Lab 31: ELISA

Question 1

what is an antigen?

Answer 1

an Ag is a molecule that stimulates an immune response

Question 2

what is an antibody?

Answer 2

an Ab is a Y-shaped protein that binds to and tags the Ag for destruction

Question 3

what are primary Abs?

Answer 3

Ab’s that directly bind to the Ag

Question 4

what are secondary Abs?

Answer 4

Ab’s that bind to the primary Ab

Question 5

what is the FAB region?

Answer 5

the two Y’s where the Antigens bind

Question 6

where is the Fc/constant region?

Answer 6

the stem

Question 7

how many Ag-binding sites are there on each Ab?

Answer 7

there are 2 Ag-binding sites on each Ab, and they are unique to each Ab

Question 8

what is ELISA?

Answer 8

Enzyme-linked ImmunoSorbent Assay

Question 9

what do we do with ELISA?

Answer 9

We use Abs tagged with an enzyme that catalyzes a color change reaction.

Question 10

what happens if there is a match with ELISA?

Answer 10

If there is a match, Abs attach and we see a color after the addition of a substrate

Question 11

what are the types of ELISA?

Answer 11

Direct: looking for the Ag
Indirect: looking for the Abs

Question 12

what is the ELISA sandwich method?

Answer 12

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)

Question 13

what is the direct ELISA sandwich method?

Answer 13

1. Add Abs to the known Ag to the well.
2. Add the specimen (Ag) where the Abs catch this Ag.
3. Add enzyme-labeled Abs of known specificity, where they bind to the antigen and remain there.
4. Wash any off that do not bind.
5. Add the substrate that changes color when acted upon by the enzyme.
6. Color development indicated a positive results

Question 14

what is the positive result of direct ELISA?

Answer 14

Color, in our case it was blue

Question 15

what is the indirect ELISA method?

Answer 15

1. Attach the known Ag to the well.
2. Add the patient’s Ab serum and wash off any that do not bind.
3. These Abs bind to the Ag.
4. Add the enzyme-labeled anti-human IgG, and wash off any Abs that do not bind.
5. Add the substrate that changes color when acted upon by the enzyme.
6. Color development indicates a positive result.

Question 16

what is our lab goal?

Answer 16

to detect the Ag in our patient’s sample

Question 17

what are the 4 steps of our lab?

Answer 17

1. Add Ag
2. An unlabeled primary Ab
3. Add HRP-labeled secondary Ab
4. Add TMB substrate
5. Observe color if positive result

Question 18

how do we ensure we detect the right Ag?

Answer 18

Because we use Abs that are very specific to the Ag. (we use the primary Ab, which is why ELISA is very specific)

Question 19

why don’t we just use a labeled primary Ab? Why do we need to use a secondary Ab?

Answer 19

Because the secondary Abs provide more signal, which makes ELISA sensitive and amplifies the signal.

Question 20

what are two important characteristics of ELISA?

Answer 20

Sensitive and Speicifc

Question 21

what are the sequence of steps used to detect a specific Ag?

Answer 21

1. Patient sample: may or may not contain the specific Ag.
2. Primary Ab specific to the Ag you want to detect.
3. Enzyme-labeled secondary Ab specific to the primary Ab.
4. Substrate

This is direct.

Question 22

what are two advantages of ELISA?

Answer 22

1. Specific: due to the specificity of Ab-Ab reactions.
2. 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).

Question 23

what is ELISA used to detect?

Answer 23

Pathogens, hormones, cytokines, growth factors, toxic agents, medications, allergens, drugs, etc.