Can you tell me whether the background caused by cell lysate matters in giving the signal? wil it be okay if I take lysate cell supernatant after short centrifugation?
The only certain way to answer this question is to carry out suitable control experiments. Carry out the assay in the presence and absence of cell lysate to see if there is interference. If this cannot be done because all cells have the analyte a non-linear response with increasing concentrations of lysate is a good indicator of matrix interference.
Hi
I totally agree with Edwin. regarding the second part of your question, we tried the cell lysate supernatant after 20 mins centrifugation and it worked fine. However, you will need to measure the amount of protein concentrations in each sample using spectro (e.g. nanodrop)
You can find an answer on your question if you add you antigen in defined concentration to a cell culture supernatant. You have to find them in the assay within the intraserial imprecisicion. If not, use a constand antigen concentration and dilute the cell culture supernant. (Detection of disturbing effects). See link to the ICH guideline.
Some general remarks about the background: You need to have an incubation buffer. For this purpose I suggest to use as incubation buffer: 0.01 mol/L phosphate pH 7.2, 0.3 mol/L NaCl, 3% v/v Gelafusal (digested gelatin with normally no cross reactions etc), 0.1% v/v tween 20, 0.001 g/L phenol red. The doubled salt (NaCl) will reduce unspecific binding and therefore the background. The inert protein (Gelafusal) is important. Cross reactions should be excluded, constant protein concentration should be given during the incubation despite of different dilutions. Gelafusal is a drug, used as plasma expander, available via pharmacy and best defined as all other drugs. Gelafusal is also free of any animal antibodies. Tween is a detergent reducing unspecific hydrophobic interactions with other proteins and the solid phase. Anyone has this in his dilution / incubation buffer and that why any blocking is nonsense and will increase background reaction and costs only.
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