elisa lab southern california biotech center miramar college
TRANSCRIPT
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ELISA LabSouthern California BioTech Center
Miramar College
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Background
Enzyme-Linked ImmunoSorbent Assay
Immunology Detecting the presence
of antibodies or antigens
Uses the binding of antigens and antibodies to see a change, such as color
Uses Pregnancy HIV West Nile Hepatitis Protein recognition
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Immunology Review
Antigen-foreign body such as virus or bacterium that infects the body, in this lab the HIV virus
Antibody- protein made by immune system in response to a antigen, in this lab what is being tested for
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How it Works
Click Here for Demo
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The Scenario
Work in a clinic Blood samples from
Patient A and Patient B were collected
Test for HIV antibodies
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Your Lab Station
4 8-well strips coated Rack with
Samples: 1 + control (Green) 1 – control (Yellow) 1 Patient A (Pink) 1 Patient B (Blue)
2 2mL 2°antibody (Clear) 2 2mL TMB (Amber)
Squirt bottle with PBS P200 micropipette and
tips
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What Has Been Done
Wells have been coated with HIV antigens (the red). This is NOT real HIV.
Wells have been blocked so that no other proteins or antigens will get stuck (the blue)
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Before Adding the Samples
Properly orient to the top of your strip
Mix the contents of each solution by gently inverting the tubes
IMPORTANT: Always change pipette tips between different samples.
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Adding the Sample
Add 100 µL of + control (GREEN) to wells 1 & 2
Add 100 µL of – control (YELLOW) to wells 3 & 4
Add 100 µL of Patient A (PINK) to wells 5 & 6
Add 100 µL of Patient B (BLUE) to wells 7 & 8
Let sit 5 minutes
100
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Washing
WHY? To rinse away the other
proteins and antibodies that are not binding in order to make sure there is not a false positive
Empty wells by flicking until no more liquid leaves
Blot dry on paper towels Fill wells to the top with
PBS buffer Empty and blot as above Do this wash 4 times
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Add the 2° Antibody
Add 100 µL of the 2° antibody (CLEAR) to each well
Empty wells and blot a 4 times (just as before)
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Detecting the Results
Add 100 µL of the TMB substrate (AMBER) to each well
Wait for a few minutes A color change to blue
indicates positive results
Record your results and answer the questions
+ + - -
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What Happened?
Substrate (TMB) making color change
2° antibody Antibody we are testing Antigen already coated
and present