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Assay timings: how flexible are they?

Background


All OMMICA assays include specified timings as part of the instructions, for each stage of the incubation process, and also, in some cases, for how long the reagents are still good for.


Adhering to them will ensure the highest possible accuracy in the results.

These timings are based on extensive development of the assays, investigation into how long the stages take, and what happens if the times aren’t adhered to. None of the results will change dramatically if the timings are off, but the accuracy may be significantly reduced. In all cases, the absorbance (therefore the calculated sample concentration) will drop.


Minimum timings


All the incubation (heating to 50 °C) timings are a minimum time to ensure complete reaction of the assay. Stopping the assay before the time specified will mean that the reaction hasn’t gone to completion, and the result obtained is an under-estimation of the true value.


Where applicable, the centrifuge timing (10 minutes) is to ensure full separation of the oil and water layers in the assay, so that the absorbance can be read accurately. The standing time for assays that don’t include oil (the water versions and the standard curves) is to ensure matching total incubation times for each.


Maximum timings


The only maximum time specified in the assays is the validity of the combined methanol reagents after they have been mixed. This is based on testing; after 2 hours or so, the effectiveness of the reagent mix drops, so results obtained will be an under-estimation of the true value.


We realise that many people who use our kits may be running many processes simultaneously, and might be unable to get back to the assay when the timer goes off. Therefore we have performed extensive testing to find out how long an assay may be left incubating/standing, after the specified time, and for the results still to be valid.


All assays: once the incubation is complete, and the tubes have been removed from the heating device,  the absorbance should be read within an hour.


Methanol assay: it’s OK if the incubation time runs up to half an hour longer than it should, but much longer than that, and the results will be less accurate, so probably worth re-running the assay.


MEG assay: This is more complex, as there are two heated incubation stages. Both have been investigated.

  • Step 1: set at 15 minutes, no issues if it runs up to an hour (i.e. 45 minutes longer than specified)

  • Step 2: set at 30 minutes. No issues for running up to 90 minutes (an hour longer than specified)


Overall guidance


Where possible, the assays should be run to the specified time, as this will ensure efficiency as well as accuracy. However, if any of the incubation stages run over by half an hour, there is no issue with the validity of the results.


Some of the assays have more flexibility than others, but this approach will allow for the same approach to be used for all OMMICA test kits.

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