To coat a plate I use a antigen solution at the concentration of 10ug/ml and I use 100ul/well (mimum protein-binding capacity of 400 ng/cm²).This is the highest concentration than you can use (according to common ELISA protocol).

If you use an insufficient amount of coated antigen you could obtain a very weak signal, but I never had problem with an increase of amount.

I hope I've helped you

To coat a plate I use a antigen solution at the concentration of 10ug/ml and I use 100ul/well (mimum protein-binding capacity of 400 ng/cm²).This is the highest concentration than you can use (according to common ELISA protocol).

If you use an insufficient amount of coated antigen you could obtain a very weak signal, but I never had problem with an increase of amount.

I hope I've helped you

Perhaps if coating with an antibody, too little will allow more random orientation of the antibody, whereas near saturation levels will 'force' antibodies to be adsorbed by their FC region due to limited spacing, thus with optimal Fv presentation?

Nobody has any ideas? Apart from Selena that you will get a lower signal? So there are no adverse effects from coating capture antibody at much less than saturated levels, other than you will capture less target?

elisa plate are made about this standard: "PolySorp® surface treament, high affinity to molecules of a hydrophobic nature

• MediSorp™ surface treament, surface chemistry between PolySorp and MaxiSorp, provides low background signal with samples containing serum

• MaxiSorp™ surface treament, high affinity to molecules with mixed hydrophilic/hydrophobic domains

• MultiSorp™ surface treament, high affinity to hydrophilic molecules"  

So you can't discriminate Fc or Fab region coating.

But if you don't want random coating and the optimal coating concentration , I suggest you to use ELISA plate biotin-pre coated so you have to you a pre-established amount of Ab strep-conjugated  as final coating

We routinely use sub-saturating concentrations of capture antibody without problems. It may or may not decrease the dynamic range, depending on the amount of background noise present. We checkerboard titrate our antibodies according to how sensitive we need the ELISA. You may get variability from too little antibody, but this is unlikely to occur with concentrations even 10-20x less than saturating (i.e.0.5-1ug/mL).  You can use spike/recovery tests to ensure that your ELISA assay is precise and accurate (not variable). Using less antibody may require you to increase incubation periods in order to achieve binding equilibrium. 

Don't worry about low concentrations. The expectable amount of protein which will be bound by 1 cm² is at 100 ng. So when you are working with coating concentrations between 1 to 10 µg/mL you have an excess anyhow ... but you need this excess due to the law of mass action. The binding to polystyrene is realized via hydrophic interactions. They need some time (over night) and slowly molecules (4 °C). Free binding sites will be blocked with the first washing when the buffer contains a detergent (like Tween 20). The hydrophocic tails of the detergent will enter any free hydrophobic binding site at the plate.

The detection limit of the assay depends not very much on the concentration of the solid phase antibody. Once again, it depends on the law of mass action: on the affinity to yourv antigen. 

i have been using 40 ug/ml for coating my antigen. i also tried 10 ug/ml and there was no significant difference between the two concentrations. However, most literature reports 10 ug/ml as an optimum coating concentration. 

If the maximal binding capacity of a plastic surface (of about 100 ng/cm²) is reached, you have the risk, that double layers which are not that much fixed as the protein dirctely at the surface. Only here you have the hydrophobic interactions between the styrene rings with the hydrophobic aminio acids. This is a hig affinit binding whre the Kd is lower than 10Exp-15 L/mol. The interactions between proteins (excpet the one between antigen and abtibody) is of a much lower affinity (Kd 10Exp-5 L/mol). So you are at risk that during the following incubations steps (with detergent and innert proteins) this interaction is broken. 

... and you are wasting the antigen at the solid phase. I very often worked even in commercial assays with coating concentrations of 1 ... 2 µg/mL.

There is one simple criteria for the optimization: The P/N-ratio. Divide the OD of the positive control by the OD of the negative contol. This ratio should be higher than 10. It's a rough estimation of the slope of cour potential curve (with you should calclualte later by the established conventional models from Rodbard etc. 

The manufacturer states, that the maximum bindung capacity of the ELISA-plates I am using is 600ng für 1 cm². Are you, Stephan, saying, that this is mostly advertisement, and not representative for coating with antibodies under routine lab conditions? That 100ng is a much more realistic value?