SimpleStep ELISA® assays: accuracy, reproducibility, and speed for COVID-19 research
Related
Abcam’s SimpleStep ELISA assays for key targets in COVID-19 have been extensively validated to support you in coronavirus research by ensuring quick and reproducible results.
Fast, reproducible results are essential to all aspects of coronavirus research. Abcam’s broad range of SimpleStep ELISAs offers validated assays for key targets in COVID-19, including receptors, cytokines, cell signaling proteins, and adherence proteins.
In this article, we will use data from our SimpleStep ELISA kits for key SARS-CoV-2 targets, such as IL1-Ra, ACE-2, and D-dimer, to demonstrate how we ensure the performance of our assays to support you in rapidly producing reproducible results for critical research areas.
If you are interested in antibodies, proteins, and kits specific for the detection and analysis of human coronaviruses, check out our range of products for SARS-CoV-2 research.
Overview
How can SimpleStep ELISA accelerate your research?
Most ELISA kits have a sandwich assay format, where two antibodies (capture, detector) are used to measure a specific target protein in complex biological samples, such as human sera or cell culture extract. Conventional ELISA assays involve 6 steps and usually take 4 – 6 hours, although many recommend an overnight capture incubation.
The SimpleStep ELISA follows a similar methodology but only involves three steps and a single wash, condensing the process to less than 90 minutes.
If you want to learn more about SimpleStep ELISA and its benefits, check out this interview with our experts.
How do we validate our SimpleStep ELISA kits to ensure optimal performance and reproducibility?
Our assays undergo extensive validation to ensure they offer the best possible assay characteristics with excellent batch-to-batch reproducibility. Here we’ll outline our validation process and how this is built to support your reproducible research, using three SimpleStep ELISAs that target known components in SARS-CoV-2 research:
- IL-1 receptor a (IL1-Rα), a cytokine receptor that is elevated in severe SARS-CoV-2 cases
- ACE-2, a receptor that allows viral cell entry
- D-dimer, a coagulation marker that is elevated in severe SARS-CoV-2 cases
Sensitivity, dynamic range, and precision: IL1-Ra
In this section, we’ll explain how we validate the sensitivity, dynamic range, and precision of our SimpleStep ELISA kits, using the example IL1-Ra.
Screening for sensitivity in SimpleStep ELISA
IL1-Rα is a cytokine receptor involved in the acute phase of inflammation and has been shown to be elevated as part of the cytokine storm response seen in severe cases of COVID-19. As IL1-Rα is normally found at low levels in crude samples, high assay sensitivity is essential for accurate quantification. Assay sensitivity (or limit of detection) is the minimum detectable level of an analyte. Our human IL1-Rα SimpleStep ELISA reports a sensitivity of 4 pg/ml (Figure 1). For comparison, IL-1Ra serum levels in healthy individuals range between 100–400 pg/ml1.
The development of each SimpleStep ELISA kit begins by screening target-specific panels of Abcam’s recombinant RabMAb® rabbit monoclonals to identify a sensitive antibody pair. All of the kit reagents are then optimized, including the blocking solution, antibody concentrations, and the tetramethylbenzidine (TMB) development solution exposure time, to generate the best possible assay. If we can’t meet the required sensitivity for the analyte, we go back to the drawing board and start over with antibody design and production.
Delivering across a dynamic range
Each kit is provided with a protein standard to determine the assay dynamic range and to quantitate the target concentration. Our standard proteins are highly purified, and where required are mammalian-expressed to ensure they accurately mimic the native biological signal.
An assay’s dynamic range extends from the lowest to the highest concentration of the target protein that can be reliably quantified. We optimize our assays to provide the broadest dynamic range in a variety of matrices. Thus, the human IL1-Rα SimpleStep ELISA shows a range between 7.8 and 500 pg/ml (Figure 1).
Assay precision for reproducible results
The reproducibility, or precision, of each SimpleStep ELISA is defined by the percentage of the coefficient of variation (CV) within the standard curve replicates and reports both intra-assay CV and inter-assay CV.
Intra-assay CV is based on the reproducibility of each point on the protein standard curve at a single experimental time point, while an inter-assay CV compares the reproducibility in signal across multiple experiments and time points. Industry standards require assay CVs of less than 20% for bioanalytical assays like ELISA. All our kits are developed to offer intra-assay and inter-assay CVs of less than 10%.
These values are important because the assay’s precision reflects its performance in the hands of the user and ensures the consistency of your results over time, so that your results are relevant and can support the transition of research to the clinic for any new coronavirus discoveries you may make. As shown in Figure 1, the IL1-Rα SimpleStep ELISA kit (ab211650) has excellent reproducibility.
Figure 1. IL1-Rα SimpleStep ELISA kit (ab211650) shows excellent sensitivity, dynamic range, and precision. The limit of detection is shown in red, and the mean background signal is shown in black.
Linearity of dilution and spike recovery: ACE-2
SimpleStep ELISAs are validated for multiple sample types, including serum, plasma, cell culture supernatant, cell and tissue extracts, milk, saliva, and cerebral spinal fluid.
As research is constantly moving forward, some assays require further validation, for example, our ACE-2 SimpleStep ELISA kit (ab235649). When we first developed the ACE-2 kit, we weren’t looking at its role in coronavirus infection, but rather its normal role in the maintenance of blood pressure. When it was discovered that ACE-2 is critical for viral entry of SARS-CoV-2 to host cells, we further validated the kit for more relevant tissue types, including testes, intestine, heart, lung, and urine.
Linearity of dilution for predictable protein quantification
An assay’s linearity of dilution determines the predictability of accurately quantifying a target protein within a sample and indicates if something in the sample matrix may be interfering with the target detection. We validate for multiple points of linearity, so you can be confident that you are getting solid, quantifiable data.
When an assay shows good linearity of dilution you can be confident in the quantification of your sample across multiple dilutions, allowing you to accurately quantify low volume.
Sensitive spike recovery in multiple matrices
Spike recovery is also an important test and related to linearity of dilution. Spike recovery determines the matrix effect on the detection of the assay analyte. Increased signal-to-noise from non-specific interactions with matrix components could result in either an over- or underestimation of the actual target concentration, and introduce an error in your research.
All calculated recovery and linearity of dilution data for our SimpleStep ELISAs conforms to industry standards and is within 20% of the analyte concentration. If the recovery or the linearity of dilution needs to be corrected for a given sample matrix, we include a specifically formulated sample diluent in each kit. Our newly validated sample matrices for ACE-2 SimpleStep ELISA all show excellent analyte recovery and linearity as shown in Figure 2, ensuring accurate results for your coronavirus research, even at very high dilutions.
Figure 2. Linearity of dilution and spike recovery tests for the ACE-2 SimpleStep ELISA kit (ab235649) demonstrate highly reproducible and sensitive results.
Cross-reactivity with homologous targets: D-dimer
If your target of interest has high homology with other proteins, we test for the possibility of cross-reaction, as in the case with D-dimer. D-dimer is a breakdown product of fibrinogen, found in the blood during the clotting response. Patients with severe COVID-19 have been shown to have higher D-dimer levels than patients with moderate disease.
As D-dimer is a breakdown product of fibrinogen, some antibody pairs may show cross-reactivity with the parent protein, fibrinogen. We found very low cross-reactivity levels of our D-dimer SimpleStep ELISA kit (ab260076) with fibrinogen, ensuring your assay signal is specific for D-dimer.
Another potential risk is the interference of other components with your assay signal. Protein binding partners, like ligands, receptors, and co-factors, can alter the measurable concentration of the analyte or alter antibody binding, which can result in an inaccurate readout. We test for interference during assay development by spiking known concentrations of potentially interfering molecules with a known concentration of our analyte. We then quantitate the analyte with and without the spiked protein. For example, our human D-dimer SimpleStep ELISA was tested for interference with TPA and other binding partners.
In addition to intraspecies homologs, some targets have high homology with proteins in other animal species. If a kit is validated for use with a serum matrix, we automatically test for cross-reactivity with human, mice, rat, and bovine serum. Some of our kits have been tested with additional species, like primates.
The data shown in Figure 3 demonstrate that the human D-dimer SimpleStep ELISA kit is specific for the human protein and shows no cross-reactivity with other species, allowing you to confidently perform your COVID-19 research in a range of samples and models.
Figure 3. Validation of the lack of cross-reactivity of the human D-dimer SimpleStep ELISA kit (ab260076) with homologous targets.
Additional validation parameters: IL1-Ra and D-dimer
SimpleStep ELISAs are calibrated against a known NIBSC WHO international standard and include a conversion factor. Calibration to an international control enables cross-comparison between data sets. This allows a comparison of kits from different sources and creates a gold standard benchmark.
The majority of our SimpleStep ELISA development and validation is performed using biological samples from pool donors, N = 50. However, we use multiple individual donor samples when validating kits for use with serum matrices to ensure broad coverage of sample backgrounds. Healthy individual donors are tested to demonstrate kit performance within the normal target analyte range, and, when possible, we compare performance in clinical samples of both the healthy and diseased states for relevant conditions. Figure 4 shows the wide biological range of human D-dimer concentrations between different donors.
Figure 4. SimpleStep ELISA validation for human D-dimer in multiple human serum matrix and serum from healthy and diseased patients (left), and for human IL1-Ra across multiple assays and sample types (right side).
Our SimpleStep ELISA kits use the same RabMAb recombinant antibodies as in our matched pairs and FirePlex multiplex products to offer consistency as your research progresses and to allow you to readily transition your research between platforms. Recombinant monoclonal antibodies offer batch-to-batch consistency and can be repeatedly manufactured to a consistent standard. The use of recombinant antibodies in all our SimpleStep ELISAs ensures reproducible assay performance regardless of the scale of your project.
Comparison of the RabMAb pair to IL-1Ra in SimpleStep ELISA, standard ELISA, and FirePlex (Figure 4, upper right) shows that the assay standard curves exhibit comparable dynamic ranges, albeit broader for the FirePlex multiplex assay, and that the determined concentrations are approximately equal across platforms.
These are just a few examples that demonstrate the extent to which we validate our SimpleStep ELISA kits. From selecting sensitive antibody pairs and optimizing all reagents, to developing assays that more than meet the industry standards and assessing in multiple relevant matrices, we ensure they perform as expected the first time and every time.
SimpleStep ELISA for coronavirus research
Abcam has a broad range of SimpleStep ELISAs that could support research into coronaviruses, including receptors, cytokines, cell signaling proteins, and adherence proteins.
Figure 5. Abcam’s range of coronavirus relevant SimpleStep ELISA kits.
References:
- Meier, C.A., et al. IL-1 receptor antagonist serum levels are increased in human obesity: a possible link to the resistance to leptin? J. Clin. Endocrinol. Metab 87, 184–1188 (2002).