Enzyme Linked Immunosorbent Assay (ELISA)

Question 1

ELISA

Answer 1

technique for detecting proteins, hormones, peptides and antibodies.
relies on the specificity and affinity of antibodies to the target antigens.

Question 2

Antigens

Answer 2

any substance that induces an immune response in the body such as: viruses, bacteria and parasites, cancerous cells

Question 3

antiboides

Answer 3

large glycoprotein molecules produced by B cells in the immune system.
found in the blood, Glycoprotein antibodies (IgG) are shaped like a “Y”
form a lock and key to from antigen-antibody complex

Question 4

Antibody-antigen interactions

Answer 4

Ab + Ag <-> Ab-Ag
Ka is the binding equilibrium, usually 10^6 10^10
is selective
bonds between antigen and antibody are weak

Question 5

ELISA techniques

Answer 5

Indierect, sandwich and competitive.

Question 6

Indirect ELISA

Answer 6

a standard of the protein to be screened is immobilized onto a plastic plate binds through charge interactions. use bovine serum albumin to block other sites.
if positive, would contain secreted antibody, secondary antibody added only binds to serum. secondary enzyme has enzyme such as horseradish peroxidase.
colour change indicates secondary antibody

Question 7

Detection of HIV by indirect ELISA

Answer 7

p24 is the core protein of the HIV virius, protein is used to detect anti p24 antibody in blood serum and determine infection.

Question 8

Sandwich ELISA

Answer 8

A capture antibody, that binds specifically to the suspect protein, is immobilized onto a plastic plate.
The remaining bare areas on the plate are blocked using a protein that is different from the target protein.
The sample to be tested is added to the plate. If the antigen (suspect protein) exists in the sample, it will bind to the antibody on the plate surface.
An enzyme-linked secondary antibody that binds specifically to another domain of the suspect protein is added.
Finally, a substrate is added to visualize the protein binding by the primary and secondary antibodies

Question 9

Competitive ELISA:

Answer 9

A primary antibody, that binds specifically to a target protein, is immobilized onto the plate and the remaining bare sites blocked.
The sample is spiked with a labeled standard of the target protein.
The labelled protein competes with the unlabeled protein in the sample (if it is positive) to bind to the primary antibody.
The higher the concentration of the unlabeled target protein in the he sample, the less concentration of the labelled protein that is retained by the antibody, and the weaker color signal.