5 key criteria for selecting the most appropriate ELISA kit to detect Human Cardiotrophin-1

Multipette and 96-well plate

Cardiotrophin-1 Factor

Cardiotrophin-1 (CT-1) is a cardiac hypertrophic factor with cardiac myocyte protective properties. CT-1 belongs to the interleukin-6 cytokine family and acts through LIF receptor ß/glycoprotein 130 (gp130)-coupled signaling pathways.

CT-1 intracellular signaling pathways enlist kinases (ERK, MAP, JAK) but also STAT and PI3-kinase/Akt systems.

Cardiac CT-1 expression is increased by hypoxia, where it protects cardiac myocytes from ischemic injury and apoptosis.

Research teams involved in cardiovascular studies regularly need to precisely quantify CT-1 levels in the sera and plasma of patients. Recent studies have shown that CT-1 may serve as a biomarker of left ventricular hypertrophy and dysfunction, myocardial fibrosis in hypersensitive patients with heart failures.

Clinicians and Life scientists are more and more looking for reliable tools to measure and monitor CT-1 levels in samples from cohorts of patients with heart failures. In addition, such tools might also be easy to use and convenient for those running the assays at the bench.

ELISA (Enzyme-Linked Immunosorbent Assay) kits are ideal for CT-1 measurements in heart-related biomarker discovery. ELISAs are in vitro immuno-assays for the quantitative and specific measurement of a biomarker in the sample of patients and culture supernatants.

Enzyme-Linked ImmunoSorbent Assays (ELISA) workflow for the quantitative measurement of biomarkers

5 criteria may be taken into consideration for selecting the most appropriate ELISA kit to detect Human Cardiotrophin-1 (CT-1) during cardiovascular and heart failure studies.

Tip #1 – The specificity of the assay.

Even if it may seem obvious, the antibodies used in the immuno-assays must be specific for Human CT-1 (Cardiotrophin-1). This also allows the user to know which species are detected by the assay.

Certain ready-to-use ELISAs are specific only for Human CT-1. Other assays are compatible only for Mouse CT-1.

Tip #2 – The absence of cross-reactivity with other related-cytokines.

Make sure that what you measure is effectively related to Human CT-1.

The antibodies employed might not cross-react with related secreted cytokines like the human Angiogenin, BDNF, BLC, ENA-78, FGF- 4, ILs, IP-10, G-CSF, GM-CSF, IFN-gamma, Leptin, MCP-1, MCP-3, MDC, MIP-1alpha, MIP-1 beta, MIP-1, MMPs, PARC, RANTES, SCF, TARC, TGF-beta, TIMP-1, TIMP-2, TNF, TPO or VEGF by example.

With this is mind, you are sure that the signal you get is really proportional to levels of CT-1 detected (colorimetric read-out for ELISA).

Tip #3 – The sensitivity and robustness of the assay.

Be it for clinical studies, personalized medicine or translational research purposes, the immunoassay used should ensure the detection of the analyte at low levels. As far as the Human CT-1 is concerned, the detection limit is now 20 pg/ml; detection range being from 20 pg/ml to 12 000 CT-1 pg/ml for the recently released Raybiotech Human CT-1 ELISA. This information is of interest for end-users as it helps to define the dilution factor of the sera (plasma) when running the assay (very often, the recommended dilution is 2 fold).

Whatever the end-user and the laboratory where the assays are run, you need to trust the results obtained. Select your immuno-assays with low variation of results (also called coefficients of variation or CV). Two types of CVs are described for ELISAs. To assess the variation of an ELISA within a data set obtained from one experiment, the term used is “Intra-assay” CV. For data obtained from repeated experiments, the CV is called Inter-assay CV. A robust ELISA will have CVs as low as possible. Human CT-1 ELISAs now guarantee Intra-assay CV below 10% and Inter-assay CV below 12%.

During clinical and multi-centric studies, the assay can be run by several end-users over a long period of time. To guarantee optimal coherence of the results, you might also look for a reliable source of procurement (sometimes a complete study can last for several years) providing you with QCd and homogeneous production batches.

Tip #4 – The format of the assay.

Usual CT-1 ELISAs are 96-well plate formats. A few CT-1 ELISAs are available with cleavable strip plates. Such a format is quite smart as it makes CT-1 quantification possible through multiple experiments (when samples are collected over a long period of time by example).Human CT-1 standard curve

Highly CT-1 specific antibodies coated on plates is not enough to get a robust ELISA to optimally measure this cytokine. Various additional reagents are needed. All these components should be rigorously selected. This is the case with the CT-1 used as the “standard” of the assay. This standard calibrates the readout of the assay with the concentration of  Human CT-1. A “Standard curve” thus obtained allows you to quantify the Human CT-1 present in the samples of your patients.

One of the reasons of the success of ready-to-use ELISAs is that they come with components optimally selected to easily perform the assay and obtain a reliable data set (recombinant human CT-1, Washing and detection reagents …).

Before running the assay, check the detailed protocols as you might like to prepare in advance the extra materials required for the test and which are not included in the kit (microplate reader capable of measuring absorbance at 450 nm, precision pipettes to deliver 2 µl to 1 ml volumes, absorbent paper, distilled or deionized water, tubes to prepare standard or sample dilutions…).

Tip #5- The type of samples analysed.

ELISAs are now compatible with plasma, cell culture supernatants and urine. Nevertheless,check the compatibility of the assay every time you select a new kit. For “exotic” liquid samples not mentioned in the datasheet, one option is to validate the assay on a small number of the collected samples. This validation step is now well accepted by ELISA providers; some of them even offer a batch reservation process (a period during which you try a plate from a provided lot number, while the complete lot of plates is reserved for you).

Want to access to robust Human CT-1 ELISA kits?

The world of immunoassays is evolving rapidly. As an example, it is now very easy to obtain a cytokine profiling of a sample and compare it to other samples for the same patient or between different patients. New antibody-based assays now even allow precise multiplex ELISA quantification of numerous analytes in one experiment (Q-Plex technology from Quansys Biosciences, Quantibody arrays from RayBiotech…). These new technologies allow researchers to rapidly identify new biomarkers and help clinicians facing new challenges (early patient stratification for personalized medicine, predictive biomarkers in drug discovery, translational research…).

Whichever technology you choose to use and its power, these 5 key points just discussed remain in any case the pillars of a reliable and specific immunoassay.

Robust Human CT-1 ELISA kits are available from RaybioTECH (cat. nr ELH-CT1-1); outsourcing to tebu-bio’s skilled lab scientists being possible upon request.

Written by Philippe Fixe, PhD
Philippe Fixe is a former Marketing Manager at tebu-bio, passionate about innovation and R&D in Life sciences, Biotechnology, Medical research, Drug discovery, and also a keen photographer.