Enzyme-Linked Immunosorbent Assay (ELISA) is a standard methodology capable of quantitating an antibody or antigen of interest with a very high degree of accuracy and sensitivity. There are four basic steps involved in the technique (i) immobilizing a ligand to a solid support (ii) binding of a sample analyte (antibody or antigen) to the immobilized ligand (iii) attachment of a conjugated analyte to the sample analyte (iv) detection. Heterogeneous assays such as ELISA involve the separation of free analyte from the bound analyte so that the sample may be quantified. Carrying out this separation via filtration has several advantages such as speed and much higher available binding capacities than the commonly used plastic (nitrocellulose has approximately five times the capacity than regular polystyrene) of 96 well microplates.
 
 

    The ELISA filter plate allows researchers to utilize the excellent binding characteristics (~40ug IgG per well) of nitrocellulose in the standard SBS 8x12 multiwell format. As well as ELISA, the nitrocellulose filter plate can also be used for other types of ligand binding applications such as hybridoma supernatant screening. The ELISA filter plate is totally compatible with automated systems, and also the filter-to-collect vacuum manifold and filtrate collection microplates. ELISA performed with the nitrocellulose filter plate takes much less time than traditional methods using regular microplates for the separation. Coating the nitrocellulose filter takes only minutes, compared with overnight procedures employed for coating polystyrene microplates. Also, the use of vacuum filtration greatly reduces the time required for washing steps, and enables quantitative collection of filtrate into a collection plate.
 

• 96 well 350µl per well, 8x12 format constructed from opaque white styrene, which is chemiluminescent-friendly.
• Individual discrete disks of Whatman Nitrocellulose (0.45um) at the bottom of each well.
• Patented drip director design for the non-crosstalk collection of filtrate.
• Compatible with many automated platforms and liquid handlers.

• High precision, accuracy and reproducibility.
• Sensitivity comparable to standard microplates.
• Very easy and fast protocol - no long, overnight, immobilization steps required.
• Adaptable to other separations based assays.

1. Place plate on vacuum manifold, prewet the membrane with 200ul of PBS. Incubate 2 min, remove by vacuum.
2. Add 50ul of coating Ab (10ug/ml) in PBS, incubate 5 min.
3. Remove by vacuum, add 100ul of 1-3% BSA/PBS block buffer. Incubate 5 min. .
4. Apply sample diluted in 1-3% BSA/PBS, 0.1% Tween. Incubate for 15 min.
5. Wash 4 times with 200ul PBS-Tween on manifold
6. Add 50ul of diluted conjugated antibody diluted in1% BSA/Tween/PBS. Incubate for 15 min.
7. Repeat washing step 5.
8. Add 100ul substrate, incubate until color develops
9. Apply vacuum ensuring to collect filtrate into an appropriate collection plate. Place in plate reader.