All of the above answers is correct and I think that 2 hours is too late but it is dependent to the environmental conditions in this 2 hours.

Light and high temperature are affect the intensity of absorbance in ELISA reader.

- Incorrect incubation times or temperature may cause of inadequate color development.

- Plate left too long before reading cause high background signal

I think that 30 min is the time´s limit.

Based on the protocol Millipore, most measurements should be done within 30 minutes. However, depending on the protocol, measurements should be done ​​within 5 minutes. Personally, I prefer to perform the measurements as soon as possible after to add the stop solution and homogenization plate, if possible within 5 minutes.

I do agree with all colleagues views, as it is a coluometric assay and one measures the intensity of the color and the colour would changed over time for sure.

You should measure the samples within the next 5-10 minutes after the colorimetric reaction was started. If you wait too long (hours) samples will reach a reaction plateau and staining differences are less significant.

I would also recommend max. 5 minutes. 2 hours is too late, for sure, because of the mentioned reaction plateau.

All of the above answers is correct and I think that 2 hours is too late but it is dependent to the environmental conditions in this 2 hours.

Light and high temperature are affect the intensity of absorbance in ELISA reader.

- Incorrect incubation times or temperature may cause of inadequate color development.

- Plate left too long before reading cause high background signal

On the other hand, couldnt one argue that if any changes, they would apply to all wells the same, therefore they would still be comparable (although with a lower OD of course)?

Don't forget the o.d. Reading needs to be in the linear region. I typically don't go above a reading of 1 o.d. Unit for 450nm. But it depends what the specs say and how the blue color develops. Kinetics of blue color formation is more accurate. You can get a slope for the reaction instead of a single endpoint.

Reading within 30 min after experiment would give the best results.

I don't understand how anyone can answer your question without knowing the stability of the colorimetric products of your reaction. If they are end products and stable you shouldn't be in trouble.

The best solution would be next time you repeat the assay (even if this is just with standards) is to read the results at several different times. You can then use this information to determine if time is important and (if necessary) adjust your 2-hour results to an earlier time.

These assays are done all the time, Michael. The stability is known for the yellow product that forms after quenching. It is in the manufacturers guidelines for most ELISA products. Typically, people measure one endpoint, ie they quench and read. The stability for the yellow product is about 1 hr at standard room temp. For the development of the color, it is completely arbitrary and depends on how much HRP is present, the enzyme that is attached to the abs. For detection. So it could take hours or seconds. That is why I recommend looking at abs 650nm so that several points can be taken to determine a slope or initial velocity for the color development. If the assay develops too far, the readings are out of the linear range, and every well has the same max. O.d. Of 2. So when it is light blue by the eyeball method, that is the time to quench. But it is better to do the readings with time if you can. And most machines if not all, even if done manually, can do this.

For an accurate result, based on the protocols to complete reaction, reading after 5 min is recommended. But as other colleagues mentioned, it should be read during 30 min after adding stop solution. I've tried for several times in different minutes ( during 30 min, 45 min, 1 hour) to read the plate. They showed different results affected on the curve.

There have been a number of good suggestions so far. I would definitely suggest a repeat Elisa with standards and test different time points of reading to assess best time. Although this is not fool proof (variables such as temperature/reagent quality etc can effect this timing) it will give you a good grounding. Leaving the plate too long distorts your standard curve since the higher concentrations reach a plateau and the lower concentrations start to catch up. Also the colour can fade and precipitates appear over time which also add to problems. We routinely read plates between 5-20minutes

Read the manufacturers instructions (if using a kit) they should be able to help and show you how you should plot your data for analysis. Remember not all standard curves give a linear response.

We always stop the colour reaction in our ELISAs with acid. In this case the plates can be kept wrapped up (to avoid evaporation) in the dark for several days without deterioration. Personally, I always run a standard curve over a large concentration range with each assay - then use the linear part of this curve to calculate concentrations in the sample (also assayed over a wide range of dilutions). I have found with this approach that results are very reproducible between assays and the time for colour development can be adapted according to how quickly it comes up in different sample sets.

2 hours is too late. Generally, Elisa assay is read 5 min by manufacture.

Don't panic early. If the TMB reaction is stopped, you have plenty of time. The limiting part in your ELISA is the enzyme you used for labeling of the e.i. secondary antibody. The HRP will use the H2O2 to oxidized TMB. This product is blue and it's will be oxidized until either no H2O2 or no unoxidized TMB is available. After a while, all wells will have the same blue color. Therefore to measurement principles are possible: kinetic or end point. In the kinetic measurement you measure the plates at least twice and calculate the slope of the OD per well. This slope correlates to the concentration. But, this way takes more time and you didn't profit from the huge procedure. Normally you are measure via endpoint measurement. Here you have to stop the reaction. Normally 1 mol/L H2SO4 is sufficient, some are using also 2 mol/L, some adding 0,05 mol/L NaSulfite. This will capture the residual H2O2.

Normally the plates should be read soon after stopping the reaction with 1 or 2M H2SO4, if you use TMB substrate. I recorded 3 readings in ELISA reader, after gap of 5 minutes each after stopping the reaction. The OD values reduced with passage of time! I agree with most you who say the plates should be read in 5 minutes

Depending on the actual assay (and environmental factors like lab the temperature), one usually finds a time when the signal is best and the assay is stopped. If one should forget to stop and/or to measure the plate in time, of course that's not nice, but as long as you have a standard curve on the plate (or at least reference values), it should not be a disaster.

I have conducted an ELISA assay experiment by measuring different plates including fixed and not fixed, at time interval (5mn-10mn-20mn-30mn-44mn-50mn-1h-2h-3h-5h-7h-12h -24h-48h).

For those fixed, the variations were significant from 5h, whereas for the plates without fixation showed a slight variation from 1h and a significant variation from 2h ..... I have also noticed effect of temperature and light and relative humidity...

I think 5-10 mints is beter for reading the ELISA plate