Dear Chriatopher

You must use BALF of mice immunized with PBS as the negative control to calculate the cut-off value. Use of blank samples would not be necessary. Nevertheless, don't forget to employ a same dilution of positive and negative samples. Besides, during the process of developing a novel indirect ELISA kit, it would be helpful to use some antigen-free microwells (coated just with blocking materials) to evaluate non-specific binding of antibodies (herein IgAs).

Good Luck

Hey,

It seems you are doing a quasi quantitative assay. You can calculate your cut off value by the following formula.

CP=average of the Matrix blanks (at least 3) +3.09*SD for 99.9% confidence level or for 95% confidence level you can use the factor as 1.645.

There are lot of statistics required for determination of cut point, but I think this will suffice for your assay.

Hope it will help you

As a good starting point use sample (BALF, serum) from animals that have not been immunized. It is best to use multiple animals, or at least multiple acquisitions of sample from a few animals. These samples should be run in several independent (from start to finish) assays. The results will become your background in the assay. Typically one sets an initial cutoff by looking at 3SD above background. This may change based on the noise in the system. One should always run the pre sample along with any post treatment samples in the same assay and on the same plate if possible. Remember, an ELISA is only semi-quantitative and will need a good standard curve if you wish to estimate relative antibody concentrations. Looking at OD ratios is not a good way to estimate antibody concentrations. End point titrations, performed in the proper diluent may give relative concentration estimates.