I hope the attachment may help you 

I think the following article may help you to increase the sensitivy of my ELISA.

I would repeatedly recommend to use an HPLC-photometric method (please see file; Insulin RP-HPLC).   Only the HPLC method uniquely has a nice calibration line inserting to the zero point, although the high-recovery analysis is the important issue in the HPLC analysis (please see file; Lysozyme by RP-HPLC and HPLC-Surfactant-SEC BIN).   This SEC method using NP-40 (nonyl-phenyl) is not giving sufficient recoveries to alpha-Lactalbumin and ovalbumin giving bent calibration curves like ELISA.   Therefore, we must have developed a satisfying SEC method using Brij-58 (cetyl/palmityl/1-hexadecanyl-non-ionic detergent) to apply onto hydrophobic membrane proteins (please see file; SEC column 300 A silica).     

On the other hand, ELISA method has a nororious bent caribration curve, which is not beautiful at all.   Further, postsynaptic density (PSD) has been found by using non-quantitative electron microscopy (EM).

By the way, Shank protein is not found in my liver specimens and Hc by using PDMD method (please see file; HepG2 fucoidan). 

Hepatoma HepG2 (without fucoidan) has Ankyrin repeat and fibronectin type-III domain-containing protein 1 at 2.0, Ankyrin repeat domain-containing protein 15/KN motif and ankyrin repeat domain-containing protein 1 at 0.83, Ankyrin repeat domain-containing protein 50 at 5.0, and Ankyrin-2/Brain ankyrin at 0.7 μg/mg of cell protein, respectively.

Healed HepG2 (with fucoidan) has Ankyrin repeat and fibronectin type-III domain-containing protein 1 at 1.7, Ankyrin repeat domain-containing protein 38/KN motif and ankyrin repeat domain-containing protein 4 at 2.5, Ankyrin-2/Brain ankyrin at 1.5, Ankyrin-3/ANK-3 at 1.4 μg/mg of cell protein, respectively.

LC tissue (No.6) has Ankyrin-R/Erythrocyte ankyrin at 6.9 μg/mg of tissue protein and HCC tissue (No.6) has it at 1.4 μg/mg of tissue protein.

Hepatoma HepG2 (without fucoidan) has PDZ domain-containing protein 2/Activated in prostate cancer protein at 2.2 μg/mg of tissue protein.

Thus, Shank protein is not linked to liver cancer.

Thanks for your answer, I am trying to detect Shank proteins in my ELISA, not the entire PSD.

I appreciate your help.

The sensitivity of sandwich-type immunoassays can be improved by several means:

1. More sensitive label (e.g. use luminol-based high-sensitivity substrates with chemiluminescence detection)

2. Longer incubation of the sample (up to 48 hrs) with shaking

3. Increase sample volume if possible

4. Use antibodies with higher affinity (particularly the capture antibody is critical).

5. Do not use samples with extreme matrix concentrations, since high viscosity will slow down the binding of the analyte. Better dilute (at least 1:2) with a suitable sample buffer. Calibration and samples should be similar in composition.

 I am a little bit confused that you want to develop an ELISA for PSD, but you are using a recombinant GFP protein as a calibration substance.

Good morning, thanks for your answer. I am trying to develop an ELISA for Shank. The protein that I use to make the standard curve is a recombinant shank-gfp protein. With my ELISA setting I am able to detect Shank in PSD samples, but not in cortex for example, and I need to increase the sensitivity of the assay, because with my standard curve I am able to detect different concentration of my shank-gfp protein but in the nanogram/ml order, not picogram/mL.

Thanks for yur time and invaluable help!!

Hello,

I'm not sure to understand the problem. Is it really a sensitivity issue or rather a specificity problem?

What exactly is the antigen of the primary antibody you use? 

Is your recombinant PSD immunologicaly identical to the native PSD found in the cortex? How is performed your PSD preparation? Are you sure that it preserves the tertiary structure of the PSD?

ng/mL is a good sensitivity in ELISA.

You did well.

CB-P

Thanks Catherine,

One of our home made antibody recognise two different fragments of the c terminal region of Shank3 and the other the hole c-term. They function very well in combination and they don´t recognizes the others isoforms of Shank by ELISA and also western blot.

We use a shank3-gfp protein as a control for the standard curve. When we use PSD fractions to confirm if we are able to detect shank, we do. But with CTX samples where shank is more diluted than in the PSD, we don´t detect shank.

We suspect that we have sensitivity problems, because we are able to build this standars curve but in the order of nanogr/ml and we need to improve our sensitive in the assay.

I don't know if our PSD fractionantion protocol preserves the structure of the PSD. Do you know how I can check it??

Best,

Carolina

Hi Carolina,

you should try ELAST Form Perkin Elmer. It is an enhancer for ELISAs. Your secondary antibody has to be HRP attached. After the last step of your ELISA don't develop directly, just add a two-step procedure that takes maximum 1hr more. I was looking for antibodies against angiogenic proteins and had problems with high background and modified my ELISA (every single step was modified at the end). I had to dilute the serum to reduce the background but without losing the signal. In my case I increased the sensitivity by 20x (see link). I was even able to see antibodies in some samples when I diluted the serum by 100x (not published).

http://clincancerres.aacrjournals.org/content/early/2015/01/29/1078-0432.CCR-14-1956

Hello Carolina,

I agree with Matthias. You can use an AMDEX secondary antibody which is bound to ten Peroxidase molecules. It improves the signal.

However, if you have different Shank protein preparations you can also use them each in turn to coat the wells of you plates and use them as fixed competitors for the free protein you want to assay. If your antibody exhibit a better affinity for the protein of your samples than for the one coated then you can get a great sensitivity.

To quickly check for the structure of your protein preparations you can use either bi-dimentional electrophoresis or PAGE in non denaturating conditions. Otherwise there are other techniques like Maldi or MS which can be difficult to set up.

Best regards.

CB-P

This a little off topic but something to think about, but for the ELISA for lyme one doctor is collecting the first urine sample after a deep massage and that seems to be improving sensitivity.  There is some belief that lyme hides in biofilm communities in the body and is difficult to detect and they just breaks it up.