0:00 elisa is short for enzyme-linked
0:03 immunosorbent sa with this
0:06 plate-based technique we can detect and
0:09 quantify
0:10 soluble substances such as proteins
0:14 during an elisa the antigen which is the
0:17 target molecule
0:19 is immobilized and specifically detected
0:22 by antibodies
0:23 which are enzyme-coupled when providing
0:27 a colorless substrate the enzyme
0:29 produces
0:30 a measurable colorful product an
0:33 antibody is attached to the bottom of
0:35 the well
0:36 this antibody is specifically designed
0:39 for binding a specific protein of
0:41 interest
0:42 another soluble antibody is provided
0:46 the antibody is also specifically
0:48 covering protein
0:49 x this antibody is conjugated to an
0:53 enzyme when we now add a substrate
0:56 the enzyme can convert it to a colorful
0:59 product
1:00 which can be measured later there are
1:03 many variants of an elisa
1:05 in the direct elisa the antigen
1:08 is immobilized on the bottom of the
1:11 plate and
1:12 the enzyme-linked antibody directly
1:15 binds to the antigen
1:16 in the indirect elisa a primary antibody
1:20 binds our antigen
1:22 and then a secondary antibody binds the
1:25 primary antibody
1:27 the secondary antibody is enzyme linked
1:30 the most common type is the sandwich
1:33 elisa
1:33 a primary antibody is immobilized and
1:37 captures the antigen of a solution and
1:40 another primary antibody
1:42 specifically binds the same antigen but
1:45 all three elisa's
1:46 have one thing in common the last
1:49 antibody binding
1:50 is always conjugated to the enzyme which
1:53 confirms binding here we provide an
1:56 example of an elisa
1:58 for this sandwich elisa we have two
2:01 wells
2:01 both coated with primary antibody
2:04 specific for protein x on the left
2:08 we provide a sample containing our
2:10 protein
2:11 on the right we have the control without
2:14 the target protein
2:15 our antigen in orange will be
2:18 immobilized
2:19 a washing step will discard everything
2:22 else
2:23 now we provide the enzyme-linked
2:25 antibody
2:26 it will bind our antigen in the left
2:29 well and
2:30 remains bound after washing whereas the
2:33 antibody is discarded in the control
2:36 in the last step a colorless substrate
2:39 is given
2:40 since the enzyme in most cases
2:42 horseradish peroxidase
2:44 is exclusively present in the left well
2:48 it produces a measurable colorful
2:50 product here
2:52 this can be investigated and also
2:55 quantified
2:55 using spectophotometry the control
2:59 remains colorless since substrate is not
3:02 converted here if you want to have a
3:05 closer look into the direct or the
3:07 indirect elisa
3:09 you may click one of these videos here
Chrome Extension