Double Quiz micro - fri

ELISA

Enzyme-Linked Immunosorbent Assay

direct assay: detects presence of antigen tests for active infection

indirect assay: detects presence of antibody tests exposure to pathogen (past infection)

capture assay: detects either antigen or antibody tests for active or past infection

STEP 1 of INDIRECT

antigen (LPS from Salmonella or E.coli) is adsorbed to the bottom of the well in a 96well plate

STEP 2 of INDIRECT

Blocking reagent (5% nonfat milk)

Block unoccupied areas of the plate wells (membrane)

• prevent nonspecific binding

• reduce ELISA background signal

• block nonspecific binding to adsorbed proteins

• stabilize proteins adsorbed to plate for better interactions

STEP 3 of INDIRECT

Primary antibody (chicken egg yolk (IgY))

3a- Egg yolk (antibodies) is added to the well

• if antibodies specific for antigen are present, they will bind to antigen

• 3b - rinse to remove unbound antibodies

STEP 4 of INDIRECT

Secondary antibody conjugated to enzyme: rabbit IgG that binds to chicken IgY

4a - Add secondary antibody conjugated with enzyme

• rabbit anti-chicken IgG + alkaline phosphate

• 4b- rinse to remove unbound secondary antibody

STEP 5 of INDIRECT

add chromogenic substrate (para-nitrophenyl phosphate pNPP)

• substrate is cleaved by enzyme linked to secondary antibody, resulting in detectable color signal that is proportional to the amount of antibodies

• this allows is to quantify the abundance of antibodies in the sample

Why and how to do the ELISA? steps

1. collect a sample from a patient that was vaccinated

2. dilute that sample between 1:20 to 1:1280

3. perform ELISA to determine if antibodies are present or not, at the various dilution levels

Interpret ELISAs : Controls

1. positive control: contains the antibody at detectable levels

   1. suggest that the ELISA procedure worked

2. negative control: do not contain antibodies

   1. suggest that there is no contamination in the experiment that would give a false positive result

titer

amount of circulating antibodies

Interpret ELISAs steps

1. find the most dilute sample that gives a positive (color change) result

2. report the inverse of that dilution

titer interpretation: which flu strain is most susceptible to a vaccine formulation?

higher ELISA titer = stronger antibody binding → greater susceptibility to the vaccine-induced immune response