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Dialogue: 0,0:00:00.00,0:00:04.00,Default,,0000,0000,0000,,(English captions by Andrea Matumoto, University of Michigan.) An agglutination assay is a simple way to\Ndetect and to measure antibodies in a clinical
Dialogue: 0,0:00:04.00,0:00:09.00,Default,,0000,0000,0000,,specimen directed against a specific antigen\Nof interest.
Dialogue: 0,0:00:09.00,0:00:15.00,Default,,0000,0000,0000,,In this animation the principles and potential\Npitfalls of the assay will be demonstrated.
Dialogue: 0,0:00:15.00,0:00:21.00,Default,,0000,0000,0000,,The main reagent used in the assay is a solution\Nof insoluble tiny beads usually composed of
Dialogue: 0,0:00:21.00,0:00:22.00,Default,,0000,0000,0000,,latex.
Dialogue: 0,0:00:22.00,0:00:28.00,Default,,0000,0000,0000,,Alternatively to measure antibodies against\Na microbial pathogen the killed bacterial or
Dialogue: 0,0:00:28.00,0:00:31.00,Default,,0000,0000,0000,,yeast cells can be used as the agglutinating\Nparticle.
Dialogue: 0,0:00:31.00,0:00:36.00,Default,,0000,0000,0000,,However in this example latex beads are used\Nand they have been prepared so that the antigen
Dialogue: 0,0:00:36.00,0:00:41.00,Default,,0000,0000,0000,,of interest coats their surfaces and they\Nare concentrated enough to produce a visible
Dialogue: 0,0:00:41.00,0:00:45.00,Default,,0000,0000,0000,,milky suspension.
Dialogue: 0,0:00:45.00,0:00:49.00,Default,,0000,0000,0000,,To measure antibodies against the antigen\Nthe particles are added to the wells of a
Dialogue: 0,0:00:49.00,0:00:55.00,Default,,0000,0000,0000,,ninety-six well microtiter plate and then\Nsera taken from different patients are added
Dialogue: 0,0:00:55.00,0:00:57.00,Default,,0000,0000,0000,,to the wells in the first column.
Dialogue: 0,0:00:57.00,0:01:02.00,Default,,0000,0000,0000,,Two fold dilutions of the sera are prepared\Nin the rows and then a known negative and
Dialogue: 0,0:01:02.00,0:01:12.00,Default,,0000,0000,0000,,positive control serum are added in the last\Ntwo columns.
Dialogue: 0,0:01:12.00,0:01:16.00,Default,,0000,0000,0000,,When the plate is allowed to incubate at room\Ntemperature the wells containing the negative
Dialogue: 0,0:01:16.00,0:01:18.00,Default,,0000,0000,0000,,control serum remain unchanged.
Dialogue: 0,0:01:18.00,0:01:22.00,Default,,0000,0000,0000,,The wells containing the positive control\Nserum have developed a visible button at the
Dialogue: 0,0:01:22.00,0:01:28.00,Default,,0000,0000,0000,,bottom of the wells and the solution in those\Nwells has changed from milky to clear.
Dialogue: 0,0:01:28.00,0:01:35.00,Default,,0000,0000,0000,,So what accounts for the appearance of the\Npositive wells and what accounts for the lack
Dialogue: 0,0:01:35.00,0:01:38.00,Default,,0000,0000,0000,,of change in the negative wells?
Dialogue: 0,0:01:38.00,0:01:42.00,Default,,0000,0000,0000,,To understand what's going on, lets take\Na microscopic look at the negative and positive
Dialogue: 0,0:01:42.00,0:01:46.00,Default,,0000,0000,0000,,wells to see what is happening in each case.
Dialogue: 0,0:01:46.00,0:01:51.00,Default,,0000,0000,0000,,When there is no antibody in the well with\Nthe beads they remain in suspension giving
Dialogue: 0,0:01:51.00,0:01:53.00,Default,,0000,0000,0000,,the well a milky appearance.
Dialogue: 0,0:01:53.00,0:01:59.00,Default,,0000,0000,0000,,However, when specific antibody is present\Nit binds to and crosslinks the beads.
Dialogue: 0,0:01:59.00,0:02:05.00,Default,,0000,0000,0000,,This causes the beads to clump and to form\Nlarge aggregates that sink to the bottom of
Dialogue: 0,0:02:05.00,0:02:07.00,Default,,0000,0000,0000,,the round bottom wells.
Dialogue: 0,0:02:07.00,0:02:12.00,Default,,0000,0000,0000,,They make the suspension clear as they sink\Ninstead of milky and the aggregates settle
Dialogue: 0,0:02:12.00,0:02:17.00,Default,,0000,0000,0000,,into a pellet or a button which forms at the\Nbottom of the well.
Dialogue: 0,0:02:17.00,0:02:22.00,Default,,0000,0000,0000,,So you will see the button at the bottom of\Nany well that has enough specific antibodies
Dialogue: 0,0:02:22.00,0:02:25.00,Default,,0000,0000,0000,,in it to precipitate the beads.
Dialogue: 0,0:02:25.00,0:02:29.00,Default,,0000,0000,0000,,But when the antibody is diluted out, the\Nbutton no longer appears.
Dialogue: 0,0:02:29.00,0:02:49.00,Default,,0000,0000,0000,,The patient's antibody titer is the last\Ndilution of serum that produces a button.
Dialogue: 0,0:02:49.00,0:02:54.00,Default,,0000,0000,0000,,But how to we explain the absence of a button\Nin the most concentrated wells of the serum
Dialogue: 0,0:02:54.00,0:02:59.00,Default,,0000,0000,0000,,with the highest titer showed by the orange\Narrow?
Dialogue: 0,0:02:59.00,0:03:04.00,Default,,0000,0000,0000,,Imagine a well that has many more antibody\Nmolecules in it than beads.
Dialogue: 0,0:03:04.00,0:03:08.00,Default,,0000,0000,0000,,In this situation the beads will be completely\Ncoated with antibody and there will be no
Dialogue: 0,0:03:08.00,0:03:12.00,Default,,0000,0000,0000,,possibility of crosslinking and precipitation.
Dialogue: 0,0:03:12.00,0:03:18.00,Default,,0000,0000,0000,,This phenomenon, which is referred to as a\Nprozone sometimes occurs in cases of syphilis.
Dialogue: 0,0:03:18.00,0:03:23.00,Default,,0000,0000,0000,,The standard screening test for syphilis is\Nan agglutination assay in which undiluted
Dialogue: 0,0:03:23.00,0:03:28.00,Default,,0000,0000,0000,,serum is added to beads coated with the antigen\Ncardiolipin.
Dialogue: 0,0:03:28.00,0:03:33.00,Default,,0000,0000,0000,,In secondary syphilis the antibody titers\Nare sometimes so high that the test exhibits
Dialogue: 0,0:03:33.00,0:03:36.00,Default,,0000,0000,0000,,a prozone and is falsely negative.
Dialogue: 0,0:03:36.00,0:03:40.00,Default,,0000,0000,0000,,So how do you think you could overcome this\Npotential problem and make the correct laboratory
Dialogue: 0,0:03:40.00,0:03:42.00,Default,,0000,0000,0000,,diagnosis?
Dialogue: 0,0:03:42.00,0:03:46.00,Default,,0000,0000,0000,,Did you think of diluting the serum and then\Nretesting it?
Dialogue: 0,0:03:46.00,0:03:51.00,Default,,0000,0000,0000,,By diluting the antibody in this situation\Nthe amounts of antibody and antigen are closer
Dialogue: 0,0:03:51.00,0:03:53.00,Default,,0000,0000,0000,,to being equivalent with one another.
Dialogue: 0,0:03:53.00,0:03:56.00,Default,,0000,0000,0000,,Then the conditions for crosslinking can exist.
Dialogue: 0,0:03:56.00,0:04:01.00,Default,,0000,0000,0000,,Now when crosslinking occurs a precipitate\Nforms and the button develops at the bottom
Dialogue: 0,0:04:01.00,0:04:05.00,Default,,0000,0000,0000,,of a tube indicating a positive test.