Questions and Answers about ELISA

Youssef Farhat, Written 8/14/13, Last updated 8/14/13


Here is a list of questions and answers about ELISA which may help clarify certain points for our viewers:

Question 1

I am planning to run an ELISA soon but I have a question regarding sample preparation.  If I don’t know the working range of my samples for the ELISA, should I just add the samples to the plate instead of diluting them and use the standard curve to measure against this?  Or should I initially run the assay with a series of dilutions  to calculate the working range, although time and expenses are a limiting factor with this method?

Answer 1

I would first run a single sample (preferably from an experimental group that you expect to have the highest levels of protein) with a few ten-fold serial dilutions of it, along with the highest and lowest dilutions of your standard curve, as well as one “blank” sample (i.e. pure water or whatever else you may be using to dilute your samples) to serve as a negative control.  This quick test can be done using only a single 8- or 12-well strip if you purchased a commercial ELISA plate, or a single, partially coated ELISA plate if you are coating and blocking the ELISA plate yourself.

Once you’ve identified the dilution of that sample which is somewhere between your high and low standards, you can dilute the rest of your samples the same amount and go ahead with the entire experiment’s samples.  I highly recommend taking this approach with new ELISAs, as the data is much more difficult to interpret/trust when you have samples with absorbance measurements outside of the range of your standard curve.

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Question 2

When you do the dilution step, do you do it only once, to make sure that you find the right concentration for the working range of the assay?

Answer 2

It is highly recommended to test a practice sample as described in Answer 1, above, to see if it is within the working range of your ELISA before doing an ELISA on precious samples from an entire experiment.  It may be that you do not need to dilute your samples at all for some ELISAs, while others may require you to dilute them 100X in order to get them within the working range of the assay.

In any case, once you have determined that your samples will be within the working range of an ELISA, you can proceed in the future to dilute similar samples similarly.  Once your experiment has changed, however, you may want to repeat the test as described in Answer 1 to verify that you are diluting your samples appropriately.

Question 3

If you gave a patient an antibody and he developed Ag/Ab complex and you want to measure the amount of antibodies in his serum related to the dose of antibodies administered, what type of ELISA can I use?

Answer 3

If your goal is to measure how much of the antibodies that you administered are still circulating in his serum, you could first coat an ELISA plate with a pure solution containing only the antigen.  Then, add serum to the wells so that any free antibody sticks to the antigen, and use a secondary antibody labeled with HRP to detect the antibody that stuck to the antigen.  You could then use solutions containing known amounts of antibody to make a standard curve and calculate the antibody concentration of your serum samples.

Click to see Calbiotech's ELISA Kits

Question 4

Why does there need to be a step to convert substrate from blue to yellow by the addition of Stop Solution?  Is the first color change from white to blue sometimes flawed?

Answer 4

The change from blue to yellow happens when you add strong acid to the reaction in the final step of the ELISA.  The purpose of the acid is actually to inactivate the enzyme which produces the blue color.  Not stopping the reaction could potentially skew your measurements, since blue would continue to get produced while measuring the samples on the plate reader.  However, as a consequence, the low pH causes the blue chemical to turn yellow due to its chemistry.

Question 5

What are ELISA plates with antigen-coated wells used to measure?

Answer 5

Plates coated with an antigen can be used to test for the presence of antibodies to that antigen in serum samples. This allows us to see if a person or animal has been exposed to or developed resistance to a pathogen, for example.

Click to see Calbiotech's ELISA Kits

Question 6

Why is BSA used during the Blocking step of ELISA plate preparation?

Answer 6

BSA covers up the empty space between the capture antibodies.  If you don’t use it, then some of the detection antibodies might stick to those empty spaces, and if that happens, you will get a signal from those detection antibodies, even though they didn’t bind to the protein of interest.  Your measurements will therefore be falsely too high.  If you didn’t watch our first ELISA video yet, this all might make more sense after you watch it (

Question 7

Do incubation times vary from kit to kit or from antibody to antibody, or are they the same for all ELISAs?

Answer 7

Incubation times are generally pretty flexible (“overnight” could be 12 hours for some people, or 18 hours for others).  However, it’s always better to follow the manufacturer’s directions to increase your chances of success on the first try at an ELISA.  There may be a good reason that incubation times vary from kit to kit.  For example, longer incubation times might be recommended for certain antibodies if they require more time to bind completely to their target, while shorter times may be recommended if a protein or reagent is unstable or degrades rapidly.

Citing this Protocol

Farhat, Y. “Protocol Title.” Protocol Place. Apr 2013. Accessed on mm/dd/yyyy. <>

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