Hello, we are trying to optimize a microplate assay using chemiluminescence for detection of ABO IgG2.

We are experiencing abnormally high background from our reagent blanks (background >20% lightemission).

We are using an indirect ELISA method, coating white polystyrene microplates in human RBC fixed w 0.05% glutaraldehyde. We are precoating the plates w 0.1mg/mL poly-l-lysine and blocking w 1%BSA overnight at 4c.

- Human serum incubates 1h 37c followed by 3x washstep.

- Biotin conjugated mouse anti-human IgG2 incubates 1h at RT. washstep.

- streptavin, Alkaline phosphatase conj. incubates 30min at RT. washstep

- Dynalight substrate incubate 10 min followed by reading on luminometer.

Background have been between 200k-600k RLU to reagent well 0.5-2^6 RLU.

Any theories? can insufficient blocking cause anti-IgG2 to bind nonspecifically to either the polystyrene or the poly-l-lysine?

We have been unable to 'wash away' the excess conjugate.

Linear relationship between background and concentration increase of both anti-IgG2 and enzyme conjugate, respectively have been observed.

Thanks in advance!

- Confused student