ELISA Frequently Asked Questions

1. Can you recommend a dilution for my samples?

Due to the large range of protein expression levels across different users and samples types we don’t recommend a specific dilution for every application.

We recommend always diluting at least 1:2 using the diluent specified in the kit (usually Sample Diluent or Standard Diluent).

The Optimal dilution is where the sample signal intensities fall in the middle of the dynamic range and should be determined by either:

• Prior to performing the full experiment, test a serially diluted representative sample from your cohort. Alternately, a small pool of several samples can also be used with this same method if sample volume is limited.

-or-

• Refer to published literature for expected concentrations and derive the optimal dilution level based on the expected dynamic range of the kit.

2. How many times can I use the kit?

The plate is 12 x 8 well strips. Place unused strips back into the foil pouch.

There are two standard vials for two runs. Do not store a reconstituted standard for later use.

Prepare only as much detector reagents as required for the immediate run.

3. I suspect my samples may contain interference factors (hemolytic/bilirubin/lipemic/blood, etc). How will this affect the assay?

The interference factors can affect both the biological components of the kit (capture/detector antibodies) as well as potentially affect absorbance readings. To mitigate suspected interference factors:

• Dilution is the best way to mitigate interference factors. Dilute samples in the specified kit diluent as much as the target analyte concentrations will allow based on the dynamic range of the kit.
• Centrifuging samples at 10,000 rpm for 10 minutes at 4°C can remove some interference factors.
• Heterophilic or animal binding antibodies must be addressed with specific anti-interference buffers.

4. Why does the pellet contained in the Standard vials appear different sizes?

This is a normal phenomenon of lyophilization. The pellet may appear small and dense or fluffy and flocculent. This is due to very small temperature and pressure variations across the lyophilization chamber during drying. This will not affect the standards provided they are thoroughly reconstituted.

5. How should I lyse my cells/tissues?

The recommended mechanical lysis method is contained in the protocol. We recommend mechanical lysis using either freeze/thaw or ultra-sonication to lyse cells. Cells should be lysed in a PBS buffer.

Chemical lysis is acceptable if:

• The final concentration of any detergents, organics solvents or reducing agents are less than 0.01%.
• The concentration of your target of interest is high enough such that it is expected to be detectable based on the dynamic range of the kit when fully diluting the lysis buffer to acceptable concentrations for assaying as above.
• At this step, please check the expected RNA-seq data for tissue/cell line, and the location the protein is expressed in the cell on Uniprot to make sure it makes sense.

6. How much sample do I need?

The kits require 100 uL, so if the minimal dilution is 1:2, 50 uL is required.

7. My sample measurements are increasing as dilute further?

This could be a couple problems. Typically, it is due to interference factors in the sample. Usually this happens with a serum or plasma, not cells/tissues/supers. The optimal sample dilution level must be determined by serial dilution. Where the sample measurements exhibit linear measurements from the back calculated linear regression from the standard curve across 2 or 3 dilutions is the optimal dilution point.

More dilution is always better in an ELISA because it mitigates any potential assay interference and provides a more accurate measurement.

8. My sample measurements are lower than the blank?

This is normal if the sample is expressing target analyte concentrations lower than the dynamic range of the assay. The blank measures higher because there is nothing in the sample, causing non-specific binding. The small amount of matrix in the sample actually reduces non-specific binding in some cases.

9. My blank is very high, my standards are low?

This can be a complex issue, either user or kit failure.

10. Can I use blood samples?

You could use this kit with whole blood, but I would not recommend it. Whole blood has a great deal of interference factors that could affect the results of the test. If you have whole blood, I would recommend processing this to serum or plasma to obtain much better/consistent results.

11. Can Kit be run on separate days?

I would recommend:

• 1. Prepare and dilute your samples just prior to class.
• 2. Prepare the standard curve dilutions just prior to class. Its best to prepare both these and the samples directly before the assay, but they will probably be alright for 30 minutes or so.

The assay should be completed within 4 hours of preparing reagents. Detector and conjugate dilutions can be prepared just prior to use during the previous step incubation.

Please note, You could probably trim the sample/standard incubation step slightly without hurting the sensitivity too much if you needed. The assay will run the same, but will just provide a little lower overall signal level.

12. Lowest standard curve is lower than the blank or blank is too high

Based on the observation that the lowest standard curve is lower than the blank, I would suspect that this is one or more of the following:

• 1. The wells were insufficiently washed.
• 2. There was a pipetting error.
• 3. There was a standard dilution error.
• 4. There was a reagent preparation error.

13. Why is this kit not detecting another commercially available recombinant protein that I purchased?

Not all of the commercially available recombinant proteins can be detected. There are inherent difficulties with antibody detection of recombinant proteins that need to be considered. Folding of the recombinant protein may be different from the endogenous native form, and may prevent antibody access to the epitope. This is particularly the case with tagged proteins.

The standards amino acid sequence may be different than your positive control sequences and expression systems.

Even if you use the same length recombinant protein, the different expression system, different glycosylation and different protein folding can change antibody binding. That is probably why, the antibodies in this kit cannot detect the recombinant NEDD9 proteins that you purchased.

14. Competitive Assay: 0 (ng/mL) well vs. blank well

For a competitive assay, the well for 0 ng/mL detects how much signal the Antigen-Biotin-Complex is reacting with the kit without the competition of your samples/standard. Since this well will only contain the Antigen-Biotin-Complex, it will have the strongest reaction with TMB, thus having the deepest blue shade. The purpose of a 0 ng/mL well is to have all the reagents and buffers in the assay in an environment without the standard so you can confirm that your standard and Antigen-Biotin-Complex is working competitively.

On the other hand, the blank for a competitive assay will be a well with no sample and no Antigen-Biotin-Complex (Step 10.4 in the manual), which will detect nonspecific binding of the kit for you to subtract from. It should only contain sample diluent.

15. Explanation of ELISA Calculations

Deriving sample measurements by regression from the standard curve can be done a few different ways. It really depends on what software you have.

The power exponent function or linear regression will work fine and is typically the easiest for users who just have software like Excel. If your standard curve is relatively straight the 4-P (four parameter logistic (4-PL)) may not be necessary.

If the curve is sigmoidal, we would always recommend doing a 4-P fit if you have some software. If you have software (such as PRISM) which can use a 4-parameter sigmoidal curve fit model, that typically is more accurate for many ELISA users, but not everyone has this software capability.

16. How many cells should I use to prepare cell lysate samples?

This will depends on the level of the target the client study. Commonly lysate cells at 1x108cell/ml will work.

17. Anticoagulants (Aka, "Will citrate work for my assay?)

For our kits, we recommend heparin and EDTA as an anticoagulant as they generally produce better results in most cases.

Below is a list of general pros and cons regarding anticoagulants for ELISA Sample Processing:

Recommend using a different anticoagulant if you have the opportunity.

18. Detection of a transmembrane protein on the exosome in xxx (plasma, serum, etc). Is protein extraction necessary?

We would recommend the following protein extraction buffers:

19. Long term storage of sample types:

Samples should be kept in aliquots at -80oC. Protein deterioration can/may occur, but this is entirely dependent on the stability of the protein. Our lab validates with fresh samples and has not done protein stability testing—we recommend running the assay with fresh samples if possible.

20. Standard Curve + Blank worked, sample type is negative.

Generally, if serum/plasma, could be caused by interference (Matrix Effect). If Sample Type is cell lysate/tissue homogenate, could be due to uses of detergent / chemical lysis. We recommend mechanical lysis to be safe.