One may be able to figure out what is the optimal protein concentration for coating plate for ELISA. For IgG antibodies (150kd), concentration at 5 microgram per ml is close to saturation for coating the 96-well plate. Excessive Ag may not increase the assay signal and sensitivity. We usually use 2 - 2.5 microgram per ml, 100 ul per well, in carbonate buffer. One may need to titrate out the optimal concentration of different size of protein for coating.

ELISA is avery sensitive method. If you like to justify your action, just compare the coatings on the plates [stored antigen and the fresh antigen] Lalitha Kabilan

Have never heard of reusing the samples but if you are measuring another protein of interest which is not cross-react with the antiboy of your ELISA plate it may work. I have not tried it myself though.

I guess as long as you get the same result after re-use. However, there may be contamination issues. Personally I would not do it.

If you found a decent concentration after 24h at 4°C you may reuse the antigen. However, if your antigen needs to be stored at -20° or -80°C you may not get the same result

I have not tried it myself, but I know that it is possible to reuse the antigen to coat another ELISA plate. The best way to determine this is to have a go... Coat your first plate, remove the antigen (do the ELISA) and use the retrieved antigen to coat a second plate overnight. Repeat the ELISA the next day and compare your results. This might be problematic if your antigen is not stable at 4degC for long... but otherwise it should be OK as normally an excess of antigen is used in coating. The ELISA results will tell you whether your experiment work and the strength of the signal will allow to decide whether to go ahead or not.

Agree with above answers. Some people including myself reused retaining antigens for second coating before, especially those precious ones. The resulting signals may reduced because the Ag concentration for coating dropped. Thus it may be good for "Yes and No" assays and may not be appropriate for high quality quantitative assays.

It depends on the conditions under which you are coating the plate, the stability of the antigen in the coating buffer at the coating incubation temperature, and the stability of the coating buffer. If the antigen is in great excess, the amount by which the concentration is decreased by coating the plate will be small. In that case, if the antigen is stable in the coating buffer at 4°C for at least 48 hours (coating of two plates overnight + time to perform the assay) then it is probably OK, but should be confirmed. However, if your antigen is in great excess, it would be better to titrate the antigen to an optimal (lower) concentration and store aliquots of the antigen in the optimal buffer and at the optimal temperature for use at a lower concentration in multiple assays. In either case, running controls with known activity and trending the data from those controls to detect problems will give you more confidence in the reliability of the method.

We tried for blood screening assays in the mid-90s, using low concentrations of radioactively labeled coating compound. We definitely decided NOT to ever re-use, we simply lost sensitivity and specificity..

Thank you for all the information. I would be soon performing the trial run using these samples. Shall update the outcome soon.

Dear Alok, for reliable results of the recovered material, you'll need to do validation. Thus, I'd only do it if the material in question is really precious and cannot be obtained easily when you need it again.

I'd expect a decrease in antigen concentration and also some loss of performance due to denaturation. But actually, as you probably will do a competitive ELISA later (or are you capturing an antibody?), you simply might measure the actual antigen concentration in the recovered buffer. If possible, compare against the originally used coating solution.

Can you provide us some more details? Is the antigen a protein or something else? What is the concentration in the coating buffer?

Dear Wolfgang, Well you have rightly pointed out. The conc. of protein-antigen used with the coating buffer was 0.6mg/ml and the conc. for the same sample after recovery was about 0.4mg/ml.

I think two things could be possibly done. Run on SDS gel with the sample retained and secondly, making up the conc. of the recovered sample to 0.5-1mg/ml to do a trial Elisa to capture the antibody binding conc. .

Few limitations I can make out ryt now would be the Antibody usage and effort.But, I believe it is worth a trial to check compare the results using recovered sample vs fresh sample.

Dear Alok, 600ug/ml is fairly high. Have you tried 50ug/ml or similar, even less should do well. Then, your protein sample will last longer and you don't have to wrack your brains (and bones) about recovering.

Thanks, I would reduce the concentration of my protein and try.

Even 50 ug/mL is high for an ELISA. Try titrating the antigen from 50 ug/mL down to 0.5 ug/mL. One of my most reliable, validated ELISAs uses the coating antigen at 2 ug/mL. Of course, your results will depend on the quality of your antigen. Have you run it on an SDS gel to check the purity or on a Western blot to look for degradation? Also, have you established controls for the assay? It is important to use controls - preferably a high, medium and low control and track the resutls to confirm the validity of each assay. Good luck!

One may be able to figure out what is the optimal protein concentration for coating plate for ELISA. For IgG antibodies (150kd), concentration at 5 microgram per ml is close to saturation for coating the 96-well plate. Excessive Ag may not increase the assay signal and sensitivity. We usually use 2 - 2.5 microgram per ml, 100 ul per well, in carbonate buffer. One may need to titrate out the optimal concentration of different size of protein for coating.

I agree with Ms Wloch, a concentration of 2 ug/mL is good enough for ELISA based assays. We do that all the time, coat 200ng/well for proteins and 100ng fpr peptides. However, if in doubts it is always a good idea to do an experimentation for checking the optimal coating concentration. It would be the best to check 0.5ug/mL, 1ug/mL, 2 and finally 5ug/mL and adding 100uL /well. Also, regarding the reuse of antigen solution previously coated, since the coating time is overnight, the exposure time is high enough to have caused any change in the antigenic epitope in that particular buffer. Therefore, it should work fine the second time at the same quantity being coated/well.

Dear Alok, if antigen is the limiting factor, then it can be recoated. Usually it works without much of a problem. However, one needs to compare the readings of the first and second batch, once, to be sure of it. Second, in your case as you are coating excess of antigen, it appears antigenic material is not really a limiting factor. So the coating concentration can be reworked and there is no need to recoat the antigen.

It will also depend on if you'll be doing a qualitative or a quantitative assay. You may have problems with the reproducibility in a quantitative assay esp with protein antigens.

If planning to saturate a plate with antigen, keep in mind that you are assuming that the binding capacity does not vary from well to well, i.e. that you are binding the same amount of antigen to each well. In my experience this is not true.

I think not variable and retained

In ELISA you need to have an excess af all reaction partners with the exception of the analyte (here in this case the antibody in a serum cell culture media, ...). Therefore you need to find an optimum which means that you will bin about 100 ng protein /cm². It's not that much so that you can re-use the coating solution. Optimum means at all, that you have to find the best concentration which gives you the excess of antigen during the solid phase binding but not more than the concentration which is required.

In general you should try this and you will find (depending on the distance to the optimal concentration) that you can ru-use the solution 1 - 10 times. This means only, that there is some optimization required.

I fully agree with Stephan. Optimization is required.

2-2.5 ug/ml is appropriate but optimization is required, in my experience high conc of coating antibody might give high blank value also.

Optimization should be done antigen (protein) by protein. There are several influencing parameters.

- MW: high WM binds much better

- Content of hydrophobic amino acids available on the surface of your protein

- Multilayers of the coating protein can be caused by protein-protein interaction (which can be influenced by the ionic strength and the pH

- temperature

- surface chemistry

- presence of competing other proteins in your solution

- competing contaminants (like detergents, lipids, lipoproteins ...)

The best way is to run a checkerboard titration in dublicates, one for positive one for negative controls ans search for the best positive/negative-ratio.