Pipetting and Enzyme-Linked Immunosorbent Assay

Question 1

P-20

Answer 1

• 2 ul / 20 ul — 1 ul = 010

- YELLOW TIP

Question 2

P-200

Answer 2

• 20 ul / 200 ul – 50 ul= 050

- YELLOW TIP

Question 3

P-1000

Answer 3

• 100 ul / 1000 ul – 10 ul= 0100

- BLUE TIP

Question 4

Immunology

Answer 4

• Study of the immune system
• How the body protects itself against foreign, potentially disease-causing microorganisms and molecules
• Has three fundamental functions

Question 5

Immunology functions

Answer 5

• recognize intruders
• respond appropriately to intruders in a way that protects the body.
• responds the next time the intruders are encountered

Question 6

Antibodies

Answer 6

• Mammalian immune systems produce these molecules (enzymes, proteins).
• recognize intruder molecules w/ specificity
• locate and attach themselves to antigens
• used in biotechnology research
• used in disease diagnosis and treatment

Question 7

Antigens

Answer 7

• Antibody generators
• a toxin or other foreign substance that induces an immune response in the body, especially the production of antibodies.

Question 8

Epitopes

Answer 8

• Antigenic determinates of the antigen

- Are regions of an antigen that are recognized and bound by the antibody (region that the antibody interacts with)

Question 9

What happens why an antibody attaches to an invading foreign body?

Answer 9

• They make the invaders recognizable to other cells of the immune system so they can be destroyed.

Question 10

Characteristics of antibodies:

Answer 10

• a Y-shaped structure
• two short, light chains and two long, heavy chains
• the chains are held together by di-sulfide bonds
• a variable region exists at the top ends of the Y, representing about 25% of the entire antibody structure
• the variable region is different in all antibodies and it is this region that interacts with the epitopes of an antigen through ionic bonds, H bonds, and hydrophobic interactions
• A constant region occurs in the bottom 75% of the antibody and is used for recognition by other components of our immune system.

Question 11

ELISA

Answer 11

Enzyme
Linked
Immunosorbent
Assay
- Used to diagnose diseases such as HIV/AIDS, malaria
- can track pathogenic agents in water, food and the air
- Used to identify genetically modified organisms
- Can trace food allergens, molecular markers of pregnancy and drug use.

Question 12

Hepatitis A

Answer 12

Contagious human disease that can be transmitted via the fecal-oral route.

Question 13

ELISA procedure

Answer 13

Part 1. Spread disease using 400 ul with 3 students (Alex, Abcde, Jym)
Part 2. Bind the antigen to the wells
Part 3. Add primary antibody
Part 4. Add secondary antibody
Part 5. Enzymatic color reaction

Question 14

Part 2: Bind antigen to wells

Answer 14

• Microplates made of polystyrene
• Absorbs proteins by hydrophobic interactions
• Label 12 well strip with 3 positive controls, 3 negative controls, and 3 with initials
• Transfer 50 ul to the positive control. This represents hepatitis A.
• Transfer 50 ul of the negative control.
• Transfer 50 ul of bodily fluids.
• Wait 5 minutes for the proteins in the samples to bind to the plastic.
• Wash the unbound sample out of each well (empty)
• Fill each well with wash buffer
• Remove wash buffer
• Repeat wash buffer and remove wash buffer

Question 15

Part 3: Adding the primary antibody

Answer 15

• Transfer 50 ul of the primary antibody into each well of the strip.
• Wait 5 minutes for the primary antibody to bind to the antigen
• Discard unbound sample
• Wash buffer, remove buffer, wash buffer, remove buffer

Question 16

Part 4: Adding the secondary antibody

Answer 16

• Transfer 50 ul of the secondary antibody into each well. The secondary antibody has the enzyme HRP (horse-radish peroxidase).
• Wait 5 minutes for the primary antibody to bind to the antigen
• Discard unbound sample
• Wash, remove, wash, remove
• Wash, remove, wash, remove

Question 17

Part 5: The enzymatic color reaction

Answer 17

• Transfer 50 ul of the enzyme substrate into all wells.

HRP (horse radish peroxidase) will reduce hydrogen peroxide into water while at the same time oxidizing 3, 3’, 5, 5’- tetramethyl benzidine (TMB) into cationic form.

TMB is colorless, but its catatonic form has a blue color.

Question 18

How did ELISA - Enzyme linked immunosorbent assay work?

Answer 18

• The antigens (HEP A) adhered to the bottom of the first 3 wells which were positive and possibly to the last 3 wells which had the test subject (bodily fluids).
• We added a primary antibody and the epitope of the antigen was bound to the variable region of the antibody.
• We added a secondary antibody which contained the enzyme horse-radish peroxidase (HRP). This Antibody bound to the primary antibody at the primary antibody’s constant region and the secondary antibody’s variable region.
• We added the enzyme substrate TMB and noted any color changes.
• The enzyme HRP (horse radish peroxidase) substrate is TMB (tetramethyl benzide) which interacts with the HRP producing a cationic TMB (TMB+).
• TMB is colorless, but when it is catatonic (TMB+) it is blue.

Question 19

Advantages of ELISA

Answer 19

• Cost effective

- Highly sensitive

Question 20

Disadvantages of ELISA

Answer 20

• Antibodies and epitopes must match to work

- Enzyme/substrate reaction must be read immediately.

Question 21

What is happening in the wells for each step of ELISA procedure

Answer 21

1. Wells 1-3 are coated with positive control, wells 4-7 are coated with negative control and wells 8-12 are coated with test sample (bodily fluids). Let the proteins in the samples bind to the plastic for 5 minutes and then wash wells by emptying all fluid. Next, add a wash buffer and then wash wells. (Do this twice)
2. The primary antibody is added to all wells. If any antigen is bound to the wells, the primary antibody will attach to the epitope. Let the primary antibody bind to antigens for 5 minutes. Repeat the double wash process.
3. The secondary antibody is added to the wells. The secondary antibody binds to the primary antibody (5 minutes) Repeat the double wash process TWICE.
4. Add the enzyme substrate TMB to the wells. The enzyme substrates will react with the HRP enzyme to produce the reactant TMB+.
   HRP reduces hydrogen peroxide into water while at the same time oxidizing TMB into TMB+. (Catatonic)