You may stop your ELISA at any time. I usually estimate the OD by eye, and if I think that the OD is fine, the plate will be stopped. You can simply use this shortened development time in your new protocol. By the way, most researchers would recommend a maximum OD of 1.0 due to linearity issues.

Another solution is to measure the absorbance several times at different times without stopping the reaction (you may have to choose another wavelength). Later you can choose the optimal measurements.

However, I assume that these low serum dilutions are unnecessary anyway. In most cases, you are only interested in high dilutions.

Dear Michael,

Thank you for your reply, very helpful. I'm wondering: when performing measurements at various timepoints, without stopping the reaction, I have to use 625 nm instead of 450. If for example it turns out that the OD625 is optimal at 6 minutes (lets say OD of 1.0), would a similar/equal OD425 be measured after stopping the reaction? In other words, are OD measurements at 625 and 450 nm related?

Yes, the values are related. As far as I remember, the absorbance would increase by about a factor of 2 after stopping it by acid. You may determine this factor experimentally quite easy.

As Michael Weller indicated you could read your plate at earlier substrate incubation times or use a wavelength with a lower optical extinction. But I think you are going about the ELISA optimization incorrectly, which may result in an unsatisfactory assay, especially in terms of reproducibility. The chessboard titration should initially be done with your positive control serum vs. the secondary antibody using your preferred substrate incubation times, wavelength, etc., but not with the antigen concentration. Generally, a concentration of antigen that is saturating for the plastic is desirable; using lower concentrations will result in your assay being affected by small differences in the amount of bound antigen. Usually [antigen] of 1-2 ug/ml is about right. Your positive control serum for this purpose should not be “highly positive”, just have a good elevated reactivity. From this analysis you should be able to establish a concentration of primary and secondary antibody that produces a desired OD in the desired substrate incubation time (usually ~1 hour). Your “highly positive” sample may then be over the range of the spec, but you can always dilute such unusual samples more than your standardized serum dilution and multiply the final OD by the additional dilution factor. Or better yet read the OD of a plate containing a highly-reactive sample at a shorter time point before it exceeds the spec capability, and multiply that OD by the average ratio of OD’s at that time point to that of the standard time using sample(s) run on the same plate that are within the spec-reading capacity. This ratio is constant (although not linear with time) regardless of the absolute antibody level. In this way you won’t compromise the sensitivity of the assay for low-level reactors. In general, you should avoid serum dilutions

Thank you for your replies! The problem was a lot of a-specific binding of Immunoglobulins. I tested various serum samples (including Ig depleted serum), various serum diluents and various blocking buffer combinations and found out the combination I was using was sub-optimal, hence leading to an overflow of the OD signal.

It seems that your reactant concentrations are not optimum. I had a similar problem. I suggest testing lower antigen concentration and higher serum dilution.