Sample Preparation For Elisa

July 9, 2020

Although the preparation of samples for ELISA must always be optimized based on the target molecule and the specific assay at hand, there are general guidelines for the correct preparation of the samples before starting the experiment.

In this post we bring you a general guide for the preparation of samples for ELISA, including those that are used most frequently in these immunoassays.

General Tips For Sample Preparation For Elisa

  • As a general rule, serum, plasma, cell and tissue extracts are used diluted 50% with the buffer.
  • The protein concentration in the extracts should be at least 1 mg / mL.
  • The preservation of the samples will be based on the expected storage period: Chilled (4-8ºC) for periods of less than 48 hours, frozen (-20ºC) for periods of less than a month and deep-frozen (-80ºC) for periods of up to 6 months.

Preparation Of Samples For Elisa Depending On The Type Of Sample

1.- Culture supernatant

  • Centrifuge the cell medium for 10 minutes (1500 rpm, 4ºC).
  • Use immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze / thaw cycles.

2.- Cell lysates

  • Wash the cells with PBS.
  • Homogenize and lyse the cells by adding the lysing buffer.
  • Centrifuge the lysate for 5 minutes (10000g, 4ºC).
  • Use immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze / thaw cycles.

3.- Extracts of tissue

  • Wash the tissue with PBS.
  • Cut into 1-2mm sections and keep them on ice.
  • Prepare the extraction buffer (can be previously prepared and stored at 4ºC)
  • Add 300uL of this extraction buffer for every 5mg of tissue and homogenize.
  • Shake the sample for 1 hour at 4ºC.
  • Centrifuge for 5 minutes (10000g, 4ºC).
  • Use immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze / thaw cycles.

4.-Serum

  • Collect the blood in a tube without additives, and keep it at room temperature for 30 minutes.
  • Centrifuge for 10 minutes (3000rpm, 4ºC).
  • Use immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze / thaw cycles.

5.- Plasma

  • Collect the blood in a tube with anticoagulant, and keep it at room temperature for 30 minutes.
  • Centrifuge for 10 minutes (3000rpm, 4ºC).
  • Use immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze / thaw cycles.

6.- Urine

  • Collect urine in a sterile container.
  • Centrifuge the sample for 1 minute (10000g, 4ºC).
  • Use immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze / thaw cycles.

7.- Saliva

  • Collect saliva with a suitable device.
  • Centrifuge for 2 minutes (10000g, 4ºC).
  • Use immediately after centrifugation or freeze the supernatant at -80ºC previously aliquoted to avoid freeze / thaw cycles.

8.- Milk

  • Centrifuge the sample for 15 minutes (1500g, 4ºC).
  • Collect the aqueous fraction and repeat the same procedure 3 more times.
  • Filter on 0.2um filter.
  • Immediately use or freeze the supernatant at -80ºC previously aliquoted to avoid freeze / thaw cycles.