Overview

  • Product name

    Human Cardiotrophin 1 ELISA Kit (CTF1)
    See all Cardiotrophin 1 kits
  • Detection method

    Colorimetric
  • Sample type

    Cell culture supernatant, Plasma
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    < 20 pg/ml
  • Range

    16.46 pg/ml - 12000 pg/ml
  • Recovery

    100 %

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 98.2 76% - 112%
    Plasma 101.2 70% - 134%

  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Human
  • Product overview

    Abcam’s Cardiotrophin 1 (CTF1) Human ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human Cardiotrophin 1 in plasma and cell culture supernatants. (Human Cardiotrophin 1 concentration is pretty low in normal plasma, it may not be detected in this assay).


    This assay employs an antibody specific for Human Cardiotrophin 1 coated on a 96-well plate. Standards and samples are pipetted into the wells and Cardiotrophin 1 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Human Cardiotrophin 1 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Cardiotrophin 1 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.


    We have not been able to detect the endogenous Human Cardiotrophin 1 in normal serum with ab99996, only in serum spiked with Human Cardiotrophin 1.

  • Notes

    Optimization may be required with urine samples.

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab99996 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

Images

  • Representative Standard Curve using ab99996.

  • Standard curve with background signal subtracted (duplicates; +/- SD).

Protocols

References

ab99996 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

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.

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Answer

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

Answer

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.

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