1 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 2 00:00:04,000 --> 00:00:09,000 specimen directed against a specific antigen of interest. 3 00:00:09,000 --> 00:00:15,000 In this animation the principles and potential pitfalls of the assay will be demonstrated. 4 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 5 00:00:21,000 --> 00:00:22,000 latex. 6 00:00:22,000 --> 00:00:28,000 Alternatively to measure antibodies against a microbial pathogen the killed bacterial or 7 00:00:28,000 --> 00:00:31,000 yeast cells can be used as the agglutinating particle. 8 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 9 00:00:36,000 --> 00:00:41,000 of interest coats their surfaces and they are concentrated enough to produce a visible 10 00:00:41,000 --> 00:00:45,000 milky suspension. 11 00:00:45,000 --> 00:00:49,000 To measure antibodies against the antigen the particles are added to the wells of a 12 00:00:49,000 --> 00:00:55,000 ninety-six well microtiter plate and then sera taken from different patients are added 13 00:00:55,000 --> 00:00:57,000 to the wells in the first column. 14 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 15 00:01:02,000 --> 00:01:12,000 positive control serum are added in the last two columns. 16 00:01:12,000 --> 00:01:16,000 When the plate is allowed to incubate at room temperature the wells containing the negative 17 00:01:16,000 --> 00:01:18,000 control serum remain unchanged. 18 00:01:18,000 --> 00:01:22,000 The wells containing the positive control serum have developed a visible button at the 19 00:01:22,000 --> 00:01:28,000 bottom of the wells and the solution in those wells has changed from milky to clear. 20 00:01:28,000 --> 00:01:35,000 So what accounts for the appearance of the positive wells and what accounts for the lack 21 00:01:35,000 --> 00:01:38,000 of change in the negative wells? 22 00:01:38,000 --> 00:01:42,000 To understand what's going on, lets take a microscopic look at the negative and positive 23 00:01:42,000 --> 00:01:46,000 wells to see what is happening in each case. 24 00:01:46,000 --> 00:01:51,000 When there is no antibody in the well with the beads they remain in suspension giving 25 00:01:51,000 --> 00:01:53,000 the well a milky appearance. 26 00:01:53,000 --> 00:01:59,000 However, when specific antibody is present it binds to and crosslinks the beads. 27 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 28 00:02:05,000 --> 00:02:07,000 the round bottom wells. 29 00:02:07,000 --> 00:02:12,000 They make the suspension clear as they sink instead of milky and the aggregates settle 30 00:02:12,000 --> 00:02:17,000 into a pellet or a button which forms at the bottom of the well. 31 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 32 00:02:22,000 --> 00:02:25,000 in it to precipitate the beads. 33 00:02:25,000 --> 00:02:29,000 But when the antibody is diluted out, the button no longer appears. 34 00:02:29,000 --> 00:02:49,000 The patient's antibody titer is the last dilution of serum that produces a button. 35 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 36 00:02:54,000 --> 00:02:59,000 with the highest titer showed by the orange arrow? 37 00:02:59,000 --> 00:03:04,000 Imagine a well that has many more antibody molecules in it than beads. 38 00:03:04,000 --> 00:03:08,000 In this situation the beads will be completely coated with antibody and there will be no 39 00:03:08,000 --> 00:03:12,000 possibility of crosslinking and precipitation. 40 00:03:12,000 --> 00:03:18,000 This phenomenon, which is referred to as a prozone sometimes occurs in cases of syphilis. 41 00:03:18,000 --> 00:03:23,000 The standard screening test for syphilis is an agglutination assay in which undiluted 42 00:03:23,000 --> 00:03:28,000 serum is added to beads coated with the antigen cardiolipin. 43 00:03:28,000 --> 00:03:33,000 In secondary syphilis the antibody titers are sometimes so high that the test exhibits 44 00:03:33,000 --> 00:03:36,000 a prozone and is falsely negative. 45 00:03:36,000 --> 00:03:40,000 So how do you think you could overcome this potential problem and make the correct laboratory 46 00:03:40,000 --> 00:03:42,000 diagnosis? 47 00:03:42,000 --> 00:03:46,000 Did you think of diluting the serum and then retesting it? 48 00:03:46,000 --> 00:03:51,000 By diluting the antibody in this situation the amounts of antibody and antigen are closer 49 00:03:51,000 --> 00:03:53,000 to being equivalent with one another. 50 00:03:53,000 --> 00:03:56,000 Then the conditions for crosslinking can exist. 51 00:03:56,000 --> 00:04:01,000 Now when crosslinking occurs a precipitate forms and the button develops at the bottom 52 00:04:01,000 --> 00:04:05,000 of a tube indicating a positive test.