人Cardiotrophin 1 ELISA试剂盒(CTF1) (ab99996)

Thank you for contacting us. Sorry to reply to in English but my Spanish colleague is currently out of the office and my Spanish is unfortunately not good enough.

The source of theCardiotrophin 1 Human ELISA Kit (ab99996)informed meit isvery accommodating to many different samples types so homogenates will most likely work.
If you decide to test this kit with homogenate samples, we recommend diluting the samples at least 5-fold with Assay Diluent B to minimize any effects of the detergents in the lysis buffer.
The samples may need to be diluted further but this would need to be determined by the experimenter empirically.

So for the original lysate, in general they would want to shoot for at least 1 mg/ml protein, preferably more, to achieve 50-500 ug/ml after dilution. Again though, this range should just be used as a starting point and the optimal concentration would need to be determined empirically.
We do not sellthe Lysis buffer separately.Ifyou would rather prepare your own,please find belowsome basic tips for sample preparation.
In brief, a lysis buffer must meet the following specifications:
A) has relatively low salt content (700 mM or less)
B) does not contain sodium azide
C) does not contain >0.1% SDS
D) does not contain >10 mM reducing agents (beta-mercaptoethanol or dithiothreitol)
This would include any buffers used for immunoprecipitations, including RIPA buffer.

Regarding the cross-reactivity with Mouse and Rat:

Cross reactivity tests for this kit have not been performed for mouse and rat; however, we do know there is 88% sequence homology between human and rat and 80% between human and mouse. The capture antibody is monoclonal and the detection antibody is polyclonal.

I have been informed that a mouse ELISA kit for CT1 is now available. Please let me know if you are interested in this kit and we will add it to our catalog.

I hope this is helpful.

Thank you for your inquiry. After reviewing your standard data, I must say the curve actually looks good (see linear and log-log plots in attachments).  As you can see, the signal strength is strong, the linearity is very nice (≥0.98), the duplicates are close, and the background is low.  There is a little saturation from the highest standard but it doesn’t seem to be affecting the linearity of the curve significantly (however this point can simply be eliminated if desired).   It sounds like you may be concerned about the standards at lower end of the curve, specifically 0 pg/ml, 16.46 pg/ml and 49.28 pg/ml standards.  In regards to the 0 and 16.46 standards, it is not surprising to see these two have similar signals as they are both below the sensitivity limit which is 20 pg/ml (see pg 11 of the manual, https://www.abcam.com/ps/products/99/ab99996/documents/Cardiotrophin-1-Human-ELISA-Kit-ab99996-protocol-plain.pdf)). As for the 49.28 pg/ml standard, the OD is around twice that of the background so I see no reason why sample ODs could not be measured near this point on the curve.  I’m not exactly sure what parameters your ELISA reader software sets at “ Read More

Thank you very much for your patience. I've finally heard back from the senior scientist, and he has the following response (I've placed it in quotes): "To be honest, I really don’t see an issue with the standard curve as it is giving strong signal responses, low duplicate CVs, nice linearity (R2 ≥ 0.98) and low background. I’ve plotted it both in a log-log and linear regression model for your reference (see attachment). Since the standard and detection components seem to be working properly, we therefore have to determine what is it about these particular samples that is causing them to read so low. The most likely explanations are either the target protein levels are simply too low in the samples, or something in the samples is causing interference, inhibiting accurate measurements. Because CT-1 is a low-abundant protein in serum samples, it would not be surprising if the reason was the former. This makes sense as looking at the serial dilution ODs of those three samples (2668, 4221, 4236), they are all below the background, even when using a dilution which decreases the chances that there is some type of interference, ie matrix effects. FYI, our recommended serum dilution for this kit is 2-fold. There are a few things the customer can try to increase their chances of getting the samples within the detectable range of the assay and I have listed those below. - Incubate sample overnight at 4 degrees C - Increase amount of biotinylated ab (by 1.5 fold or so – too much may increase background) - Incubate secondary ab overnight at 4 degrees C - Increase amount of HRP-strep (by about 1.5 fold or so – too much may increase background) - Concentrate sample (for example, using a chilled spin column) Please note: it’s best to try just one of these modifications at a time. Implementing too many of these changes at once may cause high background. If all else fails, they may want to consider using a different sample type, such as plasma or another body fluid. Ultimately they may have to report that the CT-1 concentrations in these samples are below the detection limit, which is 20 pg/ml." I hope this helps. Please let me know if you have any other questions. Thank you.