WEBVTT 00:00:00.000 --> 00:00:04.000 (English captions by Andrea Matumoto, University of Michigan.) An agglutination assay is a simple way to detect and to measure antibodies in a clinical 00:00:04.000 --> 00:00:09.000 specimen directed against a specific antigen of interest. 00:00:09.000 --> 00:00:15.000 In this animation the principles and potential pitfalls of the assay will be demonstrated. 00:00:15.000 --> 00:00:21.000 The main reagent used in the assay is a solution of insoluble tiny beads usually composed of 00:00:21.000 --> 00:00:22.000 latex. 00:00:22.000 --> 00:00:28.000 Alternatively to measure antibodies against a microbial pathogen the killed bacterial or 00:00:28.000 --> 00:00:31.000 yeast cells can be used as the agglutinating particle. 00:00:31.000 --> 00:00:36.000 However in this example latex beads are used and they have been prepared so that the antigen 00:00:36.000 --> 00:00:41.000 of interest coats their surfaces and they are concentrated enough to produce a visible 00:00:41.000 --> 00:00:45.000 milky suspension. 00:00:45.000 --> 00:00:49.000 To measure antibodies against the antigen the particles are added to the wells of a 00:00:49.000 --> 00:00:55.000 ninety-six well microtiter plate and then sera taken from different patients are added 00:00:55.000 --> 00:00:57.000 to the wells in the first column. 00:00:57.000 --> 00:01:02.000 Two fold dilutions of the sera are prepared in the rows and then a known negative and 00:01:02.000 --> 00:01:12.000 positive control serum are added in the last two columns. 00:01:12.000 --> 00:01:16.000 When the plate is allowed to incubate at room temperature the wells containing the negative 00:01:16.000 --> 00:01:18.000 control serum remain unchanged. 00:01:18.000 --> 00:01:22.000 The wells containing the positive control serum have developed a visible button at the 00:01:22.000 --> 00:01:28.000 bottom of the wells and the solution in those wells has changed from milky to clear. 00:01:28.000 --> 00:01:35.000 So what accounts for the appearance of the positive wells and what accounts for the lack 00:01:35.000 --> 00:01:38.000 of change in the negative wells? 00:01:38.000 --> 00:01:42.000 To understand what's going on, lets take a microscopic look at the negative and positive 00:01:42.000 --> 00:01:46.000 wells to see what is happening in each case. 00:01:46.000 --> 00:01:51.000 When there is no antibody in the well with the beads they remain in suspension giving 00:01:51.000 --> 00:01:53.000 the well a milky appearance. 00:01:53.000 --> 00:01:59.000 However, when specific antibody is present it binds to and crosslinks the beads. 00:01:59.000 --> 00:02:05.000 This causes the beads to clump and to form large aggregates that sink to the bottom of 00:02:05.000 --> 00:02:07.000 the round bottom wells. 00:02:07.000 --> 00:02:12.000 They make the suspension clear as they sink instead of milky and the aggregates settle 00:02:12.000 --> 00:02:17.000 into a pellet or a button which forms at the bottom of the well. 00:02:17.000 --> 00:02:22.000 So you will see the button at the bottom of any well that has enough specific antibodies 00:02:22.000 --> 00:02:25.000 in it to precipitate the beads. 00:02:25.000 --> 00:02:29.000 But when the antibody is diluted out, the button no longer appears. 00:02:29.000 --> 00:02:49.000 The patient's antibody titer is the last dilution of serum that produces a button. 00:02:49.000 --> 00:02:54.000 But how to we explain the absence of a button in the most concentrated wells of the serum 00:02:54.000 --> 00:02:59.000 with the highest titer showed by the orange arrow? 00:02:59.000 --> 00:03:04.000 Imagine a well that has many more antibody molecules in it than beads. 00:03:04.000 --> 00:03:08.000 In this situation the beads will be completely coated with antibody and there will be no 00:03:08.000 --> 00:03:12.000 possibility of crosslinking and precipitation. 00:03:12.000 --> 00:03:18.000 This phenomenon, which is referred to as a prozone sometimes occurs in cases of syphilis. 00:03:18.000 --> 00:03:23.000 The standard screening test for syphilis is an agglutination assay in which undiluted 00:03:23.000 --> 00:03:28.000 serum is added to beads coated with the antigen cardiolipin. 00:03:28.000 --> 00:03:33.000 In secondary syphilis the antibody titers are sometimes so high that the test exhibits 00:03:33.000 --> 00:03:36.000 a prozone and is falsely negative. 00:03:36.000 --> 00:03:40.000 So how do you think you could overcome this potential problem and make the correct laboratory 00:03:40.000 --> 00:03:42.000 diagnosis? 00:03:42.000 --> 00:03:46.000 Did you think of diluting the serum and then retesting it? 00:03:46.000 --> 00:03:51.000 By diluting the antibody in this situation the amounts of antibody and antigen are closer 00:03:51.000 --> 00:03:53.000 to being equivalent with one another. 00:03:53.000 --> 00:03:56.000 Then the conditions for crosslinking can exist. 00:03:56.000 --> 00:04:01.000 Now when crosslinking occurs a precipitate forms and the button develops at the bottom 00:04:01.000 --> 00:04:05.000 of a tube indicating a positive test.