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Should I run any internal controls with your ELISA kits? Where can I get internal controls? Product FAQ

Answer

Running internal controls along with the blanks and the standard curve is an excellent way to monitor the performance of any ELISA kit. Internal controls are samples containing known amounts of the analyte being measured with the kit. A key attribute of internal controls, and what differentiates them from the samples of the standard curve, is that they duplicate the composition of your actual samples as closely as possible. This is important because your samples may contain components that affect detection of the analyte differently than does the Standard Diluent provided in the kit. In other words, internal controls allow you to determine if assaying the same amount of analyte in the Standard Diluent (the standard curve) and in the sample matrix (internal controls) gives the same results. It's also important to remember that internal controls give you confidence in the accuracy of your results. Internal controls help ensure that the assay is performing consistently from assay to assay, every time you run it.

One source of internal controls is naturally occurring samples (such as serum or plasma) containing known amounts of the analyte you want to measure. Such positive control samples can be run as-is, or they can be diluted to various known concentrations, preferably with a naturally occurring negative sample of the same type. It's a good idea to run at least 2 internal controls, if you can. One usually has an analyte concentration between the lowest point on the standard curve and the curve's midpoint, while the other has a concentration between the midpoint and the highest concentration. Some researchers also run an internal control that falls close to the midpoint of the curve.

If you don't have access to naturally occurring controls, you can prepare them yourself. Let's say that you will be measuring interleukin-6 (IL-6) in human serum samples with an ELISA kit, Cat. No. KHC0061. First you will need to obtain some pooled normal human sera, which will be used as the sample matrix for your internal controls. Preferably, the IL-6 content of this serum should be negligible (but known) or too low to measure. Next you should add known amounts of human IL-6 to this serum to create the internal controls, using the human IL-6 standard provided in the kit. After you prepare the standard curve as described in the product manual, add some of the leftover concentrated IL-6 standard to the serum matrix. As described above, you can prepare controls with high and low IL-6 levels, or high, medium, and low, if you have enough wells in your ELISA plate.

Even if your sample type is not serum and you're not measuring IL-6, follow the general procedure outlined above to prepare your internal controls with your protein and sample matrix of interest. A valuable resource describing Spike and Recovery and Linearity of Dilution assays can be found here (http://tools.thermofisher.com/content/sfs/brochures/TR0058-Spike-and-Recovery.pdf). If you need more help, please contact Technical Support at techsupport@thermofisher.com.

Answer Id:: E12609

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What's the difference between the chromogen blank and the zero standard in your ELISA kits? Is it necessary to run both of these types of blanks? Product FAQ

Answer

The chromogen blank and zero standard provide you with different indicators of assay performance. The chromogen blank-comprising only chromogen and stop solution-should have an A450 of less than 0.03. The A450 of the chromogen blank can be used to blank your plate reader and, in any case, it should be subtracted from all other A450 values you obtain from the plate.

If the chromogen blank is higher than approximately 0.03, you should check the color of the chromogen reagent straight from the bottle. It can range from clear and bluish to clear and slightly yellow. If it is a stronger blue color, then it is most likely contaminated and should not be used. The most frequent cause of contamination is transfer of unused chromogen solution back into the bottle. The chromogen solution should always be transferred to a clean reservoir in the first place.

The other blank wells that you run are the zero standard wells, which are sometimes referred to as the “zero wells”. The A450 of the zero standard wells in your assay should be less than 0.35, and preferably lower than this. Although the zero standard A450 value should be low, it is usually higher than the chromogen blank. It serves as the critical first point on the standard curve, so if the zero standard value is too high, it reduces the dynamic range covered by the standard curve. Expected values for the zero standard are provided as part of typical data in the kit manual. If the zero standard value is higher than expected, this usually indicates that there is some other problem with the ELISA.

We strongly recommend that you include both the chromogen blank and the zero standard when you run an ELISA. Since we recommend that all blanks, standards, controls, and samples be tested in duplicate, these blanks take up only 4 wells. If you are short of wells for samples, you can omit the highest standard in the standard curve as well as the next-highest one, if absolutely necessary. You should still make all of the standard dilutions as instructed in the manual, but don't add the one(s) that you're omitting to the plate. This recommendation is based on the assumption that the A450 values for your samples will still fall on a standard curve drawn with the remaining points. If they do not, then you will need to run the assay again with all of the standards or dilute the samples so that they fall on the abbreviated standard curve.

Answer Id:: E12610

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I am getting an elevated background in my ELISA. What should I do? Product FAQ

Answer

Here are possible causes and solutions:

Insufficient washing and/or draining of wells after washing. Residual solution containing anti-rabbit IgG HRP or streptavidin-HRP can elevate the background if left in the well.
Wash according to the protocol. Verify the function of the automated plate washer. At the end of each washing step, invert the plate on absorbent tissue on the countertop and allow it to completely drain, tapping forcefully if necessary to remove residual fluid.
Chromogen exposed to light prior to use, resulting in a blue color.
Keep chromogen in its vial until you are ready to dispense it into the plate, and then pour it into a reservoir to prevent contamination of the vial with equipment. Do not cover the plate with foil.
Incubation time is too long or incubation temperature is too high.
Reduce incubation time and/or temperature.
Contamination of pipette, dispensing reservoir, or substrate solution with anti-rabbit IgG HRP or streptavidin-HRP.
Do not use chromogen that appears blue prior to dispensing onto the plate. Obtain a new vial of chromogen.
Blanks that have been set up improperly.
Follow the protocol when designating blank wells. Blank wells contain only chromogen and stop solution. Subtract blank well results from all other wells.
Incorrect dilution of standard stock solution or standards diluted in serum, culture supernatant, or other.
Follow the protocol instructions regarding dilution of the standard. Dilute standards only in the Standard Diluent Buffer provided in the kit.

Answer Id:: E12630

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