Hi, there are a lot of factors to check while ELISA assay is not work. Normally, I do trials first to check the right concentrations of target, Abs, and even the substrate to be used in ELISA. I found that different dilutions range of Abs (not only the manufacture recommended condition) will be able to give good colorimetric results. Most of time only  changing the subtract concentrations will help. In lots of ELISA, we found that manufacture suggestions are really suggestions only.  If you try to play different dilutions in above factors and still have plateau reading too fast, the last you can try is to use Kinetic Reading of your colorimetric results ( some ELISA reader has this function.) This will give you good understanding of when your color results reach the plateau or overboard. Say, your color development time is 30min, and actually in 10 min, your color result already past OD= 2.0. In this case, it is a need to dilute down a factor, like your IgG, Abs, or subtract to read more slowly before your 30 min development time due. In lab, we do have some protocols that color developed so fast, so our color reading stop points set at 15min. Hope this info would help.

You need to titrate each reagent one by one. 

You also need to do a time course for color development 

Hi,

I would agree with the previous answers. It sounds that the assay is a bit too sensitive for your purposes.

Several factors influence assay sensitivity:

- Agitation and shaking increase sensitivity and shorten incubation times. Typically not all laboratories have plate-shakers so the incubation is performed at RT or 37C for 60 minutes to compensate the lost sensitivity.

- Incubation temperature, higher the more sensitive. Some ELISAs benefit from ambient temperature incubation, whereas some require +37C or +4C.

- Sample dilution.

Higher sensitivity of the ELISA can lead to increased background of the assay (reagent blank) and aspecific signal from sample matrix. The matrix effect is important to check. You should have a sample of similar composition as your serum samples, but which does not contain IgG, or is from a species which your detection antibodies do not cross-react with. Ideally this material should not give any signal over a broad dilution range. In practice, high concentration of serum always gves some aspecific background. You the pick a dilution range where this effect does not contribute to the signal. 

Easiest ways to de-sensitise your ELISA is to leave the plate at RT without agitation. You still need to check whether your assay standard and sample give a proper dose dependent signa. Make sure that the curves look identical. Sometimes the matrix causes that the biological sample dose response is different from standard. If you do not want to desensitise, other option is that you just keep diluting your sample until you find the range where the linear d.r. emerges. 

one important factor is that you should verify is that  your assay can run for 30 minutes so that the reagent blank and the sample controls remain at decent absorbance levels (

It seems that you need to optimize the assay conditions, as suggested in previous answers. In my opinion and experience, rocking and shaking does not impact the reaction as much, as the antigen antibody reactions happen by the Brownian movements of these molecules.

Take your time, the immune reaction is not the fastest but will happen anyhow within minutes. The longer incubation times protects you from time gradients on the plate which leads to an increased (im)precisicion. The detection limit depends anyhow only on the affinity of the solid phase antibody to the antigen (here: IgG).