How do you choose the best ELISA test for your project ?

There are many, many ELISA tests out there to choose from, and making the best selection can sometimes be a bit daunting. In this post, let’s take a look at the key points to keep in mind.

What is your species of interest?

One of the first thing to consider when selecting your ELISA test is of course to make sure it is tested for your species of interest :=) Apart from the standard human, mouse and rat ELISA, if you’re working on canine, porcine, bovine… and many other species, you might be surprised to find that there may actually be an ELISA test readily available. At tebu-bio, we have a dedicated ELISA search engine that may be of help for this.

Some kits may even be universal for very conserved analytes among species (eg. many of the Arbor Assay hormone detection kits).

Range & Sensitivity

Another point is to check range and sensitivity of the ELISA test to make sure they will fit with the target abundance in your samples. Several sensitivity levels may exist for the same targets. The high sensitivity range may not necessarily fit your need.

Sample Type

Sample type has also to be considered: some ELISAs will have been tested for other standard samples than just serum, plasma and culture supernatant, like tissue and cell lysate , or even urine. Cell based ELISA assays allow quantification of your protein of interest directly in your cells, by seeding the cells directly into the wells.

Quality controls

As the range of ELISA brands is constantly expanding on the market, it is important to ensure ELISA test quality by checking the quality controls performed by the manufacturer: spiking results (% recovery), types of samples tested, linearity assessment, reproducibility (inter- and intra-assay CV%)..

An illustration of what you should find in terms of quality controls is given in the datasheet of this human IL-6 ELISA. Recovery was determined by spiking various levels of Human IL-6 into the sample types listed below (serum, plasma and cell culture supernatant) and measuring the mean recoveries :

In an ideal world, you would want to have 100% recovery. Results shown above between 93% to 95% recovery are excellent. These results also prove that the ELISA has been tested on samples other than just recombinant protein.

Linearity assessment is also a standard quality control that shows the consistency of the results observed after diluting the samples, versus neat. In the table below (same human IL-6 ELISA), results are shown after diluting ½ and ¼ serum, plasma and cell culture samples. Ideally, you would like to get 100% of the results being equal to the results obtained for neat samples. Results are shown as average % of the expected range (eg. 95% of sera diluted ½ fell within a range between 84% and 103% of the neat sample result).


Another feature to take into consideration in your choice is the specialization of the manufacturer in specific protein families.

PBL Assay Science  is the Interferon company with human ELISA against each type of Interferon, as well as mouse, pig and Monkey. Their kits are extensively characterized and optimized for high specificity and sensitivity. An example, the new mouse interferon alpha ELISA provides sensitive quantitation of ALL mouse IFN alpha subtypes to 2.38 pg/ml with robust matrix tolerance including serum and plasma, short incubation (~ 2 hour assay time) and reliable results with < 10% inter- and < 8% intra-assay CVs.

Raybiotech specializes in cancer, inflammation and immunology targets (cytokines, chemokines…). Cytoskeleton Inc is an expert in cytoskeleton related proteins quantification; Cedarlane laboratories have a large selection of ELISA for coagulation factors…

If in doubt, get advice

When searching through the internet for a specific ELISA test, you may end up with numerous ELISA references and the choice may be difficult. We can advise you contact as to which one to choose for your particular application. For a large project, we may advise to test the ELISA of your choice first and reserve the full quantity from the same batch for you to limit the variations that you may observe between lots.

If time is of the essence for you, a format with a compressed workflow may be best suited.

Something that may sound stupid, but make sure you check the wavelength to be used for reading your plate. Not all ELISA substrates have the same wavelength.

If you have some doubts regarding the best method to choose for your data analysis, you may be interested by the tool developed by my colleague Dimitri (check his post where you can download the excel file).

Finally, if you are looking at performing several ELISA for different targets, you may wish to consider a multiplex technology which may be more cost effective and may save some sample. Technologies like Q-Plex from Quansys or Quantibody from Raybiotech allow quantification of several targets at once (up to 20 in one plate for Quansys, a handy solution if you are dealing with a high number of samples., up to 1000 human, 200 mouse and 67 rat  targets for Quantibody).

Don’t hesitate to contact me through the form below if you have any questions about which is the best kit for your research!

See what other researchers have used

As I am aware that nothing is better than quoting real users, I have included below a few key citations for Raybiotech ELISAs from highly ranked journals:

Human IGFBP-2:
Png KJ1, Halberg N, Yoshida M, Tavazoie SF.
A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells.
Nature. 2011 Dec 14;481(7380):190-4. doi: 10.1038/nature10661.

Human IL-11:
Marusyk A1, Tabassum DP2, Altrock PM3, Almendro V1, Michor F4, Polyak K5.
Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity.
Nature. 2014 Oct 2;514(7520):54-8. doi: 10.1038/nature13556. Epub 2014 Jul 30.

Human 6-Ckine:
Mitchell DA1, Batich KA2, Gunn MD3, Huang MN4, Sanchez-Perez L5, Nair SK6, Congdon KL5,
Reap EA5, Archer GE7, Desjardins A7, Friedman AH7, Friedman HS7, Herndon JE 2nd8, Coan A8,
McLendon RE9, Reardon DA7, Vredenburgh JJ7, Bigner DD1, Sampson JH10.
Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients.
Nature. 2015 Mar 19;519(7543):366-9. doi: 10.1038/nature14320. Epub 2015 Mar 11.

Mouse bFGF:
Cao X1, Li LP, Wang Q, Wu Q, Hu HH, Zhang M, Fang YY, Zhang J, Li SJ, Xiong WC, Yan HC, Gao YB,
Liu JH, Li XW, Sun LR, Zeng YN, Zhu XH, Gao TM.
Astrocyte-derived ATP modulates depressive-like behaviors.
Nat Med. 2013 Jun;19(6):773-7. doi: 10.1038/nm.3162. Epub 2013 May 5.

Mouse VEGF:
Seimetz M, Parajuli N, Pichl A, Veit F, Kwapiszewska G, Weisel FC, Milger K, Egemnazarov B,
Turowska A, Fuchs B, Nikam S, Roth M, Sydykov A, Medebach T, Klepetko W, Jaksch P, Dumitrascu R,
Garn H, Voswinckel R, Kostin S, Seeger W, Schermuly RT, Grimminger F, Ghofrani HA, Weissmann N.
Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice. Cell. 2011 Oct 14;147(2):293-305. doi: 10.1016/j.cell.2011.08.035.

Written by Isabelle Nobiron, PhD
Isabelle is a Product Manager at tebu-bio, and also the company's ISO Quality Manager.