It depends on the plate you want to use. For most ELISA plates, you simply add your protein of interest (here your F(ab) fragment) in buffer to the plate and incubate, e.g. over night at 4°C or for 2h at RT. But as I wrote, it depends a lot on your plate and your protein. Sometimes pre-coating is necessary to orientate all your proteins in the same way. AFAIK, there is no common standard protocol that always works fine.

Hi Rehan,

I agree with Susanne on the pre-coating procedure. However, the second part of what you want to do is a bit vague. If the antigen is present in the PBMC then you should be able to do a sandwich ELISA to quantify the particular antigen without removing the cells. But it sounds like you are trying to immobilize a particular cell type onto the plate and then perform flow. If the idea is to isolate a particular cell type from PBMCs then you would want to modify the procedure used to proliferate T cells from PBMCs and perform the flow in the plate. Please see attached protocols.

Hope this helps.

Thanks Susanne and Padmaja.

The idea is to use immobilize the F(ab) fragment, which will capture mouse antihuman CD16 mAb. I intend to throw-in the PBMNCs, the NK cells of which express CD16 which will be crosslinked by the mAb in the plate. CD16 crosslinking activates NK cells and this activation is free of any target cell. We have flow cytometry based assay to enumerate NK cell activation by staining for IFN-G and CD107a (degranulation) expression. In order to be able to do that I'll have to retrieve the cells from the crosslinking assay and stain for flow cytometry.

I've read not to use cell culture plates because they are pre-coated and it would prevent the protein from adhering. So which plate should I use?

Thanks very much

I use the high affinity plates from Bio-Greiner for ELISA's, they may have something suitable for your particular needs.

Hi Rehan,

coating is quite simple: use 1 - 10 µg/mL of your primary antibody in a 0.1 mol/L carbonate buffer pH 9.6, even when you are preparing a socalled CELISA. Start with normal ELISA plates, "high-binding plates" creating often problems. You will have the bindig via hydrophobic interactions between the hydrophobic amino acids and the polystyren rings. The 1 µg/mL is anyhow in excess, you can bind 100 - 400 ng/cm² only. Somtimes you need a little bit more (up to 10 µg/mL), it's has to be evaluated by checkerboard titration.

For the the immunologic binding of the anti-human-antibody, a voncentration of max 1µg/mL is sufficient. You can incubate this antibody in PBS, 0.1% tween 20. You should use some FCS or albumin for all following washing steps to make sure that all detergent is removed. The use of the detergent is most the best way to block the plated efficientely. The cells in the alter steps have to be incubated in isotonic solution like RPMI, PBS, ... containing FSC or Albumin to make sure that the cells are not lysed until the last step. 

The lables could be HRP or AP. Check your cells before uising them in ELISA. Both activities can be present in cells!

I agree with Stephan. I usually coat with a pH 9.5 buffer if I believe that the antibodies have an acceptable stability (polyclonal antibodies are usually stable). usually I get similar binding if I coat the antibodies with PBS, pH 7.2. There is an old recommendation that the optimal coating pH is 1 pH unit above the isolectric point of the antibodies but most pH works in the pH range 7.2-9.6 as long as there are no other proteins or detergents in the buffer. I usually start with standard ELISA plates. For your purposes you do not need to achieve optimal binding so most protocols will work. 

The capture part in your assay is probably the easiest part. You may however run into problems when you have a anti-mouse IgG as a capture antibody as it may also capture your mammalian detector antibody giving a high background if you run the whole assay in the same plate. If you try to elute the cells from the surface you may induce changes in living cells. I am not sure how I would elute the cells without damaging them too much. I would probably consider rapid elution of the antibodies at på 2.8 and then neutralizing them immediately but this may have an effect on the cells. I could also try fixing the cells with 1% paraformaldehyde before eluting them to minimize changes prior to analyzing them by flow cytometry. I have not tried it on a microtitre plate, but it is likely to work.

You can coat the antibodies diluted to 1 µg/ml to a plate in PBS, pH 7.2 at RT for 2 hrs to overnight.

coating should happen better overnight. It takes some time to bind hydrophobic amino acid by hydrophobic amino acid. Sure, you have some binding already after two hours. Also PBS works well, but not for the majoritz of all proteins for coating. In some cases the pH as well as the molaritiy of the coating buffer needs to get optimized. Here we have a quite normal protein so that the carbonate buffer should work best. 

Decades ago, I used to pan out CD4+ T cells from mouse lymph node preparations. Typically, we coated petri dishes - not tissue culture plates - with anti-mouse antibody in .1M bicarbonate buffer pH - 9.5. The reason for using this pH is that the bond between the protein and the polystyrene is mediated via tyrosine residues in the protein, and this is facilitated at high pH, since the pKa of the phenol group of tyrosine is around 10. That can be done in `-2 hours. Then we blocked the plates with PBS containing 1% BSA for half an hour and rinsed with PBS. Capture antibody[anti-CD4] was added to the lymph node cells, incubated for 15 minutes, then the cells were washed and plated out on the antibody coated dish. These were incubated at room temperature for half an hour - swirl the plates every 10 minutes to spread the cells out evenly. If memory serves, we did 5 x 10^7 cells per 100 mm Petri dish. Non adherent cells were pipetted off, and the plates washed twice with PBS/.1% BSA. Adherent cells were released by pipetting on 3 ml of RPMI at pH = 3.0 [NO FBS or BSA!], gently agitating and the cells were pipetted into a 15 ml conical tube containing 12 ml of D-PBS/.1% BSA. The cells were >95% CD4+ by flow cytometry using a different CD4 antibody. They were functional in vivo [injected into lethally irradiated mice]. How that will work in your assay is anyone's guess. The low pH wash may alter the response of the NK cells. That I cannot predict.

Thanks very much Gary. This is pretty close to what I intend to do. I have two two questions:

Did you use any colorimetric assay to ascertain the coating of your anti-mouse antibody on the plate ?

What buffer did you use to bring RPMI pH to 3 (it must have been turned yellow, so did you rely on color or you measured the pH).

Thanks again.

Rehan Faridi Hi Rehan, I want to follow up on your question about the coating protocol for Fab fragment. Could you share what concentration and condition worked for you?

concentration should be between 1 and 10 µg/mL. Normally 2 to 5 µg/mL is suficcient. In 0.1 mol/L carbonate buffer pH 0.6 (50 or 100 µL/well). Over night at 4 °C. After this time, remove as much as possible of the volume. Don't wash the plates with a detergent containing buffer. Stofre the coated plaetes upside down in a freezer.

I agree with Stephan except the pH that I think should be 9.6 rather than a very acid buffer. We normally coat with 0.1 mol/L of carbonate buffer pH 9.5 or PBS pH 7.2. I usually test these two coating variants and most of the time I get similar results when using the coated plates for capturing an antigen. The theory is that the coating pH should be approximately 1 pH unit above the isolectric point of the protein that you use for coating. The pH of antibodies are usually around 6-7 with a rather broad range. Thus pH 9.5 is rather good but PBS also seem to work OK at most cases. The emptying of the plate and keep the plates in a freezer will work but you could also try to wash the plates and then block them with e.g. 1% bovine serum albumin or 1% dried milk powder under the same conditions as for the coating of the Fab fragments.

The high pH is the garantee the hydrophobic interaction of the protein with the polystyrene. In most cases, neutral pH is working as well. but not that effective.