ELISA

Question 1

What does ELISA stand for?

Answer 1

Enzyme Linked Immunosorbent Assay

Question 2

Generally, what is ELISA used for?

Answer 2

to detect small amounts of proteins (antibodies or antigens) in a sample

Question 3

What does indirect ELISA detect?

Answer 3

Antibodies in serum

Question 4

What does direct ELISA detect?

Answer 4

Antigens in serum

Question 5

What type of ELISA did we use in lab?

Answer 5

indirect

Question 6

How will the concentration of antibodies be effected after the immune system has been exposed to a virus and has launched a response?

Answer 6

the concentration of antibodies will be high

Question 7

How will the concentration of antibodies be effected during an immune response to a virus?

Answer 7

the concentration will be high

Question 8

How will the concentration of antibodies be effected after the immune system has recovered from a virus?

Answer 8

the concentration will be low

Question 9

Give examples of the kind of viruses or infections can ELISA be used for

Answer 9

HIV
Flus
West Nile Virus
Small Pox
Lyme disease

Question 10

What are 3 advantages of using ELISA for testing?

Answer 10

it is cheap, highly sensitive, and produces quick results

Question 11

What are antigens?

Answer 11

usually proteins or polysaccharides on the surfaces of invasive cells and viruses that trigger an immune response in the host body

Question 12

How do antibodies bind to antigens?

Answer 12

with great specificity like a lock and key

Question 13

What virus or infection did we simulate in lab?

Answer 13

West Nile Virus

Question 14

What is the first step of indirect ELISA?

Answer 14

purified antigens of the infectious agent are loaded into plastic microtiter wells

Question 15

What happens when the antigens are loaded into the microtiter wells?

Answer 15

the antigens bind to the bottom of the wells via hydrophobic interactions

Question 16

How many washes are there in indirect ELISA? What are the purposes of washing?

Answer 16

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

Question 17

After the first wash, what would be rinsed away?

Answer 17

any unbound material that the antigens may have been loaded with

Question 18

What is added to the microtiter wells after the antigens have been washed?

Answer 18

the patient’s blood serum

Question 19

Once added, what will happen if the patient’s blood contains antibodies for the antigens in the wells?

Answer 19

the antibodies from the patient’s blood will recognize the antigens and bind tightly to them at the bottom of the well

Question 20

What is the purpose of the second wash in indirect ELISA?

Answer 20

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

Question 21

What is added to the microtiter after the second wash?

Answer 21

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

Question 22

What is the purpose of adding the secondary antibodies?

Answer 22

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

Question 23

What is the purpose of the third wash?

Answer 23

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

Question 24

What is the last thing added to the microtiter?

Answer 24

chromagen substrate

Question 25

What happens to the chromagen substrate if there are antibodies present?

Answer 25

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

Question 26

What does it mean if the chromogen substrate changes to dark purple?

Answer 26

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

Question 27

What does it mean if the chromogen substrate changes to light purple?

Answer 27

a weak positive

the patient is likely at the beginning or end of the infection and there is a low concentration of antibodies