Cluster of Differentiation (CD) molecules are a group of cell surface glycoprotein molecules of growing interest in immunology for identification of cell differentiation and determination of cell type. These molecules are typically found on the surface of cells. However, in disease or abnormal states, these cell markers are secreted into the bloodstream. Studying these makers in the bloodstream can help researchers obtain a profile of markers to help diagnose disease and the stage of disease progression.
R&D Systems, Inc. offers ELISA kits for the quantification of many of these CD markers in their Quantikine® kits product line. These kits are colormetric solid phase ELISA kits suitable for measuring soluble CD markers in supernatants or human samples, including plasma and serum. My experience with the Quantikine® kits is limited to the CD markers group of specificities such as CD14, CD54, gp130, etc., while R&D Systems, Inc. does offer Quantikine® Kits for other target types.
The assay is a basic sandwich ELISA on a monoclonal antibody pre-coated microplate. Detection is accomplished with an enzyme linked polyclonal antibody and substrate with the color intensity being quantitatively read at 450 nm. The kit is very thorough and complete with 96 sample wells formatted as 12 strips, each with 8 wells, which is useful because 8 samples can be assayed at a time instead of requiring 96 sample wells to be used at once. Other components such as conjugate, color development reagents and wash buffers are provided in excess quantities. The kit also includes useful standards consisting of lyophilized recombinant proteins of known quantities for accurate standard curve development.
Overall, this kit is well-designed, typical of the quality one expects from R&D Systems, Inc. products. The assay procedure is thorough and simple to follow, including easy protocols for sample preparation, reagent preparation, assay procedure, data calculation and helpful technical tips. Assuming that your samples are prepared ahead of time, the total assay time is less than 5 hours, including the time required to read the sample on a microplate reader. The assay procedure is very easy to follow and requires a minimal amount of technical knowledge, making the kit available to those unfamiliar with the ELISA format or beginner technicians. I immediately obtained excellent results using the kit from the onset, without having to modify procedures or free-lance any steps to optimize results.
According to specifications, the assay kit has a linear range of approximately 1000 ng/mL to 3000 ng/mL. These numbers vary slightly depending on the sample type (serum or plasma) and the type of anticoagulant used in the sample (heparin, EDTA or citrate). Intra- and inter-assay precision range from a %CV of about 5% to about 8%. The % accuracy of recovery and linearity ranges in the mid 90%’s, again depending on sample type. When evaluating citrated plasma samples, I found these specification values to be accurate and consistent. Additionally, when evaluating normal citrated plasma samples, either from a pooled source or a commercially available normal sample, I obtained concentration values expected for these types of samples. R&D Systems, Inc. also offers human control samples for use with the kits, but I have no experience with these sample types, opting to use commercially available normal human plasma. I have not used the kits to evaluate samples other than citrated plasma and cannot comment of the reliability of the kit for these sample types. As a side-note, I compared these kits to multiplexed microarrays on a nitrocellulose thin film slide. The microarrays offered much greater dynamic linear range, about 3 logs, with very slightly higher %CV’s for consistency and accuracy. Additionally, when looking at the cost, for a similar price, the microarray offers many more data points due to the multiplexing capability compared to the single data point offered by the ELISA.
Regarding the value of the kits, I obtained 96 well kits for $460, making the per/sample cost approximately $5. Keeping in mind that several of those sample wells must be used for standard curve samples, the average per/sample cost will probably be about $10 per sample. At this cost, the kit is not prohibitive to use. The kit has a very robust and fool-proof design despite the very small dynamic linear range. I would suggest using this kit for any lab requiring a fast and easy assay which can be run by a technician level operator to obtain reliable and accurate results. Multiplexed microarray immunoassays offer more value and higher throughput. However, they are not available commercially and will be for the advanced user. Most likely within a year, these kits or services will be available commercially.