How to select the right ELISA kit

The Enzyme Linked Immunosorbent Assay (ELISA) is a widely used laboratory technique designed to detect and quantify proteins, hormones, antibodies, and other molecules in samples such as serum, plasma, saliva, urine, or cell culture supernatants. ELISA assays are used in various fields, including clinical diagnostics and research.  

Selecting the right ELISA kit is an important decision that greatly impacts the results of your experiments. By thoughtfully evaluating factors like sensitivity, dynamic range, precision, and workflow you can make a well-informed decision that aligns with your research objectives. Other parameters that are better indicators of ELISA performance should also be considered. These include type of samples to be measured, recovery, dilution linearity and parallelism (%) to measure the target protein in real samples like plasma, serum, or cell culture media.

ELISA Assay Principle

The main principle of an ELISA involves the binding of an antigen (the target molecule) to a specific antibody. The assay is mostly carried out in a 96-well plate that is coated with capture antibodies specific to the target antigen.

 

ELISA Assay Protocol- Example:

1. Coating: The wells of the 96-well ELISA microplate are coated with a specific capture antibody that binds to the target antigen.
2. Sample Addition: The sample containing the target antigen is added to the wells. Antigen present in the sample binds to coated capture antibody.
3. Detection Antibody: A secondary antibody that is linked to an enzyme, is then added to the wells. It binds specifically to the target antigen, forming a sandwich complex (capture antibody- antigen – secondary antibody).  
4. Substrate Addition: A substrate (e.g. TMB Tetramethylbenzidine) is added to the wells. The enzyme catalyzed color change of the substrate is directly proportional to the amount of target protein present in the sample.
5. Signal Measurement: The color change and the intensity of the color is directly proportional to the amount of target antigen in the samples. It is detectable with a standard microtiter plate ELISA reader (spectrophotometer). A dose response curve of the absorbance (optical density, e.g. OD at 450 nm) versus the standard concentration is generated, using the values obtained from the standards (calibrators).

 

ELISA Assay, 96 well microtiter plate

 

How to select the right ELISA kitBefore purchasing an ELISA assay, always read the kit protocol booklet (package insert). This may help to decide if the kit will be suitable for your needs.

Check as follows:

1. ANALYTE

Which protein biomarker will you be measuring? Be sure to use the correct term during your search. Some biomarker proteins have alternative names (e.g. PERIOSTIN or POSTN ELISA (POSTN is the gene that encodes Periostin).

2. SPECIES – SPECIFICITY – CROSS REACTIVITY

Verify if the assay can be used in the respective model you will be measuring- e.g. human, or preclinical samples such as rat, mouse, monkey. Often ELISA kits can be used for various species, due to high homology between species. As an example the biomarker ELISA kit for NT-proANP was developed for measurements in human samples. Due to the high sequence homology between species, the kit is successfully used to measure NT-proANP as a cardiac safety biomarker in various animal models (rat, mouse, rabbit, monkey). 

3. SAMPLE TYPE

Which sample type (matrix) will you be using (e.g. EDTA-plasma, heparin-plasma, citrate-plasma, serum, cell culture supernatants, urine..) ?

Verify if the assay is compatible for your sample type and check the information in the protocol book. Also check if the assay validation was performed in the respective sample type you will be using. These data can often be found on the website of the assay manufacturer.

Important: the analysis of some biomarkers in the “wrong” sample matrix may lead to “false” results due to a matrix effect.

4. SAMPLE VOLUME

Verify the sample volume that will be required per well, consider duplicates when measuring your samples.

5. SENSITIVTY – BIOMARKER CONCENTRATIONS TO BE EXPECTED

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.

How to select the right ELISA kit – before selecting an assay, study the validation data provided in the protocol booklet.

Some companies document the data on their respective website- product page. Documented reference values in an apparently healthy cohort and/or pathological values in serum and/or plasma of the analyte of interest may also be helpful to get an idea in which range sample values can be expected. It will be helpful to know which levels of the target protein can be expected in your respective sample. Sometimes samples require a pre-dilution step to assure that the signal falls within the dynamic range of the assay. Check the information in the protocol booklet if samples require a pre-dilution step and if the dilution buffer is included in the kit.

Of note: assays offering high sensitivity offer a different dynamic range than assays with a lower sensitivity.

    6. DYNAMIC RANGE

The dynamic range if an ELISA refers to the upper and lower range of concentrations of the target protein that the assay can accurately quantify.

Of note: the reported values given for the sensitivity and dynamic range of an assay can be misleading because they are often determined by using the standard protein (calibrators) in a assay buffer. However, this often does not represent the kinetic of detection of an endogenous protein /analyte in the biological sample.

7. ACCURACY / RECOVERY

The accuracy of 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.

The accuracy (% recovery) is determined by spiking a biological sample (= sample matrix: serum, plasma-EDTA, heparin, citrate) with a known amount of the purified target protein. The spiked sample is then measured in the ELISA and the concentration is calculated from the standard curve. This calculated concentration is compared to the known concentration of the protein which is expressed as a percentage of recovery. As an example, a 90% recovery means that the measured concentration in the sample is 10% lower than the actual concentration of spiked protein in the sample. It suggests that the proteins and other molecules in the sample type did not interfere with the quantification of the protein.

8. ASSAY PERFORMANCE – ASSAY VALIDATION

Evaluation of the assay´s performance characteristics is important in choosing an ELISA kit.

Select an assay that has gone through a rigorous validation process. Data on the following performance characteristics should be available:

• Accuracy (% recovery) – detection of a protein biomarker in clinical samples (acceptance criteria: ≥ 80% recovery).
• Dilution linearity and parallelism  – recovery of the analyte of interest in diluted samples (acceptance criteria: ≤ 20% difference from undiluted samples)
• Specificity & cross-reactivity – making sure that you detect only the analyte of interest
• Precision – within-run (intra-assay) precision and in-between run (inter-assay) precision – ensuring precise and reproducible results within an across assay lots (acceptance criteria: ≤ 15%). The CV (%) or coefficient of variability shows how consistent the assay is.
• Calibration – ensures consistent performance over the range of the assay of the calibration curve
• Sample stability – ensures the stability of the analyte of interest (e.g. exposure of real samples to multiple freeze-thaw cycles, stability at room temperature..).
• Sensitivity – depends on the analyte of interest.
• Dynamic range– depends on the analyte of interest.
• Specificity – verify reactivity with highly homologous proteins
• Calibration – ensures consistent performance over the range of the assay of the calibration curve
• Sample stability – ensures the stability of the analyte of interest (e.g. exposure of real samples to multiple freeze-thaw cycles, stability at room temperature..).

 

9. COMPONENTS of the KIT

Verify if the content of the ELISA assay kit includes all the necessary components e.g. controls, assay dilution buffer. Consider storage requirements such as temperature sensitivity and expiration date.

10. REFERENCES &CITATIONS

Verify for available citations of the specific ELISA kit. These publications can usually be found on the manufacturer´s website.  

11. PRODUCT ORIGIN

Check the originator of the ELISA kit – who developed and manufactured the assay?  More and more kits are repacked and are sold under different brands, although it is always the same kit.

The originator of the ELISA kit will more likely give you qualified support as they “know” their product (e.g. availability of additional calibrators, controls, buffers.., technical know-how on the kit..).

12. CUSTOMER SUPPORT

Verify if the kit provider can provide timely and helpful customer service.

 

 

 

 

Literature

Characterization of a sandwich ELISA for the quantification of all human periostin isoforms.Gadermaier E, Tesarz M, Suciu AA, Wallwitz J, Berg G, Himmler G. J Clin Lab Anal. 2018 Feb;32(2):e22252. doi: 10.1002/jcla.22252. Epub 2017 May 11. PMID: 28493527; PMCID: PMC6816904.