Tips for ELISA Kits

Rockland offers a wide variety of ELISA kits in sandwich assay formats. This extensive portfolio includes over 800 complete, ready-to-use ELISA kits for the detection of specific growth factors, cytokines, chemokines, hormones, glycoproteins, and neurotrophic factors that are common targets of research interest. Each enzyme-linked immunosorbent assay is rigorously tested to ensure high precision, accuracy, sensitivity, and specificity. Each ELISA kit is optimized for accurate quantitation of targets in various sample types, including serum, plasma, saliva, cell lysates, and cell culture supernatants.

In order to get the best performance from your ELISA kits, consider the following:

• Kit Compatibility

  When choosing an ELISA kit, it is important that you understand whether a given kit is known to be compatible (or incompatible) with the nature of your sample (matrix). As kits are developed, their performance is evaluated in different matrices and often reported in the instructions manual and other accompanying documentation. Make sure you understand how well the ELISA kit has been characterized in the matrix that is relevant to your samples (serum, plasma, tissue culture, urine, etc.). The fact that a kit has not been tested in your matrix of interest does not mean it will not work but rather that you would have to run additional validation assays to determine its performance.

• Understanding the Assay Before Starting

  Although most commercial ELISA kits follow the same sandwich ELISA format, differences in a range of parameters including the reporter system, type of conjugates (detection antibody), recommended incubation times, and temperatures are not unusual among kits detecting different targets, even when they come from the same manufacturer. It should not be assumed that because a particular brand of ELISA kit against one target has been used before, the same conditions will apply to a different kit of the same brand against another target. Because the nature of each analyte and the reagents used is unique to each target, it is imperative that you review and understand in advance each particular protocol, prepare accordingly and follow all directions carefully.

• Reproducibility

  Once you fully understand the proper handling and the optimal conditions and parameters for your ELISA kit, follow these steps to avoid irreproducibility issues:

  • Before running the assay, allow about 30 minutes for all the kit reagents to reach room temperature. Frozen samples should also be allowed to thaw completely before being used and repeated freeze-thaw cycles should be minimized, less than three times preferable.
  • Make sure environmental conditions including temperature and humidity are kept constant throughout the assay and between assays.
  • Work only with properly calibrated equipment including pipettes, plate washers, and readers.
  • Always confirm proper aspiration, withdrawal, and addition of reagents by direct visual inspection of tips and wells throughout the assay.
  • Always use freshly prepared substrate solutions and don’t hold those for prolonged periods of time.

  Altogether, these recommendations should favor more consistent binding kinetics and color development between assays of the same type.

• Accuracy

  In addition to having a properly generated standard curve with replicates, samples should also be tested in at least duplicate or triplicate, if possible. Whenever possible, prepare samples at several dilutions to ensure at least one of them will follow within the linear range of the assay. Samples with optical densities (OD) above or below the linear range of the standard curve will result in target concentrations to be underestimated or overestimated, respectively. Following these guidelines, every time you use your ELISA kit will result in improved accuracy of quantification.

• Standard Curves

  ELISA kits require the generation of a standard curve for the quantification of the target molecule. Because assays can never be identical but rather kept within an acceptable range of variation, the accuracy of your data will result in having a standard curve obtained under the same conditions your sample is measured. This means that a standard curve should be produced for each set of samples assayed. Avoid using the standard curves generated from other plates or other days. Make sure the standard curve is generated within the dilution range recommended by the manufacturer and that you have enough data points along such range. Run standards in duplicates or triplicates.

• Maximizing Samples

  Valuable samples are often available only in limited quantities and the ideal situation of running multiple dilutions in duplicates or triplicates is not feasible. In this regard, it is advisable not to use all your samples (especially the most valuable) until you have run a couple of preliminary assays including standard curves and one or two control samples at several dilutions that will give you a more reliable idea of the optimal dilutions to use when running your real samples. Always plan in advance the layout of your plate, keeping in mind the number of samples and dilutions to use and decide the most effective way to run your assay. This will maximize not only your samples but also the use of your kit.

• Cross-Contamination

  Avoid cross-contamination of samples or reagents by changing tips between sample, standard, and reagent additions, and using new disposable reagent reservoirs for each transfer. When planning your experiments as described before, determine the amount of reagents to be used in the planned assay to ensure you will only prepare enough reagent as required and avoid having an excess of unused reagent since you will have to make sure is discarded appropriately and never returned back to stock containers. Similarly, avoid reusing any wash or reagent reservoirs or trays.

• Data Analysis

  Make sure you understand how data should be used for calculations. ELISA kits usually indicate how correction factors for background should be applied, how to plot the data and the type of integration required to generate an appropriate standard curve. Once you decide the best way to run your assay and plate layout, prepare in advance a spreadsheet template that can generate your standard curve automatically and calculate sample protein concentrations.