ELISA

Question 1

in what percentage of people do allergies occur in?

Answer 1

20%

Question 2

when will an allergic reaction occur?

Answer 2

when the immune system overreacts to a foreign material, known as an antibody

Question 3

what are common antigens for allergies (3)?

Answer 3

• infectious agents like bacteria or viruses
• chemicals
• various environmental materials such as pollen and food

Question 4

briefly describe the cause of an immune response

Answer 4

-once an antigen enters the body, it triggers white blood cells to produce antibodies, leading to a swift immune response

Question 5

what are antibodies?

Answer 5

specialized proteins that are used by the body to identify and eliminate oathogens

Question 6

describe the structure of an antibody

Answer 6

-four polypeptide chains, two heavy chains and two light chains
-chains are linked together by disulfide bonds to create a Y shape
-at each tip of the U is a highly variable region made up of 110-130 amino acids that gives the animo acid its specificity to binding antigens
-each antibody molecule can bind to two antigens, one at each tip

Question 7

what does the binding of an antibody to an antigen do?

Answer 7

-neutralizes the antigen
-forms an insoluble complex through a process called immunoprecipitation

Question 8

what do all allergies begin with?

Answer 8

sensitization

Question 9

what is sensitization?

Answer 9

-starts when a normally non-hazardous antigen enters the body and encounters a lymphocyte cell
-the lymphocyte cell registers this non-harmful particle as threatening, which triggers the production of novel immunoglobin E antibodies
-the specific immunoglobin E antibodies attach themselves to immune cells (like mast cells and basophils) that circulate throughout the body

Question 10

how long can the sensitization process take?

Answer 10

6-10 days

Question 11

what happens after sensitization

Answer 11

-immunoglobin E antibodies in the bloodstream can rapidly bind to their antigen, triggering immune cells to release mediator compounds such as histamine and proteoglycans into the body
-once the person has become sensitized, small amounts of antigen can trigger a full allergic reaction

Question 12

what are some symptoms of allergic reactions (3)?

Answer 12

-sneezing
-itchy eyes
-anaphylaxis

Question 13

what is anaphylaxis?

Answer 13

a severe whole-body reaction to an allergen

Question 14

about how many people in the united states are affected by food allergies?

Answer 14

15 million people

Question 15

what is the most common food allergy i nchildren?

Answer 15

Cow’s Milk Protein Allergy (CMPA)

Question 16

what is CMPA? what is it caused by?

Answer 16

-cow’s milk protein allergy
-when the immune system mistakenly attacks one or more milk proteins, present in food products
-the immune response can be immediate, delayed, or both

Question 17

what are common symptoms of CMPA?

Answer 17

-vomitting
-wheezing
-eczema

Question 18

how much protein does most milk contain?

Answer 18

3%

Question 19

how is milk classified?

Answer 19

-based on the presence or absence of phosphorous
-caseins contain phosphorous and will coagulate or precipitate at a pH of 4.6 (basis of cheese curd formation)

Question 20

what proteins is milk made up of?

Answer 20

-3 or 4 casein proteins, making up approximately 82% of the proteins
-other proteins in milk lack phosphorous and are grouped together as whey or serum proteins; make up the other 18% of proteins

Question 21

what are the major whey proteins in milk (2)?

Answer 21

• beta-lactoglobulin
• alpha-lactalbumin

Question 22

what are the msot common allergens (8)?

Answer 22

-milk
-eggs
-fish
-shellfish
-peanuts
-wheat
-soybeans
-tree nuts

Question 23

how do food companies detect allergens in their products?

Answer 23

-use PCR, mass spectrometry, or immunological assays

Question 24

how do immunological assays detect allergens?

Answer 24

-identify a particular substance by its ability to bind an antibody

Question 25

what is a commonly used immunological assay?

Answer 25

enzyme linked immosorbent assay (ELISA)

Question 26

what do ELISAs do?

Answer 26

determine the presence and concentration of antigens in complex solutions

Question 27

how do ELISAs work?

Answer 27

-relies on the ability of an antibody to recognize and bind to specific antigens -use a primary antibody that is specific to the antigen of interest -use a secondary antibody that recognizes the antigen-antibody complex and is coupled to an enzyme that reacts with a substrate to produce a signal

Question 28

what are ELISAs used for (4)?

Answer 28

-used to determine serum antibody concentrations which helps doctors to diagnose viral, bacterial, and parasitic infections -identify genetically modified organisms -trace drug use -confirm pregnancy

Question 29

how do you perform a traditional ELISA?

Answer 29

-done in transparent microliter plates made of polystyrene or polyvinyl chloride -sample is deposited into small wells -proteins present in the solution, including the target antigen, will stick to the plate -proteins/antigens are allowed to bind to the plate during a short incubation period -wells are washed to remove unabsorbed antigens -a solution that contains the primary antibody is added to the wells -if the antigen is present in the wells, then it will bind to the antibody to form a complex -after a second wash, the solution with the enzyme-linked secondary antibody is added to the wells which will bind to the andigen and primary antibody complex -do a final wash, and add the colorless substrate solution containing 2,2'-azino-bis (3-ethybenzothiazoline-6-suphonic acid) aka ABTS -if there is any secondary enzyme bound to the well then the linked enzyme horseradish peroxidase will catalyze a reaction that produces a colored product from ABTS

Question 30

in what ways is an ELISA qualitative?

Answer 30

they indicate whether or not an antigen is present

Question 31

in what way is an ELISA quantitative?

Answer 31

they measure the antigen concentration

Question 32

what is required for a quantitative ELISA?

Answer 32

-several wells set aside as standards that contain a solution where the concentration of the antigen of interest is already known

Question 33

how are quantitative ELISAs analyzed?

Answer 33

-the signal intensity in each well is measured and the values are plotted to create a standard curve -the intensity of the unknown samples can then be compared to the standard curve to determine an approximate protein concentration

Question 34

give some examples of diseases that the ELISA is commonly used for

Answer 34

-WNV, HIV, TB, infectious mononucleosis

Question 35

what is an epitope?

Answer 35

a particular location within an antigen

Question 36

what does polyclonal mean?

Answer 36

heterogenous mixture

Question 37

what does monoclonal mean?

Answer 37

directed against a single epitope

Question 38

what is casein protein?

Answer 38

-contains phosphorous -coagulates at pH of 4.6 3 or 4 different casein proteins in mammalian milk -82% of milk protein

Question 39

what is whey protein?

Answer 39

-lack phosphorous -includes β-lactoglobulin and α-lactalbumin -18 % of milk proteins

Question 40

describe the process of performing an ELISA

Answer 40

1. the sample is added to the wells of the microtiter plate, where it adheres to the plastic through hydrophobic and electrostatic interactions 2. after washing away any unadsorbed sample, the wells are "blocked" with a protein-containing buffer to prevent non-specific interactions 3. the primary antibody is added to the wells, where it recognizes the antigen and binds through electrostatic interactions. Excess antibody is washed out of the wells 4. the secondary antibody, which recognizes the primary antibody, is added to the wells. If the antibody can bind the antigen, it will remain in the well after the wells are washed 5. the enzyme-lined secondary antibody allows us to detect the presence of the antibody-antigen complex when the substrate is added to all the wells (through chromogenic detection or fluorogenic detection)