ELISA Flashcards
What does ELISA stand for?
Enzyme Linked Immunosorbent Assay
Generally, what is ELISA used for?
to detect small amounts of proteins (antibodies or antigens) in a sample
What does indirect ELISA detect?
Antibodies in serum
What does direct ELISA detect?
Antigens in serum
What type of ELISA did we use in lab?
indirect
How will the concentration of antibodies be effected after the immune system has been exposed to a virus and has launched a response?
the concentration of antibodies will be high
How will the concentration of antibodies be effected during an immune response to a virus?
the concentration will be high
How will the concentration of antibodies be effected after the immune system has recovered from a virus?
the concentration will be low
Give examples of the kind of viruses or infections can ELISA be used for
HIV Flus West Nile Virus Small Pox Lyme disease
What are 3 advantages of using ELISA for testing?
it is cheap, highly sensitive, and produces quick results
What are antigens?
usually proteins or polysaccharides on the surfaces of invasive cells and viruses that trigger an immune response in the host body
How do antibodies bind to antigens?
with great specificity like a lock and key
What virus or infection did we simulate in lab?
West Nile Virus
What is the first step of indirect ELISA?
purified antigens of the infectious agent are loaded into plastic microtiter wells
What happens when the antigens are loaded into the microtiter wells?
the antigens bind to the bottom of the wells via hydrophobic interactions
How many washes are there in indirect ELISA? What are the purposes of washing?
3 washes
Each wash rinses away material that has not bound to the wells or the antigens or the primary antibodies so that the substance in the wells is pure
After the first wash, what would be rinsed away?
any unbound material that the antigens may have been loaded with
What is added to the microtiter wells after the antigens have been washed?
the patient’s blood serum
Once added, what will happen if the patient’s blood contains antibodies for the antigens in the wells?
the antibodies from the patient’s blood will recognize the antigens and bind tightly to them at the bottom of the well
What is the purpose of the second wash in indirect ELISA?
the second wash will remove any antibodies from the patient’s blood that did not bind to the antigens - this helps in the accuracy of the results
What is added to the microtiter after the second wash?
secondary antibodies (recognize human antibodies as an antigen) are added to the microtiter and bind to the primary antibodies that are already bound to the antigens
What is the purpose of adding the secondary antibodies?
they contain a conjugate enzyme which can facilitate the colour change of the chromagen substrate when it is added - this allows us to visualize the results of the ELISA
What is the purpose of the third wash?
it will rinse off any secondary antibodies that did not bind to the human antibody-antigen complex
this will allow for accuracy in the results so that there’s no conjugate enzymes to change colours where there is no human antibodies
What is the last thing added to the microtiter?
chromagen substrate
What happens to the chromagen substrate if there are antibodies present?
if the secondary antibody bound to a human primary antibody, the conjugate enzyme on the secondary enzyme facilitates the colour change of the chromagen substrate from clear to purple which gives us a positive result for infection
What does it mean if the chromogen substrate changes to dark purple?
there are lots of antibodies present and the patient is likely in the midst of fighting off the infection = they have an infection
positive for the infection
What does it mean if the chromogen substrate changes to light purple?
a weak positive
the patient is likely at the beginning or end of the infection and there is a low concentration of antibodies
