Hi All,
I am optimizing concentration for detection 1' and 2' Antibody for a sandwich ELISA using the standard checkerboard titration method. (See attachment for experiment layout and specific Ab concentrations.) Basically, I used 5-fold dilution series for both 1' and 2' Ab, and included three lysate concentrations (64, 16, and 4ug) + blank lysis buffer for each Ab condition.
And some more info:
- Capture antibody is 1ug/ml in 100ul PBS per well, using Nunc MaxiSorp plate.
- The capture Ab / detection Ab pair was chosen from a checkerboard experiment with several different antibodies prior to this experiment. So I'm pretty sure this Ab pair works.
- TMB incubation time is ~15 minute
I have attached the results below:
Table A: raw reading at 450nm
Table B: blank subtracted value, with value less than 0.1 highlighted
Table C: relative value of 64ug and 16ug (normalized to value of 4ug), calculated from Table B.
My overall question is: How to use the data I got to determine which is (are) the optimal 1', 2' Ab condition?
And here are some specific questions, which again are kind of related to each other:
(1) Since lysate amount is 64, 16, and 4ug, I expect at least some conditions will give results with 4-fold change, if they fall in the linear range. But that doesn't seem to be the case (see Table C for ratio). Is that normal?
(2) Is this a good/clean experiment? According to Table A raw reading, all blank controls are below 0.1; and the biggest value is ~2.5. I should be in the linear range for all wells, right?
(3) After blank subtraction, what's the number below which you don't trust? For example, here (in Table B), I arbitrarily set the threshold at 0.1 (since the "dirtiest" blank, well A4, is around 0.1)
(4) Now that the results are nothing close to the theoretial "4-fold" ratio, can I still use the results to determine an optimal Ab condition? Or did I do something wrong in this experiment and how should I improve?
I have never developed an ELISA before so any thoughts or comments will be greatly appreciated.
Thanks! Zhizhou
Consider what combination of antibody concentrations gave a large signal:background (S:B) ratio (at least 3) with 4 µg of lysate (columns 3, 7 and 11 versus columns 4, 8 and 12). This approach is helpful because it minimizes the problem of going beyond the dynamic range of the detection reagent or the plate reader, which may be happening with some of the high absorbances you measured. Also, if the antibodies are precious you may want to use as little as possible. Some good combinations are
(0.2, 100) (well B7, S:B = 5.6)
(1, 20) (well A11, S:B = 4.0)
(0.2, 500) (well B3, S:B = 5.1).
If the antibodies are not so precious, then you could use
(1,500) (well A3) or (1,100) (well A7).
All of these combinations give large signals with 4 µg of lysate. The next thing to do is pick at least one of them and titrate the lysate amount in linear fashion (e.g. 1, 2, 3, 4, etc.) µg to generate a response curve.
It would be a good idea also to include a negative lysate control, i.e. a lysate that does not contain the antigen, if possible. The signal from this negative lysate should then be subtracted from the signal from the positive lysate at each lysate concentration.
You should include 2 controls, in each of which one of the antibodies is omitted. This is to find out whether the signal is due to background or is really due to detection of the antigen.
- Badges
- Science topic
- Similar topics
- Higher Education
- Universities