weird linearity of dilution ELISA results
Posted 19 May 2011 - 01:24 AM
I'm attempting to optimize an in-house sandwitch ELISA for myeloperoxidase. I've already run out a grid experiment and found the optimal concentration of capture (mouse MAb)-detection (rabbit PAb)-secondary HRP. Then, I did a linearity of dilution experiment and I found out weird results which I think are due to a matrix effect. If I dilute the sample 10, 20, 30, 50, 80, 100 and 150 times I have a decrease in the OD but is not linear! In fact, if I correct the values obtained from the interpolation with the standard curve by the dilution factor I have increasing values of analyte. Also, I found out poor recoveries from the spike and recovery experiment.
Finally, if I dilute the samples with two different buffers, BSA 1% in PBS-Tween20 0.05% or only PBS, I had completely different results. Here are the obtained results not corrected for dilution:
BSA 1% PBST
dilution factor ng/ml interpolated
dilution factor ng/ml interpolated
What I noted is that BSA seems to have a kind of interference, or at least this is what I think.
My standard is diluted in BSA 1% in PBST.
Is there anyone that can help me?
Posted 19 May 2011 - 07:07 AM
Posted 19 May 2011 - 11:23 PM
Posted 20 May 2011 - 05:13 AM
Next you observe poor results with spike and recovery. This could be several things.. the matrix used for spiking, concentration, differences between native and purified ag or specificity of the antibodies.
Posted 23 May 2011 - 06:57 AM
I repeated the dilutions experiment testing also other dilution buffers and I think that I was obbserving the so-called "hook effect". In fact, if I dilute the sample at least 150 times I have linear results. By the way, I also tried other diluents with or without tween 20 (diluting both standards and samples in the same diluent) and I had higher readings for the samples in presence of tween. I think that this is due to a matrix effect but I'm not sure at all...
Posted 23 May 2011 - 10:15 PM
Posted 24 May 2011 - 12:05 AM
Posted 24 May 2011 - 02:49 AM
Suggestions: 1 Use zero calibrator, serum, etc for diluting your samples so you have a uniform matrix between sample and standard/calibrator regardless of the dilution factor.
2. Your assay should include a step where the samples/calibrators are diluted at least 1:5 -1:20 with buffer during the first binding reaction to minimize matrix effects. Include some rabbit/mouse serum or non specific rabbit mouse IgG in the assay buffer and conjugate diluent. This addition will help in blocking any heterophile ab in the samples. You can test for this in your sample by diluting samples (and calibrators as controls) and pre-incubating them for 15 min 37C to remove any heterophile activity. You should see no change with the calibrator but may see a significant change with the sample.
3. Insure that your samples are within the linear range of your dose response curve...test them by a reference method before analysis in your system.
There are companies that sell stripped serum/plasma for calibrators/controls and also heterophile blocking agents.
Posted 24 May 2011 - 04:29 AM
Posted 25 May 2011 - 03:04 AM
An alternate step is to pre-mix samples with the assay buffer (containing the nonspecific IgGs blocking reagents) and pre-incubate for 10-15 minutes BEFORE placing the samples in your reaction.
More information on heterophile antibody reactions can be found on-line. Scantibodies, Omega Biologicals, and several companies make proprietary blocking reagents.
In summary you need to:
1. have at least 1:2 dilution step with sample using assay buffer
2. include heterophile blocking agentS (non specific IgGs maybe also some animal serum proteins)in your assay buffer
3. Include the same blocking agent(s) in your conjugate diluent
4. (if needed) preincubate samples with blocking agents
5. use your 0 calibrator as your matrix for doing serial dilutions (the calibrator matrix should be the SAME or EQUIVALENT to your samples that you analyze)