Hi,

for sure, there are differences, but not that much between the brands, much more between different lots. In the diagnostic industry we reserved defined lots of polystyrol where the plates were made from.

If you would like to know which plate can be the best for your purpose, clear some simple questions:

1. Are you try to insolubilze a protein with a MW > 10 kD, you can use all kind of ELISA plates, medium binding (and normal price) is fine. You need the hydrophobic styrol rings from the plate (and there are really many) and the hydrophobic rings from the amino acids in your protein. The lager the protein is, the better the binding will be. Use a 0.1 mol/L carbonate buffer, pH 9.6. The optimal concentration is between 2 - 10 µg/mL. Avoid higher concentrations, this will create double layers which will removed during the different washing steps and incubations.

2. Are you try to bring a peptide on the plates surface, than it's helpful to check different plates of different brands. If you find one working, try to stay with the same lot.

3. Are you try to bring carbohydrates or nucleic acids (or others) on the surface, use methylated-BSA (mBSA) (5 µg/mL, 0.1 mol/L carbonate pH 9.6)

Incubate for coating always overnight at 4 °C, in the case of using mBSA wash with PBS (without any detergent) add a second round of coating).

In high affinity / high binding plates you will have sometimes background problems. Blocking can become important but creates unexpected side reactions as well.

If you would use the coated plates in your own lab only, there is no blocking required. Store the plates after removing of the fluid simply frozen. If you have to store them at room temperature, then you need some BSA (1% in PBS + sugar [glucose, mannose]). Those plates should be heat-sealed together with a desiccant bag.

Avoid cell culture plates, the manufacturing process is different. There are different mold release agents in use.

Stephan

Hi Theodore, I did quite a bit of different ELISA in former times and had to switch plates in between as well. My opinion is that it does not matter which plate you use. I would just suggest that you are using the same plates within one experiment, like determine IgM concentration in 100 samples or so. As long as your negative control which should be on each individual plate is negative and your are subtracting the background intensity from your sample values, it should still be fine. I hope this helped! Good luck with your research!

Theodore,

as -hopefully rational scientist - I do not think that there is the "One ELISA Plate with Magic Properties". Of course, there are different functionalizations and methods of functionalization of the surface and hence different binding properties which make the plates not simply interchangeable on a one-by-one basis. But I would not expect that any assay only will work with one brand's binding plates. An important aspect however might be the well-to-well variations which probably should be the most relevant aspect when choosing plates for highly sensitive assays in order to detect small deviations from reference value.

Anyone knows by chance a (independent! :) study where plates of different types and from different manufacturers have been compared?

Wolfgang Schechinger, can you recommend a brand of 96 well plate for tissue cultures with low well-to-well variations, please? I have very high variation in MTT assay between the replicates.

Hi Natalia,

sorry, I can't. Probably the highest contribution to the error is the cell number that you are seeding into the wells. Personally, I like the plates from TPP, but actually, any should do. When you are using adherent cells, maybe additional pre-treatment of the plates (eg coating with collagen, poly-lysine or gelatin) could improve your results.

Also, an assay system with less steps (eg XTT, lactate measurement) or that is independent of the actual cell number (eg FACS analysis of cell cycle distribution) might be an alternative.

As mentioned by you both plates are very good. But, as a researcher I would suggest that, if you perform with one type of plate that will be better to avoid any minute errors with regard to plate. If you start with one type of plate and in the middle if you shift another type then, my suggestion again perform ELISA with all samples with new plate. That only gives accurate results by avoiding any error with regard to plate.

We should avoid error at all stages...Our experience was that grainer protein estimation plates are poor than NUNC immusorb plates...However your standard curves and are good in both plates, you need not repeat...otherwise try to keep one positive control and negative control always whenever you do ELISA...We used to keep three types of control...Positive control, negative control and one more control which gives OD around 0.4....

Hi!

I did an experiment last week in wich i compared different ELISA plates for my assay, to see wich plate gave the best results for my assay.. I compared: Corning high & medium, Greiner high & medium. There were variations in my results and I actually now switched plate brand for further experiments for my assay.

Hope this helps :)

Hi!

I agree with Gaurav Soman.

The brand doesn't matter much as long as you are using same lot for a particular experiment. please try and get your plate reader tray cleaned and calibrated periodically for wavelengths to get good consistency.

Hi,

for sure, there are differences, but not that much between the brands, much more between different lots. In the diagnostic industry we reserved defined lots of polystyrol where the plates were made from.

If you would like to know which plate can be the best for your purpose, clear some simple questions:

1. Are you try to insolubilze a protein with a MW > 10 kD, you can use all kind of ELISA plates, medium binding (and normal price) is fine. You need the hydrophobic styrol rings from the plate (and there are really many) and the hydrophobic rings from the amino acids in your protein. The lager the protein is, the better the binding will be. Use a 0.1 mol/L carbonate buffer, pH 9.6. The optimal concentration is between 2 - 10 µg/mL. Avoid higher concentrations, this will create double layers which will removed during the different washing steps and incubations.

2. Are you try to bring a peptide on the plates surface, than it's helpful to check different plates of different brands. If you find one working, try to stay with the same lot.

3. Are you try to bring carbohydrates or nucleic acids (or others) on the surface, use methylated-BSA (mBSA) (5 µg/mL, 0.1 mol/L carbonate pH 9.6)

Incubate for coating always overnight at 4 °C, in the case of using mBSA wash with PBS (without any detergent) add a second round of coating).

In high affinity / high binding plates you will have sometimes background problems. Blocking can become important but creates unexpected side reactions as well.

If you would use the coated plates in your own lab only, there is no blocking required. Store the plates after removing of the fluid simply frozen. If you have to store them at room temperature, then you need some BSA (1% in PBS + sugar [glucose, mannose]). Those plates should be heat-sealed together with a desiccant bag.

Avoid cell culture plates, the manufacturing process is different. There are different mold release agents in use.

Stephan

In my direct/indirect ELISA I always use maxisorb Nunc plates, they seem more sensitive than polisorp Nunc and really I am afraid to change

I appreciate your suggestions

Just stay with the plates if the assay is already in place. You should know only something about the items behind in the case you have any trouble.

Good luck.

Hi,

I agree with Stephan Kiessig.

There are differences, but not that much between the brands, much more between different lots.

You should use same lots to reduce the variations.

In Indirect-ELISA the type of plates you use are very important. in DAS-ELISA like you use it don't matter much but you should use all the same type of plates in all the experiments.

We have been manufacturing antigen capture immunoassay kits for nearly three decades and find that 96 well plates are the most troubling aspect of our work. There is variability between and even within lots of 96 well plates. Also, lots found to work well can degrade in performance during storage while awaiting entry into the production stream. This is a particularly vexing problem that we continue to explore. In the case of a research laboratory, I agree with the thread of the responses to your question - stay with the plate brand that works for you. Also try to purchase enough plates from a recently manufactured lot to get you through your project. Since we see plate to plate variation under the best of circumstances, we recommend including a standard curve on each plate to control for this. Sorry I'm so late with this response, I just happened upon this question today. Good Luck!

Dr. Bess’s experiences and some of the preceding suggestions involving careful attention to the measuring equipment involved in doing ELISAs are remarkable. I suspect that much of these reproducibility problems derive from one underlying vulnerability: the signal generated in these ELISAs was limited by the amount of primary reagent bound to the solid phase. As a result small differences in the amount of e.g. capture antibody among wells manifest as signal variability. Ideally, the ELISA should be designed so that the analyte being measured is the only component that is limiting. This is relatively easy to achieve for ELISAs for antibody by binding excess antigen to the solid phase and by diluting the sample sufficiently. For antigen capture ELISAs in which antibody is on the solid phase, excess antibody may be difficult to ensure because of its cost, purity, affinity, etc. However, overcoming these latter issues should reduce interassay variability.

I agree with Dr. Ruben's comments. In the case of capture antibody, we titrate it and select a a concentration that is in excess of that required to obtain good signal (i.e. we are not on the cusp where a small change in the amount of capture antibody available will result in significant changes in OD obtained when samples are tested). To ensure that the wells in each plate are coated with the same amount of capture antibody we use a robotic pipetting station (with a validated CV of 2% or less) to apply the capture antibody solution to 96 well plates. Even with this level of care, we see plate to plate variability which varies from lot to lot that remains unexplained.

To Stephan: Why are people usually using carbonate buffer and not simply PBS (as it's not susceptible to CO2 from the air)? The styrene interaction should not be pH dependent at all. Carboxylic groups should be completely deprotonated at pH >6 and lysines should be protonated until pH of about 9 and arginines won't care at all.

Is it just legacy from literature (and nobody bothered to question it) or is there a real need for this pH, at least in some cases?

Probably a heretic question, but I have had good experience with PBS as solvent for coating larger proteins (25kD and up) and never had the need to compare PBS against carbonate. Smaller proteins never (not yet) tried. I am open for advice!

Wolfgang:  You are completely right in my opinion.  Carbonate buffer as the preferred solvent for coating polystyrene plates seems to have become entrenched in the ELISA lore. This has never made much sense to me, not only for the reasons you mention, but also because  macromolecules are vulnerable to denaturation at high pH or bind to PS in a non-native form.  Like you, I have always used PBS.  I am surprised that Stephan Kiessig, who otherwise provides great advice, would recommend this solvent for coating plates.

To Wolfgang and Robert: Proteins conjugate to polystyrene via tyrosine residues, which is facilitated at pH>8.5. At this pH, PBS is not a buffer [pKas of phosphate are 2.3,  6.8 and 11.5 IIRC]. The reaction at neutral pH is much slower. This may or may not be a problem, but is the reason for carbonate buffer for coupling proteins to the plates.

That being said, typically I use protein in sterile PBS O/N at 4oC, and have not have had any problems with antigen sticking.

To Theodore; I have used a plethora of plates from different manufacturers and not found significant differences in their utility. There used to be ELISA lore that the edge wells in plates were unreliable due to how the plates were made, leading people to set up their assays using the central 60 wells. I never found it to be an issue.

Regarding the carbonate buffer issue, if the idea is that one wants to maximize hydrophobic interactions between soluble proteins and styrene residues on the solid phase, the only amino acid that would (theoretically) be positively affected by changing pH from ~7 to 9.5 is lysine, deprotonating the epsilon amino group.  A pH >10 would be needed to de-protonate the phenolic hydroxyl of tyrosine, and this would make it more hydrophilic.   So, the basis for using high pH carbonate buffers for binding proteins to polystyrene cannot be explained on theoretical grounds, as Wolfgang pointed out.   I suspect this suggestion came from empirical observations that more of some protein mixture bound to PS at high pH, but this was probably due to protein denaturation, exposing hidden hydrophobic residues. It is also quite possible that some antibodies  react better with denatured epitopes. 

As far as I can see - Both Carbonate (pH around 9) and PBS (pH around 7) have been used for initiating binding between well plates and antibodies. But one thing I have observed here in both cases the incubation times are different. 

Lets just say, I have a norma well array plate and IgG antibody,  and I would like to bind them both for ELISA. I was wondering what would be the ideal incubation time periods for both buffers in order to establish a proper bonding ?.

Thanks in advance