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Trouble Shooting
Plate Layouts - Liquid Phase
ELISA
Neutralisation Test
Karber Titres -Liquid Phase
ELISA
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Calculation of results
1. Calculate the median optical density (OD) of the four antigen control wells G,H 11-12 as follows: a) identify the highest and lowest OD readings and ignore these two values. b) Calculate the average OD of the remaining two wells. This is the median OD of the reaction (antigen) control, which represents the maximum OD value for the test, i.e., the 100% control value.
2. EITHER divide the median OD by 2. This is the OD representing 50% inhibition of the reaction control. Score wells positive if the OD is equal to or less than this value (i.e., 50% inhibition or greater). Score wells negative if the OD is greater than this value (i.e., less than 50% inhibition).
OR estimate the percentage inhibition of all wells thus:
Percentage inhibition = 100 - (OD of test serum well) x 100
(median OD of antigen control)
Acceptance criteria
Plates are assessed on an individual basis and are considered valid when:
• The reaction control has an OD greater than 1.0
and less than the maximum (plateau) value established by
previous titration of the assay (this will vary according to
antigen batch and local conditions).
• The strong positive reference serum (at 1/32 final dilution)
has a mean percentage inhibition greater than 85%.
• The weak positive reference serum has a mean percentage
inhibition between 55 and 85%.
• The negative reference serum has a mean percentage inhibition
less than 40%.
Interpretation of results
1. Spot test
Sera are considered positive if both wells show 50% inhibition or more compared to the reaction control median, i.e., have a titre of >1/32. If only one well shows this reduction in OD (i.e., titre = 1/32), the serum is considered inconclusive and is repeated. If both wells show less than 50% inhibition the serum is considered negative.
2. Titration
The end-point is defined as that dilution at which half of the wells show 50% or more inhibition compared with the OD of the median reaction control (Karber Titres for Liquid Phase ELISA) (Karber, 1931). Test sera having titres of up to and including 1/32 are considered negative, and those of 1/45 or greater, positive. If there are problems tests may have to be repeated. (Trouble Shooting)
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