You need to have some idea about the concentration of your analyt in the sample. Than you need to know the lower detection limit. Choose the dilution in a way that you are at least 2 - 5 times above the detection limit.

Now some tricks. You need to have some dilution with a sample buffer. Urine is a very variable body fluid. Especially the pH and the salt concentrations can disturb your immune reaction. So it's could be helpful to switch to an sample buffer with an higher buffering capacity. Normally 0.01 mol/L phosphate buffer with 0.15 mol/L NaCl is used. Switch to 0.15 mol/L Tris HCl same pH 7.2.

Very important is the affinity of your antibody. This determines the lower detection limit. Therefore there is not that much margin to vary the assay conditions.

Prolongation of incubation times is sometimes helpful. Avoid temperature gradients over the ELISA plate (make sure that all ingredients are at room temperature. Adding of 2% w/V PEG (Polyethylene glycol) can help also. Prolongation of the substrate reaction as well. And at least, you can switch to a kinetic measurement.

Any experiments to increase the concentration preanalytically will increase the variation coefficient (the imprecisicion) of the assay. Thry to avoid this until you haven't checked all tricks in the ELISA.

Kadryn,

I’m using commercially available immunoassay for quantitative measurement of L-FABP.

Plate is pre-coated with antibody specific to L-FABP. I’m adding urine samples of unknown concentration. To this I’m adding biotin-conjugated antibody specific to L-FABP, next avidin conjugated to HRP, and next TMB substrate. I stop the reaction with sulfuric acid.

My problem is that obtained OD of some samples is very low around 0.18 while blank is 0.17, and in that case the software that I use is not able to calculate the concentration of unknowns. I was wondering if I may fix this problem somehow..

Hi Hanna; Have you looked at normal urine samples? Is your L-FABP present at detectable levels? You might try spiking a normal urine with L-FABP as Sofia suggested. It is possible that the urine samples are inhibiting the ELISA. Check the ph. Is this human urine?

I would try one of 2 things.....

I would initially try incubating the urine on the plate with only the capture agent before binding the biotinylated secondary antibody (it sounds like you're incubating them together?). You can also adjust the incubation time and/or enhance it by putting the plate at 37 deg.

Do you use a blocking step? If so, maybe eliminate it or change the concentration of the blocking solution. This might increase your background, but if it increases your signal, then you might have a greater net difference.

Is there any data on the expected concentration of this protein in urine samples? This all may be a moot point if the expected concentration is only 10% of your lowest standard, but you might be able to weasel out the data if it is 50% of your lowest standard.

Hi Hanna,

There is one thing that is called control dilution. What you have to do is add a known concentration of your standard to each of your samples before running the ELISA. When you get the result you have to standardize your data for that amount/concentration of standard that you added. Hope this helps.

Hello, Hanna. Have you considered centrifugal concentration devices like the Amicon Ultra centrifugal filters? You can concentrate all your proteins that are above 3 kDa or 10 kDa, depending upon which filter you use. I've used these before, and they work pretty well, concentrating large molecules without concentrating salts. You'll need to pellet out any cells that have are in the urine before loading the device. I've never concentrated urine proteins before, so I can't predict how much you can concentrate your samples before precipitation problems occur. It might be worth a try, though.

Hanna:

1) I agree with Charles Hammond; use an Amicon or similar device over a Speed-vac. In my experience with speed-vacs, the samples often heated up (because of a leak in the sea, perhaps) and the protein may have been denatured. I recommend using the Minicon® Clinical Sample Concentrator (see attachment). If you use Amicon centrifuge concentrators, I recommend doping the membrane first with a blocking agent and a quick spin.

2) How do you use blocking agent? I recommend using BLOCK ACE if you can get it. it is purified milk protein, and seems to do better than 1% (w/v) BSA or skim milk. We dilute the detector Ab and the secondary Ab or streptavidin-HRP in _0.5% (w/v)_ BLOCK ACE, _then_ block with 5% (w/v) BLOCK ACE. The run takes longer, but we seem to get better results. When we diluted Streptavidin-HRP-conjugated 2° Ab for one plate in 0.5% and for the other plate in 5%, we lost sensitivity but not specificity with the 5% BLOCK ACE.

3) If you must use a biotin-streptavidin system, try doubling the concentration of the streptavidin-HRP given in the protocol. We improved our signal thus.

4) If all else fail, try doing an ECL assay. I realize that may require purchase of a different reader.

Good luck!

Wales

Hi Hanna

you could use PEG to concentrate your sample, put the sample in the dialyse bags and then put it in a plate full of PEG till the volume of your sample become half of what it was.

Charles Hammond's suggestion as well as those of Wales Nemotollahi appear to me be more practical.You may please use the membranes having molecular cut off depending upon size of the desired protein,then elute and concentrate by molecular speed vac and go for ELISA,also try egg albumin in blocking agent. A.M.Jana

did you consider the IMMUNO-PCR option?in these way you can couple the classic elisa with a pcr and increasing the sensitivity of your test.

Good morning. I had a similar issue and like the reseacher below changed the blocking agent. In my case I used a 4% albumin in 0.85% saline which completely eliminated the background.

additional option for precipitation (assuming that your sample is purified and does not contain non relevant proteins) is using Ammonium Sulphate. Good Luck

You need to have some idea about the concentration of your analyt in the sample. Than you need to know the lower detection limit. Choose the dilution in a way that you are at least 2 - 5 times above the detection limit.

Now some tricks. You need to have some dilution with a sample buffer. Urine is a very variable body fluid. Especially the pH and the salt concentrations can disturb your immune reaction. So it's could be helpful to switch to an sample buffer with an higher buffering capacity. Normally 0.01 mol/L phosphate buffer with 0.15 mol/L NaCl is used. Switch to 0.15 mol/L Tris HCl same pH 7.2.

Very important is the affinity of your antibody. This determines the lower detection limit. Therefore there is not that much margin to vary the assay conditions.

Prolongation of incubation times is sometimes helpful. Avoid temperature gradients over the ELISA plate (make sure that all ingredients are at room temperature. Adding of 2% w/V PEG (Polyethylene glycol) can help also. Prolongation of the substrate reaction as well. And at least, you can switch to a kinetic measurement.

Any experiments to increase the concentration preanalytically will increase the variation coefficient (the imprecisicion) of the assay. Thry to avoid this until you haven't checked all tricks in the ELISA.

While I like both Tamar's and Stephan's suggestions, I have small disagreements with them. Both ammonium sulfate and PEG are used to "salt-out" proteins from solution, usually by flocculation. However, some proteins become irreversibly denatured by use of such agents. My experience with PEG was disastrous when I tried to purify a plant cell wall protein with it (I got something like glue), but every protein is different. Also, a given protein will not salt out below an certain concentration of either one; that concentration must be found empirically. Of course, if one succeeds, the patients benefit and, secondarily, the results are publishable.

Stephan, I am curious about the mechanism of the effect of PEG in an ELISA. How does the PEG improve the results of the ELISA? Also, while the imprecision of gthe test is increased by preanalytical concentration, sometimes that is the price one must pay simply to detect measurable levels. The device I recommended has been in use in clinical biochemistry laboratories in the US at least since the late 1980s., especially with more dilute body fluids like CSF.

Tamar and Stephan, I would appreciate any information you two could provide the rest of us on your successes.

Thank you,

Wales

Thank you Wales.

I used Ammonium Sulphate for precipitating flagella, a 56KD protein from Salmonella and E coli, indeed, a different % is suitable for each protein, this will have to be experimentally determined. In the initial Q, keeping the functionality was not required so this may not be relevant for Hanna.

Thank you for your observation, Tamar. I could have made myself clearer. When I stated "irreversibly denatured", I was thinking about antigenicity as well as native functionality. Some precipitates lose antigenicity as well.

Hi Hanna

This is Vidhan. Well i think you are working with urine sample (is it? ). Your target is any fatty acid binding protein? (please correct me if i am wrong). Concentration technique should work along with reducing background (please share your OD of blank). But i have new suggestion of using rolling circle amplification technology where you use third antibody that bound to biotin tag with a small DNA molecule that gets amplified when incubated with a polymerase and biotin dNTP. please see attached literature.

thanks