How to Adapt DuoSet ELISA Kits to 384-well Plate Format

The following is a suggested protocol that can be used to adapt our DuoSet ELISA Kits from their traditional 96-well plate format to a 384-well plate format. This transition increases the capacity for sample analysis, allowing more than 160 samples to be analyzed in duplicate per plate, compared to 40 samples analyzed in duplicate per plate with the traditional 96-well assay. Additional advantages of the 384-well plate format include smaller sample volume requirements and the ability to run all samples on the same plate, which enhances sample comparison accuracy by eliminating inter-plate variability.

Additional optimization may be required depending on your sample type.

Adapting DuoSet ELISA to 384-well Plate Format Protocol

Required Reagents

• Reagents included in any analyte-specific DuoSet ELISA Development System: Capture antibody, detection antibody, recombinant standard, Streptavidin-HRP
• Reagents included in the DuoSet ELISA Ancillary Reagent Kit 2 (Catalog # DY008B with 384-well microplates): 384 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and reagent diluent concentrate 2.

Note: These reagents may also be purchased separately. Clear Strip-well Microplates (Catalog # DY990), ELISA Plate Sealers (Catalog # DY992), Substrate Solution (Catalog # DY999), Stop Solution (Catalog # DY994), Plate-coating Buffer (Catalog # DY006), Wash buffer (Catalog # WA126), Reagent Diluent (Catalog # DY995)

Plate Preparation

1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 384-well microplate with 25 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (100 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
3. Block the plates by adding 75 μL of Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

1. Add 25 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
3. Add 25 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
5. Add 25 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
6. Repeat the aspiration/wash as in step 2.
7. Add 25 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
8. Add 12.5 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.