Maybe due to contamination in or wrong choice of the blank?

@Andreas Eisenreich What do you mean by wrong choice of the blank ?

Dear Rishabh Tomar ,

it is very strange that when you read an ELISA will give you negative values since any diffraction of the wavelength it will read as a positive value, even just adding pure water it will positive. after you perform the analysis of the ELISA and you subtract the average of the zeros from all your samples, your actual values could become negatives, meaning that the concentration of you samples is below the concentration of the minimal value detected by the ELISA. in that case your ELISA needs to be more sensitive to be able to detect those lower values.

Hi Rishabh Tomar,

Obtaining negative values of OD HCV is something not usual in ELISA assay especially with HCV kits, the usual OD of the blank is (0.02-0.05), the negative control (OD < 0.1) and the positive control (OD > 0.8) with BIORAD kit. Let's show some known mistakes and the should be done checklist during the all ELISA tests maybe you mist one:

If all of this was checked try to use another commercial kit.

Those are the most know mistakes, for more infos you can refer the guide provided in the kit.

Good luck.

In my opinion it's a glitch in either preparation of blank or may be wrong selection of balnk slot during taking OD readings in an instrument.