Leinco Technologies validates many of their products using ELISA (Enzyme-linked immunosorbent assay) methods. An ELISA is used to detect the presence of an antibody or antigen in a sample. This tool is used heavily as a diagnostic tool in medicine but, is mainly used as a quality control test at Leinco Technologies.

This protocol provides an initial set of conditions; however, further optimization may be required on an individual basis. If there are any questions regarding the use of Leinco antibodies or proteins please call our technical support team at (800) 538-1145.

Coating the Plate with Antigen

1. Dilute antigen to a final concentration of 1-20 μg/mL using PBS or carbonate/bicarbonate buffer (pH7.4).
2. Add 50 – 100 uL of the antigen solution to the wells.
3. Seal the plate and incubate for 1-2 hrs at room temperature or overnight at 4°C.
4. Wash the plate three times with PBS/Tween and blot on paper towels after last wash.
   

Blocking the Plates

1. Block the remaining protein-binding sites in the well by adding 200 uL blocking buffer (Leinco Prod. No. B396).
2. Seal the plate and incubate for 2 hours at 37°C  or overnight at 4°C.
3. Wash the plate three times with PBS/Tween and blot on paper towels after last wash.
   

Incubate with Primary Antibody

1. Add 100 uL of the appropriately diluted primary antibody to each well.
   
2. Seal the plate and incubate for 1 hour at 37°C or 2 hours at room temperature.
   
3. Wash the plate three times with PBS/Tween and blot on paper towels after last wash.
   

Incubate with Secondary Antibody

1. Add 100 uL of the appropriately diluted HRPO conjugated secondary antibody to each well.
2. Seal the plate and incubate for 1 hour at 37°C or 2 hours at room temperature.
   
3. Wash the plate three times with PBS/Tween and blot on paper towels after last wash.
   

Detection

1. Add 100 µL of TMB Microwell Substrate (Leinco Prod. #T118) solution to each well (substrate solution should be at RT prior to use).
2. The estimated incubation times for the enzyme-substrate reaction range from 20 to 30 minutes. Add 50 μl of Leinco Technologies stop solution (Prod. No. T125). After stopping the enzymatic reaction the plate should be read at 450 nm.
   

Solutions

Note: Do not use sodium azide in any buffers or solutions as sodium azide inactivates the horseradish-peroxidase enzyme. All solutions should be at ambient temperature prior to use.

• PBS: Dilute 10 x PBS to 1 x PBS in sterile water.
• Wash Buffer: Leinco Prod. No.: W101
• Block Buffer: 1Leinco Prod. No.: B396
• Diluent: 0.05% Tween-20, 0.1% BSA in PBS*

*Sterile filter and store at 4ºC for up to 1 week.