Enzyme-linked immunosorbent assay (ELISA) is considered the gold standard of immunoassays for the high specificity and sensitivity it provides. Although researchers may choose to develop an ELISA in-house, ready-to-use ELISA kits can save time and reduce the risk of errors—provided they have been properly validated. By basing its ELISA validation process on four core performance indicators, HUABIO ensures that its ready-to-use ELISA kits deliver precise and reproducible results that you can trust.
ELISA is a microplate-based method for detecting and quantifying analytes in solution. The most widely used format, the sandwich ELISA, involves coating the microplate wells with a capture antibody before blocking and then incubating with the test samples and a reference standard (added across multiple wells to produce a concentration curve). After washing to remove any unbound material, a detection antibody is introduced and the resultant signal is measured with a plate reader. The analyte concentration in the test samples is then calculated.
Before an ELISA can be used for sample testing, it must be validated to confirm that it accurately detects the analyte of interest without artifacts or interference and produces a standard curve that closely mirrors antibody-antigen binding in the test samples. To ensure confidence in its ready-to-use ELISA kits, HUABIO bases its validation process on four core performance indicators, as described below:
• Sensitivity
Sensitivity is typically reported as the minimum detectable dose (MDD), which is calculated by adding two standard deviations (SD) to the mean value obtained from replicates of the zero standard. A comparison between HUABIO’s ready-to-use ELISA kits for human IL-6, TNF-α, IFN-γ, IL-17A, and IL-4 and those of a leading competitor indicates the HUABIO products to have consistently higher sensitivity, as shown in Table 1, spanning a 2.5-fold improvement for IL-6 to an increase of over 300-fold for IL-4.
Table 1. Sensitivity comparison for HUABIO and a leading competitor
0.7
0.26
6.23
0.21
5.69
0.36
15
10
0.03
• Stability
Stability is defined as the ability to maintain constant product performance, which is critical for generating consistent results over time. Testing shows HUABIO’s ready-to-use ELISA kits to have a shelf-life of ≥ 18 months at 4 ℃, and that storage at 37 ℃ for one day is equivalent to storing at 4 ℃ for 48 days, demonstrating the high stability of these products.
• Precision
Precision is the degree of dispersion of different measurement values in the same sample. It is determined by testing samples of known concentration multiple times on the same plate and calculating the coefficient of variation with the formula % CV = (SD/average) x 100. An assay is generally given a pass if the % CV is ≤ 15, while % CV values of ≤ 10, ≤ 8 and ≤ 5 are respectively associated with good, outstanding, and excellent precision. Representative data for HUABIO’s IL-6 ready-to-use ELISA kit are shown in Table 2 and exhibit the highest precision levels.
Table 2. Precision testing for the Human IL-6 ELISA kit (EH0001), n=22 for each concentration tested.
• Accuracy
Accuracy refers to the consistency between measured values and true values, which is best determined by running a spiking experiment. This involves introducing a known concentration of the analyte into the assay and calculating the recovery coefficient with the formula ((measured value - sample background value)/spiked theoretical value) × 100%. A value of 50–150% is considered a pass, while a value of 80–120% indicates excellence. Testing of the five HUABIO ELISA kits mentioned previously demonstrates excellence across the panel, as shown in Table 3.
Table 3. Accuracy testing for a panel of HUABIO ready-to-use ELISA kits.
84–115
95–105
89–113
80–112
88–105
Besides its rigorous validation process, HUABIO safeguards researchers’ results by incorporating several helpful design features into its ready-to-use ELISA kits too. These include color-coded kit components, to avoid confusion, and provision of ready-to-use working solutions, to save time and minimize the risk of dilution errors. Other notable features of HUABIO’s ready-to-use ELISA kits are their concise protocols, short run times (just 120 minutes from start to finish), and miniaturized packaging that facilitates storage and transportation. In addition, the use of laser-engraving instead of traditional printed labels simplifies product recognition by resisting wear.
To learn more about HUABIO’s ready-to-use ELISA kits and how they can benefit your research, visit huabio.com/elisa-kits