Elisa Henderick I-human case with complete solution all
correct rated A+
ELISA - ANSWER: Enzyme Linked Immuno Absorbent Assay
Types of ELISAs - ANSWER: Indirect
Direct
Sandwich
Cell based indirect/direct
Why use an ELISA? - ANSWER: They allow rapid screening of large numbers of
samples for the presence of an antigen or antibody.
What does an ELISA do? - ANSWER: uses antibodies to detect antigen-antibody
interactions to determine the presence of a disease agent (viruses, bacteria,
parasites)
Relative antigens present
Real world Examples - ANSWER: Pregnancy test, disease detection (HIV)
Antigen - ANSWER: Harmful bacteria/protein
Antibody - ANSWER: Proteins that recognize antigens and bind tightly to them
Primary antibody - ANSWER: Recognizes the antigen
Secondary antibody - ANSWER: Recognized the primary antibody and binds to them.
This is conjugated with HRP enzyme. Turns blue when the substrate is present.
Positive control - ANSWER: Sample known to be positive for a disease.
Expresses the protein you are detecting
Negative control - ANSWER: Sample that does not contain disease agent
It does not express the protein you are detecting.
Does not contain the antigen but does contain proteins that will bind to the wells.
What happened to the proteins in the plastic well if the sample contained the
antigen? What if they did not contain the antigen? - ANSWER: It did not matter if the
sample contained the antigen or not because the proteins still binded to the wells
regardless.
, Why do you need to wash the wells after every step? - ANSWER: It was important to
wash the wells after every step, sometimes multiple times, in order to remove any
proteins and antibodies that are unbound. This ensures that there will be no false
positive results.
When you added the primary antibody to the wells, what happened if your sample
contained the antigen? What if it did not contain the antigen? - ANSWER: If it was
negative for the antigen, then the primary antibody was flushed out through
washing. If it was positive, then the primary antibody bound to the antigen and was
not washed away during the washing step.
When you added secondary antibody to the wells, what happened if your sample
contained the antigen? What if it did not contain the antigen? - ANSWER: The
secondary antibody bound to the primary antibodies if it was positive for the
antigen. If it did not contain the antigen, the secondary antibody was unbound and
washed away during washing.
What are the 6 steps to an ELISA? - ANSWER: 1. the experimental wells are coated
with antibodies specific to target antigen
2. sample of extracted cell is added to the wells
3. if present, antibodies immobilize antigen by binding to it
4. unbound proteins are washed away
5. an enzyme-linked antibody that also recognizes the antigen is added to the wells
6. the wells is filled with solution which indicates detection of the enzyme based on a
color change (no color change=protein absent)
Blocking agent - ANSWER: skim milk, extra step that further coats wells where
purified antigen may have missed.
What component that must be absent in a negative control solution? - ANSWER:
Primary antibody in patient much be absent
Possible causes of positive results from negative control? - ANSWER: improper
washing of wells leads to remnants of antibodies not being test for in patient sample
(aka those which did not combine to primary antigen) in wells which then could
combine with secondary antibody and cause false positive.
Could also have improper adhesion between primary antigen and well walls, so a
random antibody from patient serum could combine to that etc and then test
positive.
What would be the consequence over washing the Primary Antigen? - ANSWER:
Overwashing: antigen could be removed from wells of wall, making results skew as
other things could attach instead.
correct rated A+
ELISA - ANSWER: Enzyme Linked Immuno Absorbent Assay
Types of ELISAs - ANSWER: Indirect
Direct
Sandwich
Cell based indirect/direct
Why use an ELISA? - ANSWER: They allow rapid screening of large numbers of
samples for the presence of an antigen or antibody.
What does an ELISA do? - ANSWER: uses antibodies to detect antigen-antibody
interactions to determine the presence of a disease agent (viruses, bacteria,
parasites)
Relative antigens present
Real world Examples - ANSWER: Pregnancy test, disease detection (HIV)
Antigen - ANSWER: Harmful bacteria/protein
Antibody - ANSWER: Proteins that recognize antigens and bind tightly to them
Primary antibody - ANSWER: Recognizes the antigen
Secondary antibody - ANSWER: Recognized the primary antibody and binds to them.
This is conjugated with HRP enzyme. Turns blue when the substrate is present.
Positive control - ANSWER: Sample known to be positive for a disease.
Expresses the protein you are detecting
Negative control - ANSWER: Sample that does not contain disease agent
It does not express the protein you are detecting.
Does not contain the antigen but does contain proteins that will bind to the wells.
What happened to the proteins in the plastic well if the sample contained the
antigen? What if they did not contain the antigen? - ANSWER: It did not matter if the
sample contained the antigen or not because the proteins still binded to the wells
regardless.
, Why do you need to wash the wells after every step? - ANSWER: It was important to
wash the wells after every step, sometimes multiple times, in order to remove any
proteins and antibodies that are unbound. This ensures that there will be no false
positive results.
When you added the primary antibody to the wells, what happened if your sample
contained the antigen? What if it did not contain the antigen? - ANSWER: If it was
negative for the antigen, then the primary antibody was flushed out through
washing. If it was positive, then the primary antibody bound to the antigen and was
not washed away during the washing step.
When you added secondary antibody to the wells, what happened if your sample
contained the antigen? What if it did not contain the antigen? - ANSWER: The
secondary antibody bound to the primary antibodies if it was positive for the
antigen. If it did not contain the antigen, the secondary antibody was unbound and
washed away during washing.
What are the 6 steps to an ELISA? - ANSWER: 1. the experimental wells are coated
with antibodies specific to target antigen
2. sample of extracted cell is added to the wells
3. if present, antibodies immobilize antigen by binding to it
4. unbound proteins are washed away
5. an enzyme-linked antibody that also recognizes the antigen is added to the wells
6. the wells is filled with solution which indicates detection of the enzyme based on a
color change (no color change=protein absent)
Blocking agent - ANSWER: skim milk, extra step that further coats wells where
purified antigen may have missed.
What component that must be absent in a negative control solution? - ANSWER:
Primary antibody in patient much be absent
Possible causes of positive results from negative control? - ANSWER: improper
washing of wells leads to remnants of antibodies not being test for in patient sample
(aka those which did not combine to primary antigen) in wells which then could
combine with secondary antibody and cause false positive.
Could also have improper adhesion between primary antigen and well walls, so a
random antibody from patient serum could combine to that etc and then test
positive.
What would be the consequence over washing the Primary Antigen? - ANSWER:
Overwashing: antigen could be removed from wells of wall, making results skew as
other things could attach instead.
