Coating ELISA plates at room temp

[mauer]

Hi all,

by accident I left ELISA plates for coating in incubator at 22 degrees C instead at 4 degrres overnight. What effect this would have on results?

I intend to repeat the experiment anyways, but I am just wandering if I should proceed with processing of these plates that were coated overnight at 22 degrees.

thanx all

[danfive]

You should be fine (especially if you covered/sealed the plate).
Most protocols say a couple of hours at RT or 4C overnight.
But even the R&D ELISA development guide says RT overnight as std protocol for plate coating.
See p.4 ELISA protocol ---> [Only registered users see links. ]

[mauer]

Danfive,

Thankx for your response and the link to the document. Platers were sealed, it's just that we usaully do overnight coating at 4 degrees and I was concenred if diffrences in results will be attributed to difference in coating temperature. (perhaps one can always argue that, but i just wanted to double check that i am not processingplates that shouldn't be processed)

[danfive]

Originally Posted by mauer

Danfive,

Thankx for your response and the link to the document. Platers were sealed, it's just that we usaully do overnight coating at 4 degrees and I was concenred if diffrences in results will be attributed to difference in coating temperature. (perhaps one can always argue that, but i just wanted to double check that i am not processingplates that shouldn't be processed)

Coating differences in regard to temperature affect you if:

1) Antibody is not stable at room temp, i.e. it degrades. You can check this out on the product info. Sera (sometimes sold as Ab, always labeled sera, though) may pose this problem, purified antibody usually does not.

2) If pH is not stable, overnight at room temp. pH does affect binding, you should be fine with pH > 8, but acidic pH would remove bound antibodies.

Most likely you are fine because:

1) Surface coating is determined by surface chemistry and usually results in a geometric spacing of the antibody (or protein) across the polypropylene well. (the greater the conc of Ab the closer the spacing, the less Ab the greater the spacing; you see a pattern either way).

2) Either way RT overnight or 4C overnight--you are probably binding the max amount of Ab. Therefore less likely to see a difference due to greater/lesser capture Ab on your wells.

[Zagami]

Coating process on solid-phase : the rate and extent of the coating depends on these factors : 1. Diffusion coefficient of the attaching molecule; 2. Ratio of the surface area being coated to the volume of the coating solution; 3. Concentration of the substance being adsorbed; 4. Temperature; 5. Time of adsorption. These factors are linked.
As can you see, temperature factor is variable independent, because the rate of the hydrophobic interactions, between coating macromolecule (antigen or antibodies) and well surface MTP (microtiter plate), depends on the temperature : the higher the temperature, the greater the rate. There are many variations on incubation conditions. It must be remembered that all factors affect the coating and, thus a higher concentration of protein may allow a shorter incubation time as compared to a lower concentration of the antigen for a longer time. The most usual regimes involve incubation at 37 °C for 1 - 3 h or overnight at 4 °C, or a combination of the two, or incubation (more vaguely) at room temperature for 1- 3 h.
In my experience I’ve noticed that different macromolecule, to identical concentrations, has different temperature and time for optimized coating process. In addition, for every macromolecule it is necessary to perform a series of experimental tests to get the thermal optimization condition, besides respect of the other factors, to obtain a standardized regime. Increasing the temperature may have a deleterious effect on antigen(s) in the coating stage, and this may be selective, so that certain antigens in a mixture are affected whereas others are not. Insofar, I believe that, if molecule from you used, with coating process at 4 °C in overnight according to the manufacturer's directions, that was the procedure for the maximum coating, and whatever variation, can influence the results of the samples processed.

[Zagami]

Coating process on solid-phase : the rate and extent of the coating depends on these factors : 1. Diffusion coefficient of the attaching molecule; 2. Ratio of the surface area being coated to the volume of the coating solution; 3. Concentration of the substance being adsorbed; 4. Temperature; 5. Time of adsorption. These factors are linked.
As can you see, temperature factor is variable independent, because the rate of the hydrophobic interactions, between coating macromolecule (antigen or antibodies) and well surface MTP (microtiter plate), depends on the temperature : the higher the temperature, the greater the rate. There are many variations on incubation conditions. It must be remembered that all factors affect the coating and, thus a higher concentration of protein may allow a shorter incubation time as compared to a lower concentration of the antigen for a longer time. The most usual regimes involve incubation at 37 °C for 1 - 3 h or overnight at 4 °C, or a combination of the two, or incubation (more vaguely) at room temperature for 1- 3 h.
In my experience I’ve noticed that different macromolecule, to identical concentrations, has different temperature and time for optimized coating process. In addition, for every macromolecule it is necessary to perform a series of experimental tests to get the thermal optimization condition, besides respect of the other factors, to obtain a standardized regime. Increasing the temperature may have a deleterious effect on antigen(s) in the coating stage, and this may be selective, so that certain antigens in a mixture are affected whereas others are not. Insofar, I believe that, if molecule from you used, with coating process at 4 °C in overnight according to the manufacturer's directions, that was the procedure for the maximum coating, and whatever variation, can influence the results of the samples processed.