Some people are allergic to peanuts. Your boss wants you to develop an ELISA to
ID: 16034 • Letter: S
Question
Some people are allergic to peanuts. Your boss wants you to develop an ELISA to identify such kids.(The ones with sever allergy to peanuts dont need your test, the test would help paranoid parents and those who are not sure what it is they are a little allergic to.)Hint: You want to identify individuals whose sera contain trace amounts of IgE to peanut proteins. You can use an aqueous extract of peanuts( which serves as a crude but adequate preparation of the antigen)
Warning: This is not as straightforward as it seems to be at first blush.
Outline all steps of your protocol for submission of the FDA:
1. Coat a microtiter plate with...
2. Wash the plate with...
3. Add....
4. .....
.
.
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Help as much as you can.Thank you
Explanation / Answer
elisa steps 1) add recommended amount(around 100micro liter) of antigen with recommended dilution (peanut extract) onto the 96-Well Microtiter Plates. 2) the microtiter plates have very high protein affinity, they can bind to any protein, so to block the non specific binding of the serum antibodies, run a blocking buffer (0.1%BSA) which fills the gaps left over by the antigen 3) incubate the plates for 24 hours 4) collect sera from different people and dilute it with blocking buffer. 5) use a control(positive or negative), for a negative control, use a non specific protein to peanut extract, i.e. sera of a non allergic person. 6) run the diluted sera from the microtitre plates. 7) we need a secondary antibody to identify the amount of binding. the secondary antibody is specific for the IeE 's Fc region. the secondary antibody is an Alkaline phosphatase-conjugated secondary antibody or peroxidase-conjugated secondary antibody. 8) the color change can be measured(optical density) to find out the amount of binding. all the people who show high optical density are allergic to peanut extract
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