F G G H H 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 Figure 1-1: Well numbering on a standard microtitre plate Many serological tests are performed on doubling dilutions of serum. The lowest dilution of sera being tested is usually put into wells in the first row (horizontal row H) of a microtiter plate and buffer is put in all other wells of the plate. Serial transfer of serum across the plate results in doubling dilutions of serum being made across the plate. A typical protocol for the serial dilution of sera could be: Pipette 50 l of a 1in 4 dilution of each of 11 sera to be tested into wells H1 to H11, and 50ul of positive control serum in well H12. Place 25 l of buffer in all other wells. Transfer 25 l from each well in row H to the corresponding well in row G, mix well and transfer 25 l from each well in row G to the corresponding wells in row F. Continue this process to row A and discard 25 l from each well in row A. Serum in row H has a dilution of 1 in 4, serum in row G is diluted 1 in 8 etc. until finally in row A the dilution is 1 in 512. This type of dilution protocol can be modified as required to suit different situations and test methods. This serum dilution step is common to many of the methods and is simply described as "making doubling dilutions from row X to row Y".
Serology: General introduction to serological testing 14 | P a g e Dispensing reagents into the wells can be done with single or multiple tip hand held pipettes of the Oxford type ( Figure 1-2 ) or by sophisticated automated dispensers/diluters. The system used will depend on the number of sera to be tested and the financial resources. Figure 1-2: Single and multiple head dispensers used for dispensing and diluting sera in microtitre plates. Test formats For every plate that is set up one of the sera tested should be a positive control serum. This serum is a standard positive reference serum of known end titre. It should always give the expected result within the allowable limits defined for the particular test, otherwise all the results for all the sera on the plate are invalid and the tests should be repeated. It is preferable to have a negative control serum on each plate, but when large numbers of mainly negative sera are being tested negative control sera are tested on only a few plates for any batch of tests. Protocols for some tests require the testing of a number of positive controls of different expected end titres. This is especially common for tests where sera are tested in single wells and thus large numbers of sera are tested on each plate. In ELISA tests it is common to have up to 6 negative sera and duplicate tests of 3 or 4 dilutions of the positive control serum on each plate. Alternatively a low medium and high positive serum may be included on each plate. Positive control sera should have been standardised against an international or national reference serum.
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- Brucellosis, Sera, ELISAs