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Serially diluted

Figure 17.2 Quantitation of Immunologic Tests (a) Microtiter plates used to quantitate immunoassays. (b) Hemagglutination inhibition tests done in a microtiter plate. Agglutinated cells form a rough pattern over the bottom of the well, while unagglutinated cells fall into a small button at the bottom. Eight tests (A-H) can be done in a single plate.

Controls

Agglutinated

Agglutinated

Unagglutinated

Unagglutinated reaction is observed in the dilution 1:256 but not in 1:512, the titer is 256. In any such test, controls must always be included to be sure all the reagents are reacting properly. A positive control contains known antigen and known antibody. A negative control contains the antigen with no antibodies, to check for false positive reactions.

Serology tests can be set up in test tubes, but many tubes and large quantities of reagents would be required. Therefore, the tests are usually done using plastic microtiter plates (figure 17.2). Proteins, either antigen or antibody, adsorb to the tiny plastic wells. These proteins can then be treated with reagents and washed without being removed from the plastic surface. The volumes used in each well are a mere fraction of the volumes needed for even a small test tube, so that tests can be done on very small samples. A standard microtiter plate has 96 wells, eight rows of 12 wells each; a single row is used for each sample tested, and 8 samples can be tested on a single plate. The 12 wells in a row allow two controls and 10 dilutions of sample. Thus, dilutions of 1:4 through 1:2,048 can be examined in a single test. Special equipment allows rapid dilution and mixing of reagents, and accurate reading of results.

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