I'm working on detecting different fragments of cardiac Troponin I (Not using a kit) in the patients' sera of different cardiac diseases.
I'm using the best antibodies recommended by Hytest (19c7 for capture, and 560, M18, and MF4 for detection, each at a time), papers indicate that those antibodies can detect down to 0.052 ng/mL, but I can't get them to work!!!!
I use the international human troponin standard for my standard curve.
The M18 gives the best reading with the serial dilutions down to 0.3 ng/mL in the best days, but the other two always gave me worse results (like reading only to 1 ng/mL).
Now, we bought new 560 and MF4 which are a lot worse. This could be an affinity issue, but what was published is very different from my results, they hardly detect down to 2.5 ng/mL !!
I usually biotinylate the detection antibodies myself with a biotinylation kit, then test them right after , even before aliquoting, and they worked fine for the first time.
After aliquoting, I directly incubated them in a serial dilution, compared them to my previously used M18, they were fine, BUT as soon as I started incubating my troponin in serial dilutions, they are 100 times weaker less sensitive, tried the whole sandwich experiment (incubating the capture), and the results were even worse.
For some reason, I can't get them to work as I did in the first time. Could they be degrading? I don't think so, as I'm adding azide to 0.1% as the manufacture suggested.
I prepare fresh buffers, didn't get any better.
Increased their concentration to 10 ug/mL, got a bit better but not even close to my previous results (I usually use both the detection and the capture at 2 ug/mL)
To me, I'm using the best components, can't get the expected results (which I got previously)
Can someone help ?
Thanks
Do you suspect that the problem is with biotinylation? In that case you could use HRP conjugated secondary antibody against the detection antibody.
Thanks Mike for your response.
I tested the biotinylation by directly incubating the detection antibodies to the plate. The biotinylation was fine and comparable (even better) to my control.
I'm suspecting the affinity of the antibodies to the epitopes. The papers published about those MF4 and 560 antibodies, indicate that they could detect down to 0.052 ng/mL, and I can maximally get it down to 2.5 ng/mL. So that's basically my question.
Why on earth would I use the best recommended antibodies and standard troponin, and not get the reference results.
Thanks
Zahran,
Some differences in sensitivity of detection within your experiments (batch to batch variation) are not uncommon. But 10-100x difference is not something you should accept. I expect that you have already tested increasing number of washes etc (with detergent containing buffer) - helps reduce the non-specific binding, thereby increasing signal to noise ratio.
As for what someone else published, this is where it gets a little tricky. It could be because your conditions are not optimized well enough - this is one extreme. The other extreme scenario is the published procedure represents the best results they ever got, and readers get the impression that they represent typical results. ;)
If you must have higher sensitivity, there are many ways to achieve it. All of them will require fair bit of testing, as you test by changing one variable at a time, be it concentration of 1º, 2º antibody, number of washes etc. There are also possibilities on the detection end - for example, try a conjugate where you can use chemiluminescence or fluorescence.
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