What are your assay conditions? I have had good success determining the quantity of murine mAbs in "conditioned" supernatant samples using ELISA and a standard curve generated using the appropriate IgG subclass. Is the antibody purified, or is it in serum or culture supernatant? Are you trying to quantify the concentration of a mAb or pAb?

Hey Hanif, is this in serum or is it a monoclonal? I have lots of experience with serum antibody titres, and we're at the same university! Let me know if you think I can help

Hi Greg. thanks for your answer, it is purified monoclonal antibody, my antibody is called g-12 Flightless antibody. i am trying to quantify mab concentration. I have determined that ELISA only picks the concentration difference from 10 ug/ml. but i still cant get a linear standard curve.

Hi Natalie

my antibody is monoclonal, i have tried few optimisation but still cant get a linear standard. Any suggestions would be helpful. 

What are you using to quantitate? Anti-mouse antibodies for coating and detection? Or antigen for coating? In principle these assays should be possible.

Hanif competition ELISA normally give a good inverse relationship between OD and mAb concentration. Put your data in Excell and then use the Excell analysis to draw a trendline.  Check R (given by the program). I have found that most of my competition ELISAs gave me a inversely proportional logarithmic curve.  For DAS I get an area with linearity. However at very high concentrations reaches a plateau and sometimes even get dip/prozone.  Results obtained depends on how indirect ELISA is done. 

You might consider sharing your data so that we can better understand the issue you are experiencing

Agree with Greg.  Also the exact method used ie. coating, detection, conjugate.

Maybe your antibody is not one clone.  I would try a competitive assay.

i have attached the protocol and some results. is there any optimisation that can be done to overcome the saturation of the antibody at higher concentration. 

Hi Hanif, again I mostly perform polyclonal assessments, but I do use HRP and OPD to develop. Here are some pointers to try to see if you get improvement:

It looks as though your peptide coating concentration is 40ug/mL -  I have worked with full proteins, not peptides, but to me this seems like heaps! I would routinely use 2-5ug/mL of purified protein for coating. 10ug/mL would be a good starting point.

I also generally block first without tween, I think it enables more binding to the plate and potentially reduces background. Though as your BSA is so higly concentrated you should still get high binding.

I think your antibody concentration is too high. I usually start at around 100ng/mL for the top of my standard curve, and do 1 in 2 or 1 in 4 dilutions, and this can get maxed out even for the top 2-3 wells. If you start from a much lower concentration you can develop the plate for a little longer (I dont have a set time, I wait for the start of colour to develop in the blank wells, or until all my standard wells show something, and then I stop). If you do closer dilutions your quantitation will be much better. For serum I was routinely diluting the samples 1:50,000 to get a good concenration, using 5ug/mL of coating protein.

I analysed your data and displayed it with a log(x) axis. It looks like you have acheived a linear curve between the concentrations of 10-500 ng/mL (0.01-.5 ug/m). I guess the question is do you need an ELISA that can detect several orders of magnitude difference in concentration? If your samples really could be that different you could always run the ELISA with a standard curve from 1-500 ng/mL and just run a couple dilutions of your samples (say a 1 in 10 and a 1 in 100) as one is likely bound to be in range.

Also I noticed you are reading at 450. With OPD I would always 'stop' the colour change reaction with the addition of acid (50uL 3M HCl per well) and the colour goes orange. Then I would read at 495. I found I got more sensitive detection this way.

I made a graph! Note the log axis on the bottom. You can see the linear relationship here, and you can use nonlinear regression to fit a cuve to the transformed data

Thank you Natalie for taking your time to help me out. I have been running few other assays but still struggling to get linear standard curve. I have tried narrower range of concentrations but couldnt achieve what i was demanding. Here is the ELISA standard curve. 

Most Elisa's curves can only accurately cover a log.  Also I have never seen a linear curve, I always use best fit from excel..  

thank you judith, are you saying that using logarithmic curve is fine ? 

Hi Hanif, I think using a logarithmic curve is standard practice. This will be fine for your methods (estimating antibody concentration) but I wouldn't go over a log or two in concentration for the curve.

So it means your ELISA worked!

Once again thank you Natalie. unfortunately, for my anlytical method i need to generate a reasonable standard curve with high R value. I can use the logarthmic curve to quantify my samples but it would cause problems down the track and would certainly raise questions in my thesis.