Hi Johana,

One problem may be that some of your peptides are too short or not hydrophobic enough. We routinely coat microtiter wells with peptides that are 15-25 aa long. Another buffer to try diluting your peptide in is a 0.1 M sodium bicarbonate buffer, pH 8-9. The ELISA plates we typically use are Immulon 2HB or 1B, depending on the assay. Here is a quick reference guide to the differences between the types of plates we use and their properties: http://www.nuncbrand.com/en/frame.aspx?ID=12001 . Not all plates are the same and the type of plate can greatly affect your results. Plate binding properties can also vary from manufacturer to manufacturer. So another factor may be that the high binding plate you are using is contributing to the high background binding with your IgY's.

We've had to test a number of different ELISA plates to get the optimal adsorption of peptide and low background. It would be worthwhile to try out plates with different binding properties.

When coating peptides we usually make a 100 ug/ml solution in sodium bicarb (or PBS for some peptides that we use) and add 50 uL/well to give a final peptide concentration of 5 ug/well. Coating is done overnight at room temperature. One wash with your coating buffer should be sufficient before blocking. We make up a 3% BSA blocking solution in the same coating buffer that the peptide was made in. In between incubation steps, we wash 3x by adding 200 uL/well of TBS or PBS containing 0.1-0.05% Tween-20 (you can adjust the amount of tween to see if it helps with the high background.) Hope some of these suggestions help.

Hi Johana,

One problem may be that some of your peptides are too short or not hydrophobic enough. We routinely coat microtiter wells with peptides that are 15-25 aa long. Another buffer to try diluting your peptide in is a 0.1 M sodium bicarbonate buffer, pH 8-9. The ELISA plates we typically use are Immulon 2HB or 1B, depending on the assay. Here is a quick reference guide to the differences between the types of plates we use and their properties: http://www.nuncbrand.com/en/frame.aspx?ID=12001 . Not all plates are the same and the type of plate can greatly affect your results. Plate binding properties can also vary from manufacturer to manufacturer. So another factor may be that the high binding plate you are using is contributing to the high background binding with your IgY's.

We've had to test a number of different ELISA plates to get the optimal adsorption of peptide and low background. It would be worthwhile to try out plates with different binding properties.

When coating peptides we usually make a 100 ug/ml solution in sodium bicarb (or PBS for some peptides that we use) and add 50 uL/well to give a final peptide concentration of 5 ug/well. Coating is done overnight at room temperature. One wash with your coating buffer should be sufficient before blocking. We make up a 3% BSA blocking solution in the same coating buffer that the peptide was made in. In between incubation steps, we wash 3x by adding 200 uL/well of TBS or PBS containing 0.1-0.05% Tween-20 (you can adjust the amount of tween to see if it helps with the high background.) Hope some of these suggestions help.

Hi Johana

Firstly I suggest you check the type of card you're using ELISA, as there is a big difference in the binding of peptides to board depending on the type and in some cases it is impossible to distinguish between samples and control. Another step to check is the buffer to dilute peptides and coat the plate, I use a Sodium Carbonate buffer pH 9.8. The coated plates can be stored at 5 ° C for 12h and washed with PBS pH 7.4 with 0.5% tween 20. I hope some of the suggestions is helpful.

Hi Johana, when some of your peptides are highly hydrophobic, they may adsorb "flat" to the surface of your plates and be not accessible to the antibody.

Actually, are you sure your anti"serum" contains antibodies against all of your peptides?

One way to overcome the problems associated with short hydrophobic peptides might be coating your plates with eg BSA and then covalently coupling the peptides to the BSA (the chemistry to be applied depends on the sequence of the peptides, i.e. which amino acids are present or not etc. ). Another option could be synthesizing longer peptides with a common strongly hydrophobic part and your sequence).

I suppose that your peptides have free N-termini. Then you also might try to attach a Biotin with a spacer (search for Biotin-X-Osu etc.) and immobilize on a streptavidin coated plate. With peptides containing internal lysines, you might get false negatives, however, in that case.

Quantification versus directly coated antibody is not really accurate (in terms of comparing coated amount (how much really has bound? and is the coated AB really accessible to the secondary as it would be in the sandwich?). However, you may use this setup as a reference. However, you might get more reliable results, when you coat a standard and titrate the standard with a specific antibody. Means, make your standard as close as possible to the samples you want to measure.

Hi Johana,

I concur with all of Wolfgang Schechinger’s comments and suggestions. My only additional thought is not to dwell on trying to produce a standard curve. Directly adsorbed IgY may be a useful positive control and may be able to give you an estimate of the sensitivity of the detection system (in your case very sensitive, probably due to low dilution of the detecting antibody). But it cannot really be used to calibrate the indirect ELISA for several reasons. In addition it is very difficult to reproducibly deliver nanogram amounts of protein in the volumes you are presumably using.

The plates used for ELISA are usually having affinity to protiens / peptides. But, using carbonate - bicarbonate buffer pH 9.6 for coating overnight and blocking buffer with BSA or Skim milk powder will help in overcoming the problems you are facing. As you have mentioned there is no difference between 60 ng and 10 ng signal, certainly there is problem with blocking and hence the conjugate might be binding to plate directly, and giving signal. So, coating, blocking and conjugate buffer also must be prepared with skim milk powder or BSA, i hope will solve your problem.

hi guys, i will tell the , try to buy APTES (amino propyl triethoxy silane), prepare 5 % solution and incubate the plate with APTES for 3 hrs, then wash with PBS, after that coat peptide and block the surface with blocking buffer containing BSA, then tell it's work or not

Hello Everyone,

Thank you very much for your suggestions, I will try changing the coating buffer to sodium carbonate and preparing blocking solution with BSA, since most of you seem to agree on that.

Wolfgang Schechinger: Well, I have been using serum from eggs of 3 different hens against each peptide. Each group of hens was innoculated against a single peptide so there is no way of telling whether I have specific antibodies for all of them (I was hoping to figure that out from the ELISA, but the results don't seem very reliable yet).

Unfortunately, I don't have access to a real control system of peptide/AB for the indirect ELISA, and I am aware that making my standard curve directly with the adsorbed IgYs is not really a control for the assay (as Robert Rubin mentioned), but I'm mostly doing it, as you said, as a positive control, and to check that the assay is working in general. However, the problem with the detection of quantities above 10ng is troubling: Is it possible that it is not only a problem with blocking, but also due to the low dilution of my conjugate AB?

Thanks again to all of you who have joined the discussion.

"no difference between 60 ng and 10 ng signal"

- how do you measure the signal? Do you see no difference from the beginning of color development or do you just get max signal from the MTP reader when you measure after the usual 15mins?

Color development begins very rapidly right after I add the ABTS solution and it reaches a max value after around 10 to 15 minutes. I measure with a plate reader at 415nm wavelength.

Hi Johana,

There are probably two reasons there is no difference between 10 and 60 ng directly adsorbed IgY: 1) difficulty in accurately delivering such small amounts of protein – non-specific adsorption to surfaces will confound this process, and 2) the concentration of anti-IgY detecting Ab you are using is probably very high – the amount of bound peroxidase via 10 ng IgY may be in excess wrt ABTS. If the latter is correct you are probably pushing the assay to be overly sensitive, and I am surprised you don’t have a problem in background binding. [You should stick with gelatin as a blocking agent (without tween) – BSA is not as good in my experience.] Based on this reasoning, the specific antibody being detected with some of the peptides was facilitated by the high assay sensitivity. This implies that either there was not much specific antibody raised in the chickens or there is not much peptide on the plates, even with the positive results. Ultimately, I think you need to embrace the excellent suggestions of Wolfgang Schechinger – bind peptides to the plate indirectly. Also relevant to this sensitivity issue is whether you might get a stronger anti-peptide response by immunizing with peptides conjugated to a macromolecule.

Johana try coating streptavidin first then coat the peptides in 20mm pbs . And use high binding plates from coastar or greneir

Hydrophobic peptides will be bound to polystyrene surfaces mostly without any problems. The can loose sometimes their antigenic properties, if the epitope on the peptide is the same site as the binding site for the MTP polystyrene. To overcome this problem, use longer peptides (> 15 AA).

There is a very easy way to bind even hydrophilic peptides to plastic surfaces is the binding via an "interlayer". Methylated BSA is quite helpful. Coating by standard procedure: 2 µg/mL in a 0.1 mol/L carbonate buffer at 4 °C overnight.

.. and now: wash the plates with PBS (no detergent, no protein!) 3 times and do the second coating with your peptide in the buffer of your choice (concentration 1 - 5µg/mL). The best pH is adjusted to the isoelectric point of your peptide. Then it's will have no charges and will be at the most hydrophobic condition ... and will bind to the mBSA.

Now continue with the normal ELISA procedure. Remove the coating. No blocking, no washing with detergent containing buffer. Just store the plates frozen (upside dwon) until use. Just before use, wash the plates with the washing buffer (0.01 mol/L phosphate buffer pH 7.2, 0.3 mol/L NaCl). Incubation buffer = washing buffer + neutral protein (BSA, hydrolysed gealtine, ...)

Standardization of the antibodies: There will be no internatial standard. So define that one IgY containg solution (where you have enough volume availabel) a t a defined dilution have 100 arbituary units/mL. That's your standard.

Hi Stephan, what exactly is methylated BSA? How is it made? And how stable is the interaction with the peptides one adds? Is there any advantage over using eg streptavidin coated plates and N-terminally biotinylated peptides?

Hi Wolfgang, mBSA is available from SIGMA-Aldrich:

http://www.sigmaaldrich.com/catalog/product/sigma/a1009?lang=en®ion=US

You can use it also for the binding of DNA or RNA in ELISA plates.

The biotinlytation of peptides is also an excellent alternative, but works better in longer peptides (> 15 AA).