TiterZyme® EIA Kit – Human IL-8 From Assay Designs

Assay Designs’ motto is “simplify your science” and that is indeed what they accomplish with the many ELISA and detection kits they offer. They offer a wide range of immunoassay and detection kits to identify the following from human, mouse, and rat: cyclic nucleotides, cytokines, eicosanoids, proteins and peptides (including the only non-radioactive COX kit available), signal transduction, and steroids. They also sell antibodies and various other recombinant proteins. In addition, they offer custom services which include custom plate coating and custom kit manufacturing. The kits used in our lab include a variety of human and mouse TiterZyme® EIA (enzyme immunometric assay) Kits that detect cytokines. I have used the human IL-1 beta, IL-6, TNF-alpha, and IL-8 immunoassay kits. This review will focus on the Human IL-8 ELISA because I have used it the most, but all the cytokine kits have roughly the same procedure. I have utilized the IL-8 kit off and on for about 2 years now.

The Human IL-8 EIA Kit contains everything that is needed to perform the assay except for the sample (100 uL total volume), a microplate reader capable of reading at 450 nm, graph paper (or Microsoft Excel), a shaker, and a multichannel pipet (not required, but makes washing a lot easier). The kit comes with 96 wells that are break away so you don’t have to waste wells when a whole row is not needed; the kit also includes a plate to put the wells in. Each well contains an immobilized monoclonal antibody to human IL-8. The antibody binds to the IL-8 in the sample and to the standard that is provided in the kit. After an 1-hour incubation with the sample and standard, the wells are washed and a rabbit polyclonal antibody to IL-8 is added and incubated for another hour. This antibody binds to the human IL-8 bound to the plate. Another wash is performed and a donkey anti-rabbit IgG conjugated to horseradish peroxidase is added and incubated for 30 minutes; the antibody-HRP conjugate binds to the polyclonal antibody. Another wash is performed and then the substrate is added for 15 minutes. Once the reaction is stopped, the absorbance of each well is read using a plate reader at 450 nm. The measured optical density is directly proportional to the concentration of human IL-8 in the samples and standards. I then use Microsoft Excel to determine the standard curve and use the equation from the curve to determine the concentration of each of the samples. I have detected IL-8 using this kit from cell lysates and also from cell secretions into the media. From the cell lysates (H441 cells), I used 40ug/well and from secretions I used a 1:100 dilution. These both gave good results that were within the standard curve. The protocol also states you can use serum and plasma with this kit to detect human IL-8.

This ELISA kit is extremely easy to use and provides high sensitivity: down to 8.2 pg/mL. They provide a small, detailed booklet as the protocol, but also provide a chart that maps out when and where to add each reagent. I especially like this feature because it is so easy to read and understand; too many times protocols from companies are so wordy that you must re-write it so you can easily follow it at the bench. They also provide a sheet with 96 grids in the shape of the microplate where you can write what sample is in each well. The antibody and conjugate are also dyed yellow and blue, respectively. This adds to the ease of the kit so you know exactly what step you are at. The only optimizing of this kit you have to do is to figure out how much of your sample to use. This is true for any new protocol, but since this kit is expensive, money can be wasted rather easily in the beginning if you don’t know how much IL-8 is in your sample. Instead of trying duplicates or triplicates of your sample at first, only try one of each sample to determine if you have chosen the correct amount to be within the standard curve. Then, you will not have to waste wells optimizing the conditions that suit your needs.

Overall, this kit has been helpful in our research studying host effects from certain toxins. The ease with which this kit provides reliable and consistent data is refreshing since the field of scientific research is filled with so much frustration due to inconsistent results that are obtained from labor intensive protocols

Jamie Fritz
Research Assistant
Conceptual MindWorks, Inc
Air Force Research Laboratory/Human Effectiveness Directorate