pathogens, hormones, cytokines, growth factors, toxic agents, medications, allergens, drugs, etc.
