You can store coated plates for very long times (years) when you follow some rules:

1. after yoating remove as much of your coating solution

2. don't wash the plates with a detergent containing buffer. The detergent in the washing buffer is highly hydrophobic and will slowly but surely between the hydrophobic amino acid residues and the styrol rings. This will reduce the bound protein during stoage time.

3. store the coated plates upside down in a freezer (-20 °C or below) 

4. If you need storage at room temperature (like in commercial assays):

- wash the plates wit a blocking buffer (for the normal blocking is a kind of voodoo)

- Blocking buffer: inter protein (BSA or Gelafusal [it's an hydrolysed gelatin used as plasma expander). Don't use milk in any form. It's anything but a defined reproducable solution and wil create often unspecific reaction (e.i. in patients with milk allergies). Add some sugar (1 - 3% Mannose or glucose). That's required for the binding of some residual water, otherwise the protain at the solid phase willbecome denatured. 

- remove the blocking buffer after 10 - 60 min, dry the plates under defined conditions (30 °C over night at a low humidity

- seal the plates in airtight plastic bags

As you can see, the first variante is much more easier ... and requires much less steps which needs some validation.

@Lianfa & Andrea: I have no idea who came up with this blocking using a detergent containing buffer. It's completely contraproductive and non of the commercial supplier would do this. Just try this, compare plates stored frozen vs. plates washed and blocked. You need only the concentration of your solid phase protein which is outside the maximal binding capacity.

1. During the first washing of an ELISA plate just at the beginning of the test with PBS Tween will give due to the strong hydrophobic interaction with the plate (if there would be really any binding site free) a sufficient blocking. 

2. Normally the coating concentration is between 1 - 10 µg/mL. The binding capacity of an ELISA plate is at 100 ng/cm². So you are coating in a huge excess. There is nothing free anymore. 

I used to keep antigen coated ELISA plates at 4C until 4 to 5 weeks, and they worked fine for me.

Hi sumayia, do you wash your ELISA plate before ?

Hi Faez, you mean after coating antigen and before using them for next step? Yes - I used to wash them 3 times with wash buffer (0.05% tween in 1 x TBS)

yes Sumayia

Thank you very much

No problem !

as long as you keep it hydrated it should work fine.

after the washing step

There are not a general answer to your question since depends on the stability of your antigen. However, in most of the cases, it woulld work well if you block your plates after coating with antigen, wash them with a buffer without detergent and let drying at room temperature overnight. Then , you can keep your plates (protected from light and wet) for several weeks.

I agree with Luis Mata in that it depends on your antigen that you coated. However, conservation of several weeks should work out fine. If you have any doubt about your plates maybe do a little evaluation experiment: do the coating with 2-3 plates, do the ELISA directly after with one of the plates, do the ELISA after your desired time and compare with the results you obtained from your first plate.

I agree with the previous comments. It strongly depends on the antigen you are using but also buffer formulations and the type of ELISA plates. Please look out for comercially available pre-coated ELISAs using your antigen. This should give you a hint at storage stability. However, you will need to evaluate for yourself, especially if you are using unique/prototype antigens.

Thanks for the answer, i have to keep my plate for a week and i am using venom antigen.

keep the plates sealed with a plastic tape and dry.

depending upon the antigen they can be kept for months but you have to test it

Hi Faez. I usually keep the plates coated/blocked (PBS1X-1%BSA)/washed (PBS 1X-0.05%Tween)/dry/sealed (ELISA plate sealers or scotch tape if were are out of them) at 4° for up to 1 month. Before the next step i wash them once, leaving them with the washing buffer for a couple of minutes. As someone already suggested you, you should try if this work out fine for your pair of antigen/antibody. You can try it only with the standard curve (to avoid using your samples until you are sure you have the optimal conditions).  

The upper persons have given  good guides. 

Thank you all

You can store coated plates for very long times (years) when you follow some rules:

1. after yoating remove as much of your coating solution

2. don't wash the plates with a detergent containing buffer. The detergent in the washing buffer is highly hydrophobic and will slowly but surely between the hydrophobic amino acid residues and the styrol rings. This will reduce the bound protein during stoage time.

3. store the coated plates upside down in a freezer (-20 °C or below) 

4. If you need storage at room temperature (like in commercial assays):

- wash the plates wit a blocking buffer (for the normal blocking is a kind of voodoo)

- Blocking buffer: inter protein (BSA or Gelafusal [it's an hydrolysed gelatin used as plasma expander). Don't use milk in any form. It's anything but a defined reproducable solution and wil create often unspecific reaction (e.i. in patients with milk allergies). Add some sugar (1 - 3% Mannose or glucose). That's required for the binding of some residual water, otherwise the protain at the solid phase willbecome denatured. 

- remove the blocking buffer after 10 - 60 min, dry the plates under defined conditions (30 °C over night at a low humidity

- seal the plates in airtight plastic bags

As you can see, the first variante is much more easier ... and requires much less steps which needs some validation.

@Lianfa & Andrea: I have no idea who came up with this blocking using a detergent containing buffer. It's completely contraproductive and non of the commercial supplier would do this. Just try this, compare plates stored frozen vs. plates washed and blocked. You need only the concentration of your solid phase protein which is outside the maximal binding capacity.

1. During the first washing of an ELISA plate just at the beginning of the test with PBS Tween will give due to the strong hydrophobic interaction with the plate (if there would be really any binding site free) a sufficient blocking. 

2. Normally the coating concentration is between 1 - 10 µg/mL. The binding capacity of an ELISA plate is at 100 ng/cm². So you are coating in a huge excess. There is nothing free anymore. 

Dr Kiessig, i will try the first variante, because it seems to be really easier. Thank you a lot

The easiest way around this is to coat a large number of plates and then store ALL OF THEM at -80C containing the coating solution.  Then thaw the required number of plates and follow the normal ELISA procedure.  

I work on protein modifications which vary from very stable to quite unstable.  Using this method I have found that storage of at least 3-6 months is possible without appreciable loss of antigenicity.

For the storage of coated ELISA plate for the longer time period better to contact commercial company for specified coating and storage protocol that also deals with the precautions that should be taken for coating and storing. For quick use normal coating procedure is ok.

@Arvind: the recipe I suggested based on a long lasting commercial experience. 

well after 15 years immunoassay, the answer is it depends. ... on the coated antigen, plates, humidity, blocking and stabilizing proteins and seals/foil. IVD manufacturers use to optimize this and have standardized procedures with include stabilizing proteins for long shelf life and a direct drying and vacuum sealing in plastic or aluminium bags together with dry packages. Did you do this? If not, you will have humidity inside your plate athmoshere and with all proteins this starts to spoil and you will dramatically loose sensitivity/specificity.... nobody may control what then happens on your plate. You should implement a standardized procedure with sealing and test your selected plates for functionality and variation after several times, e.g. 1, 2, 3, 6, 9, 12 month before starting experiments with samples. I know this is hard and often people will start directly, but  belive me, it is worth it. The easier way would be to get in contact with an experienced immunoassay/IVD company and use devices, which are produced under controlled conditions and regularly tested for stability. With this you can be sure and will get reliable results. 

Thank you Oliver, indeed it seems to be hard but necessary

i guess ideally not more than 24 hours, as expression of surface proteins (if bacterial cells are antigens) would start to change /degrade and if antigen coating, then they would loose their binding affinity..

I always keep them for not more than a month without preservatives at 4C and every time controls were optimal.  

Unless the nature of antigen (glycolipid, glycoprotein, polypeptide, heteromer protein,peptide), nature of plates (polystyrene, polypropylene, polycarbonate) and the source of plate and the dose and length of radiation given to plastic plates) are known, one can not give a generalized answer. You have to empirically assess. (see my paper in Immunological Methods and in Ann. New York Acad. Science, for further clarification of my answer. 

Good Question Raised by Ravindranath that could be important for preserving ELISA plate for longer time period. I agree with his answer. However, paper details with title should be given for convenience. Thanks to Ravindranath.

I agree with everyone who says it depends on antigen, preservatives, plate etc etc.

But my experience is that for proteins coated with bicarb buffer on maxisorp nunc (and similar plates), good reactivity after ~4 weeks at 4C. (Left unintentionally but became convenient)

For intentional extended storage, I would add sucrose after blocking with 1%BSA, flick out excess sucrose, air-dry at RT for about 10mins, then dry for 3hrs at 37C, then store in air-tight pouches with dessicant.

My plates are NUNC maxisorp Khayriyyah. Thank you

Hi there. At 4C you should be able to keep it fr about a month, whereas at -20C you an keep it for about 4-6 months if it is dried carefully.

Best wishes

Thank you Nikos

This reply is in response to Dr. Anand Kumar's answer. I am attaching the titles of the papers here and if he need the reprints, I can send them to him if he mails [email protected]. Thank you Anand of BHU.

Ravindranath MH, Ravindranath RM, Morton DL, Graves MC. Factors affecting the fine specificity and sensitivity of serum antiganglioside antibodies in ELISA. J Immunol Methods. 1994 Mar 10;169(2):257-72.

Ravindranath MH, Muthugounder S, Saravanan TS, Presser N, Morton DL. Human antiganglioside autoantibodies: validation of ELISA. Ann N Y Acad Sci. 2005 Jun;1050:229-42.

Did coate the plate yourself or did you get the plate already coated from manufacture?

@Diallo: i coat myself the plate

I Have a question when you storage plate at 4 or at -20, are proteins stil  solubles in carbonate buffer?

I think it may be useful to determine a storage period using accelerated shelf life testing techniques (e.g. Crowther J.R. (ed.), 2009. The ELISA Guidebook. 2nd edition, Springer Protocols: Methods in Molecular Biology, Vol. 516 (J. Walker, Series ed.) Humana Press, NJ, USA pp 323-325)

I agree with dear Seif ,I think so, one month is enough for hand made plate too,But if you