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(English captions by Andrea Matumoto, University of Michigan.) An agglutination assay is a simple way to[br]detect and to measure antibodies in a clinical

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specimen directed against a specific antigen[br]of interest.

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In this animation the principles and potential[br]pitfalls of the assay will be demonstrated.

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The main reagent used in the assay is a solution[br]of insoluble tiny beads usually composed of

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latex.

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Alternatively to measure antibodies against[br]a microbial pathogen the killed bacterial or

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yeast cells can be used as the agglutinating[br]particle.

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However in this example latex beads are used[br]and they have been prepared so that the antigen

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of interest coats their surfaces and they[br]are concentrated enough to produce a visible

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milky suspension.

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To measure antibodies against the antigen[br]the particles are added to the wells of a

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ninety-six well microtiter plate and then[br]sera taken from different patients are added

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to the wells in the first column.

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Two fold dilutions of the sera are prepared[br]in the rows and then a known negative and

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positive control serum are added in the last[br]two columns.

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When the plate is allowed to incubate at room[br]temperature the wells containing the negative

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control serum remain unchanged.

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The wells containing the positive control[br]serum have developed a visible button at the

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bottom of the wells and the solution in those[br]wells has changed from milky to clear.

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So what accounts for the appearance of the[br]positive wells and what accounts for the lack

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of change in the negative wells?

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To understand what's going on, lets take[br]a microscopic look at the negative and positive

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wells to see what is happening in each case.

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When there is no antibody in the well with[br]the beads they remain in suspension giving

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the well a milky appearance.

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However, when specific antibody is present[br]it binds to and crosslinks the beads.

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This causes the beads to clump and to form[br]large aggregates that sink to the bottom of

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the round bottom wells.

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They make the suspension clear as they sink[br]instead of milky and the aggregates settle

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into a pellet or a button which forms at the[br]bottom of the well.

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So you will see the button at the bottom of[br]any well that has enough specific antibodies

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in it to precipitate the beads.

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But when the antibody is diluted out, the[br]button no longer appears.

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The patient's antibody titer is the last[br]dilution of serum that produces a button.

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But how to we explain the absence of a button[br]in the most concentrated wells of the serum

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with the highest titer showed by the orange[br]arrow?

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Imagine a well that has many more antibody[br]molecules in it than beads.

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In this situation the beads will be completely[br]coated with antibody and there will be no

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possibility of crosslinking and precipitation.

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This phenomenon, which is referred to as a[br]prozone sometimes occurs in cases of syphilis.

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The standard screening test for syphilis is[br]an agglutination assay in which undiluted

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serum is added to beads coated with the antigen[br]cardiolipin.

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In secondary syphilis the antibody titers[br]are sometimes so high that the test exhibits

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a prozone and is falsely negative.

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So how do you think you could overcome this[br]potential problem and make the correct laboratory

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diagnosis?

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Did you think of diluting the serum and then[br]retesting it?

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By diluting the antibody in this situation[br]the amounts of antibody and antigen are closer

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to being equivalent with one another.

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Then the conditions for crosslinking can exist.

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Now when crosslinking occurs a precipitate[br]forms and the button develops at the bottom

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of a tube indicating a positive test.