ENZYME-LINKED IMMUNOABSORBENT ASSAY

Question 1

•It is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. •Some examples include: diagnosis of HIV infection, pregnancy tests, and measurement of cytokines or soluble receptors in cell supernatant or serum

Answer 1

enzyme-linked immunosorbent assay (ELISA)

Question 2

In elisa, various ____ combinations are used

Answer 2

Antigen-antibody

Question 3

ELISA always including an ___ label antigen or antibody and an activity is measured _____

Answer 3

enzyme
Colorimetrically

Question 4

The enzyme activity is measured using a ____ ___ ___ when modified by the enzyme

Answer 4

substrate changes color

Question 5

Light absorption of the product formed the after ___ addition is measured and converted to ____ __

Answer 5

Substrate
numeric values

Question 6

Depending on the antigen-antibody combination the essay is called:

Answer 6

Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA

Question 7

General components

Answer 7

Microtiter plate
Coating antigen or antibody
Blocking agent
Sample or analyte
Detection antibody
Wash buffer
Substrate solution
Stop solution (optional)

Question 8

Serves as individual reaction chambers for specific pending interactions can be measured

Answer 8

Microtiter plate

Question 9

Acts like a hook which capture the target

Answer 9

Coating antigen or antibody

Question 10

• Crucial for accurate results
• large inert molecules that binds to empty spaces to prevent unwanted molecules from attaching
• improves sensitivity amd reliability of the ELISA

Answer 10

Blocking agent

Question 11

• Can be blood serum or plasma

Answer 11

Sample analye

Question 12

• binds to target molecule

Answer 12

Detection antibody

Question 13

Colorless compound that the enzyme convert into colored product

Answer 13

Substrate solution

Question 14

Remove unbound molecules after each incubation steps

Answer 14

Wash buffer

Question 15

• Host the enzyme activity

Answer 15

Stop solution

Question 16

Analytical steps

Answer 16

1. Captured the antibody
2. Target antigen
3. Enzyme labeled detection antibody
4. Substrate

Question 17

Analytical steps (?)

Answer 17

1. Label the microtiter plate and the transfer pipettes
2. add 100 microliters of the antigen solution to all of the wells
3. incubate for 5 minutes at room temperature. At this time the antigens will man specifically adhere to the plastic through hydrophobic and electrostatic interactions
4. remove all liquids from the wells using a transfer pipette
5. Wash each well by adding approximately 8 drops of PBS buffer than remove PBS from the wells. Be careful to not spill the buffer into adjacent wells.
6. Add reagents. Remember to use a clean micropipette tip for each reagent.
7. Incubate the plates for 15 minutes at 37°C
8. Remove the liquid from the wells using the appropriate labeled transfer pipettes
9. Wash each well with fresh PBS, and then remove the liquid with its appropriate transfer pipet.
10. Add 100 microliters of the secondary antibody to each of the wells.
11. Incubate for 15 minutes at 37 degrees Celcius.
12. While the samples are incubating, prepare the detection substrate.
13. Remove the secondary antibody solutions from the wells using designated transfer pipets for each sample. Wash each well once with fresh PBS buffer. Remove the liquid using the transfer pipet designated for each sample.
14. Add 100 microliters of the substrate solution to each of the wells.
15. Incubate at 37 degrees Celcius for 5 minutes.
16. Examine your results. Differences between negative and positive samples will be obvious, with most positive samples turning into brown in color. If color is not fully developed after 5 minutes, incubate again at 37 degrees Celcius for a longer period time.

Question 18

The antigen is immobilized on the surface, and an enzyme-labelled antibody directly binds to the antigen.

Answer 18

Direct ELISA

Question 19

In direct elisa, the ___ is immobilized on the surface, and an enzyme-labelled antibody directly binds to the antigen.

Answer 19

Antigen

Question 20

The antigen is immobilized on the surface, and an unlabelled primary antibody binds to the antigen. Then, an enzyme-labelled secondary antibody binds to the primary antibody

Answer 20

Indirect ELISA

Question 21

In indirect ELISA, the antigen is immobilized on the surface, and an unlabelled primary ___ binds to the ____. Then, an enzyme-labelled secondary ___ binds to the primary antibody

Answer 21

Antibody
Antigen
Antibody

Question 22

A capture antibody is immobilized on the surface, the antigen binds to this antibody, and then a second, enzyme-labelled detection antibody binds to the antigen.

Answer 22

Sandwich ELISA

Question 23

In sandwich ELISA, a capture ___ is immobilized on the surface, the antigen binds to this antibody, and then a second, enzyme-labelled detection ___ binds to the ____

Answer 23

Antibody
Antibody
Antigen

Question 24

The sample antigen competes with an enzyme-labelled antigen for binding to an antibody. The amount of enzyme-labelled antigen bound is inversely proportional to the concentration of the sample antigen.

Answer 24

Competitive ELISA

Question 25

In competitive ELISA, the sample ___ competes with an enzyme-labelled ____ for binding to an ___. The amount of enzyme-labelled antigen bound is inversely proportional to the concentration of the sample antigen.

Answer 25

Antigen
Antigen
antibody