I am running ELISAs to measure antibody levels against a Plasmodium protein across three Plasmodium species.

My optimized conditions are:

1. Coating antigen concentration: 2ug/ml, coating done with 50ul/well for 12 hours at 4 degrees Celsius.

2. Washed 6X (200ul/well) with PBS-T (0.05% Tween-20) and blotted dry.

3. Blocked with 200ul/well 0.2um filtered 3% BSA prepared in PBST for 1 hour.

4. Washed as in bullet 2.

5. Primary antibody:

- Samples: 1:1,000 dilution from serum (dilution buffer: 1% BSA, 0.05% Sodium Azide, in PBST).

-Standard: 1:200, 2-fold dilution across a replicated of 7 wells.

Primary antibodies are incubated at room temperature for 2 hours covered in aluminum foil.

6. Washed as in bullet 2.

7. Secondary antibody: 1:10,000 dilution and incubated at room temperature for 1 hour; 50ul/well.

8. Washed as in 2.

9. Reaction is developed with 100ul 1-Step Ultra TMB-ELISA from thermo scientific for 2 minutes in the dark.

10. The reaction is stopped with 100ul 0.27N H2SO4. The plates are read at 450nm wavelength using an ELISA plate reader.

Since the aim is to compare antibody levels against the same antigen in three species, the plates are run in a stack of three (each for one antigen) for the same group of samples at a time.

This protocol has worked very well.

For a few samples left; two of my antigens pass for a successful experiment in terms of blank ODs especially. However, for the same samples; the last antigen fails completely; the plates turn completely blue, even in the blanks moments upon TMB addition.

I have repeated this severally changing different batch reagents, plates etc but this does not get resolved.

I have attached some of my results.

I am grateful for your time and support.