Hello,

I'm setting sandwich ELISA to measure amount of target protein in human serum.

I have recombinant protein and checked 2 kinds of antibodes(different epitope) were suitable to perform sandwich ELISA.

(Detection range is minimum 200pg)

However, in human serum, I have some problem.

1. To check matrix effect and influence of serum components on the ELISA, I added recombinant protein (200pg to 200ng) in serum diluent(1:50, 1:100, 1:200, 1:500).

But, in serum diluent, OD value were about 0.6 (200ng) to 0.3(200pg). Max OD value is too low, and min OD value (background) is too high.

(without serum, OD value of recombinant protein in 1% BSA buffer was about 2.0 to 0.1-2)

Do I have to dilute more? or change the dilution buffer?

(Actually, I added EDTA and Nacl to diluent buffer to reduce non-specific binding, result was same)

2. Despite I washed 10 times (5min X 10 times) by using 1XPBST before add TMB solution, OD value of blank (only serum diluent. without recombinant protein) was too high (0.2 to 0.4).

What can I have to do decrease background detection?

Thank you and have a great day