- The ELISA protocols do not recommend shaking during incubations. Have you tested shaking and decided against it or is it unnecessary?
We tested our protocols with and without shaking during incubations and determined that there is no difference between the two approaches. Therefore, we believe that shaking is not necessary.
- Is your ELISA kit suitable for use with tissue lysates? If so, what are the protocols?
Theoretically, our ELISA kit can work with tissue lysates. Our general sample preparation protocol for tissue lysates is as follows:
- Rinse the tissue with PBS to remove excess blood.
- Chop the tissue into 1-2 mm pieces.
- Using a tissue homogenizer, homogenize the samples in PBS or lysate solution such as the Mammal Tissue Protein Extraction Reagent (Boster Bio Catalog Number AR0101) at a ratio of 10 mL lysate solution to 1 g of tissue.
- Centrifuge the homogenates at approximately 5000 x g for 5 min.
- Assay immediately or store the homogenates at -20°C (avoid repeated freeze-thaw cycles).
- My samples contain very low cytokine concentration. What is the minimum concentration that can be measured with confidence using your ELISA kits?
Low cytokine concentrations are typical for many biological samples. When determining the minimum concentration that can be reliably measured by ELISA, consider the following:
- The standard curve: As the concentration range of ELISA is typically 0 – 1000 pg/mL, the data points on the standard curve for this range correspond to 0, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 pg/mL.
- The assay sensitivity: Boster’s Picokine ELISA kits typically have a reported sensitivity of 10 pg/mL. The concentration detected in many biological samples will fall between the 0 and 15.6 pg/mL data points of the standard curve. As long as the value detected is above the statistical sensitivity of the ELISA, (e.g., 5 pg/mL or greater), the value is statistically significant. Results below this detection limit are of questionable validity.
- Is your ELISA kit suitable for use with tissue homogenates?
For most cases, yes. If there is enough target protein present in the tissue of interest, the ELISA kit will work. Also, if there are a known alternative processing of the protein in a specific tissue that results in protein reactivity change to the kit, we will note it in our product datasheet.
- Is your ELISA kit suitable for use with any non-validated sample types?
In order to use an ELISA kit with a non-validated sample type, it is necessary to perform a spike and recovery study to determine if a non-validated sample type will work with a particular kit. To do this:
- Divide the sample into two aliquots.
- In one of the aliquots, you should “spike in” a known amount of the kit standard.
- Perform a dilution series to compare the spiked to the unspiked sample.
Generally, samples with expected recovery and linearity between 80-120% are acceptable. This method can be used to validate any sample type that has not been previously evaluated by Boster.
- Can I extend the standard curve?
No one can guarantee the assay accuracy once the concentrations outside the specified range within the curve are used. A specific range is generated to provide the statistical confidence for the assay accuracy.
- What causes high variability between sample duplicates?
The two main reasons for high sample variability in an assay are inconsistent pipetting and washing. Thus, it is important to perfect these techniques. However, some of this variability is unavoidable — this is the rationale for calculating the average results from sample duplicates. Another possible culprit for high variability is the “edge effect” in which the outermost wells of the plate are more vulnerable to drying out due to evaporation. Plate stacking will also cause variability because temperature will be unevenly distributed across the plates.
- What are the differences between the sandwich ELISA and competitive ELISA?
Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for a different, non-overlapping part (epitope) of the antigen molecule. A first antibody (known as capture antibody) is coated to the wells. The sample solution is then added to the well. A second antibody (known as detection antibody) follows this step in order to measure the concentration of the sample. Higher signal output reflects higher concentration of the target antigen in the sample.
The key event of competitive ELISA is the process of competitive reaction between the sample antigen and antigen bound to the wells of a microtiter plate with the primary antibody. First, the primary antibody is incubated with the sample antigen and the resulting antibody–antigen complexes are added to wells that have been coated with the same antigen. After an incubation period, any unbound antibody is washed off.
The more antigen in the sample, the more primary antibody will be bound to the sample antigen. Therefore, there will be a smaller amount of primary antibody available to bind to the antigen coated on the well, resulting in a signal reduction.
- Why do my wells turn green after I add the stop solution?
The green color is a result of incomplete mixing between the substrate and stop solution. After adding the stop solution, gently tap the plate or place it on a shaker until the mixture in the wells turns yellow.
- Why does a brown or orange-brown precipitate appear in my wells after adding the stop solution? How can I resolve this issue?
The precipitate is a result from insufficient washing after incubation with the HRP- labeled detection antibody. To resolve this issue, perform a 30-second soak during each wash step followed by a complete removal of all liquid in the wells.
- If I don’t use all the wells from a microtiter plate for my current ELISA assay, how can I preserve the unused wells for future use?
The microtiter plate typically has removable strips of wells. Unused wells may be removed from the plate, returned to the foil pouch containing the desiccant pack and stored at 2-8°C for up to one month.