Hello everyone. please assist me in the following issue. I am currently developing a custom sandwich ELISA for the detection of antigen x. I am using two monoclonal antibodies with a secondary detection antibody which is HRP conjugated. In the development protocol, I am using serially diluted known concentrations of antigen x and a blank control well which is coated with mAb then incubated with 0pg/ml antigen x, blocked, then the rest of the procedure....first of all should this be my control? ..(What if I use uncoated but blocked well with 0 antigen x as my control?)
Secondly (if the first one is my control) how do i calculate/determine my background noise value? so that i know whether it is high or low and optimize the other parameters accordingly? I am measuring absorbance at 450nm so I do not get the interference signal presented by 450/630nm reading.
Thirdly, If I decide to use 450/630nm in reading absorbance, should I consider the 630nm values as background? Is the background noise(unspecific binding) the same as interference (from possible scratches on the wells ,630nm value)
Finally, any information on how i can determine antibody-antigen dynamic range?
Your Input and advise will be highly appreciated.
I think, if you use PBS as your diluent, you could just transfer PBS in, let's say, 24 wells, read the plate at the same wavelength as you are supposed to read your reaction, calculate the average, StDev and CV, and subtract that average from your true reads (meaning after you add the substrate and get the color reaction). But in my experience these values are so small you can just ignore them.
Why do you get scratches on wells?
The dynamic range implies ACCURATE determination of your analyte/antigen using the standard curve. You need to determine higher limit of quantitation and lower limit of quantitation while your curve remains linear.
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