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How to Obtain Reproducible ELISA Assay Results
ELISA (Enzyme Linked Immunosorbent Assay) tests are one of the most used biomedical methods for quantifying analytes in biological samples. Apart from their sensitivity and specificity, they are characterized by their simplicity and cost-effectiveness compared to many other analytical methods. However, the easiness in performing an ELISA assay may mask some of the more complex aspects when measuring biological samples.
One of the most common ELISA techniques is the Sandwich ELISA which is used to measure the amount of antigen between two layers of antibodies. Sandwich ELISAs have the limitation that the antigen (e.g. protein in the sample) must have at least two antigen binding sites so that at least two different antibodies can bind. However, these assays are useful when the concentration of the antigen in the sample is low or high concentrations of other antigens contaminate a sample.
Principle of a Sandwich ELISA Assay
Typically a 96-well microtiter plate is pre-coated with a specific “capture antibody” (monoclonal or polyclonal antibody). After adding the sample to the microtiter plate and during an incubation period, the protein of interest present in the sample binds to the pre-coated antibody in the well. Next, the “detection antibody” is added to the well, which forms a sandwich (capture antibody – target antigen – detection antibody).
Figure 1: ELISA Assay Principle
The results can be quantified by adding an enzyme conjugated antibody specific for the second epitope, catalyzing an enzymatic color reaction. The amount of bound enzyme conjugate in the well is detected after following a short incubation with an appropriate substrate. The resulting signal (color change in the well) is detectable with a standard microtiter plate ELISA reader measuring the absorbance (optical density) – the greater the signal, the greater the concentration of the analyte in the sample that is measured throughout the range of the standard curve.
Figure 2: Typical standard curve (using 7 calibrators) for a sandwich ELISA assay, showing the relation between the signal and the analyte concentration
How to Obtain Reproducible ELISA Assay Results
To obtain reliable results it is recommended to choose an ELISA Assay that has gone through a full validation protocol. Results (validation data) must be provided that include data on specificity, sensitivity (LOD, LLOQ), accuracy/recovery, parallelism and dilution linearity, precision (within-run and in-between run), calibration, stability, and lot-to-lot consistency.
CORNERSTONES OF AN ELISA ASSAY VALIDATION
1.SPECIFICITY
Ensuring accurate measurement of the protein of interest
The performance of an ELISA is linked to the quality of the antibody pairs used for the analyte/biomarker detection.
The supplier (assay developer ) needs to :
-select antibody pairs with high affinity and specificity with mapped binding sites
-optimize the ELISA kits to quantify biomarkers in both healthy and pathological samples
2.SENSITIVITY
Enabling the detection of low levels of the analyte of interest
The sensitivity of an ELISA assay refers to the lowest limit of detection (LOD) of the protein that can be detected with the antibody pair used in the ELISA kit. The sensitivity depends mainly on the affinity of the solid phase antibody (coating antibody). Therefore, using a high affinity antibody can increase sensitivity.
-Analytical sensitivity – limit of detection (LOD) is the lowest concentration that can be measured (detected) with statistical significance by means of a given analytical procedure. This concentration is calculated as the background +/- 2 standard deviations.
-Functional Sensitivity – lower limit of detection (LLOQ) is the lowest concentration at which the analyte can be reliably detected.
3.ACCURACY / RECOVERY
Ensuring the correct presence or absence of the protein of interest in samples with different matrices (serum, plasma)
The accuracy in an ELISA assay correctly identifies the presence or absence of the target protein / antigen (biomarker of interest) in a specific sample, which excludes matrix effects that may interfere with the measurement of the analyte of interest.
-Ideally, the accuracy should be determined in all of the sample types (e.g. serum, EDTA plasma, heparin plasma..) where the analyte of interest will be measured. For this reason, the various sample matrices are spiked with known amounts of the recombinant analyte. Samples are analysed against the standard/calibration curve of the assay and then compared with the nominal value.
4.PARALLELISM / DILUTION LINEARITY
Ensuring that samples can be measured with confidence / Ensuring that serial dilutions of samples are quantitated accurately
-Parallelism determines whether real samples with high endogenous analyte concentrations yield the same level of detection in the standard curve after sample dilution. This procedure highlights differences in the binding affinity of the antibodies to the endogenous analyte and the analyte used for calibrating the ELISA. Parallelism in real samples containing endogenous levels of the analyte should always be tested during assay validation.
-Dilution linearity determines the recovery of the analyte in samples that are spiked with the recombinant analyte, used for calibrating the assay (standards).
5.PRECISION
Ensuring precise and consistent results within and across lots
-Within-run precision (repeatability) – evaluation of the precision of the ELISA that measures the variation between multiple determinations of one sample in one single test run.
-In-between run precision – evaluation of samples that are tested several times within one ELISA assay lot guaranteeing accurate results when using ELISA kits that derive from different kit lots.
6.CALIBRATION
Enabling the accurate quantification of the protein of interest
-The accurate quantification depends on the linearity and the reproducibility of the standard curve. During the optimization process of the ELISA kit, low variability between the results of the calibrators must be ensured. Whenever available, standard calibration with standards calibrated to NIBSC standards or WHO reference should be employed to ensure a harmonized standardization.
7.STABILITY
Enabling stability of the analyte of interest in respective samples as well as the stability of the reagents in the ELISA kit
During development, stability of all assay components as well as the stability of the analyte of interest in the respective sample matrices (serum, plasma) should be tested.
-The stability of the analyte can be tested in samples when e.g. samples undergo repeated freeze-thaw cycles or if samples are exposed to room-temperature for a prolonged (defined) period of time.
-Assay kit stabilty (e.g. stability of the kit reagents) should be tested at various temperature settings for a defined period of time.
-Shelf-life of the kit: the real-time stabilty of kit reagents should be tested during the shelf life of the kit. e.g. when kit reagents are stored at 4°C for 6/12/18 months.
8.LOT-TO-LOT CONSISTENCY
Ensuring that consistent results are obtained with different ELISA kit lots
The use of “in-house controls” (serum and plasma samples) containing the endogenous analyte as well as samples that are spiked with the recombinant analyte are tested in every kit lot batch to evaluate whether the data are within the set quality control ranges.
How to Obtain Reproducible ELISA Assay Results
- BIOMEDICA´s ELISA kits undergo a full validation following international quality guidelines making sure that your precious samples only detect the analyte of interest.
Learn more about how to obtain reproducible ELISA assay results by clicking on the link:
WHY CHOOSE ELISA KITS from BIOMEDICA?
Specific – accurate biomarker detection with characterized antibodies
Reliable –validation using clinical samples
Reproducible – guaranteed lot to lot consistency
Further reading
A Practical Guide to Immunoassay Method Validation. Andreasson Uet al., Front Neurol. 2015 Aug 19;6:179. doi: 10.3389/fneur.2015.00179. PMID: 26347708; PMCID: PMC4541289.