Elisa Sample Preparation Guide

The procedure below provides general guidance for the preparation of commonly tested samples for use in ELISA assays. Please check with the literature for experiments similar to yours for your new assay development. In this article, the following samples are covered:

General Tips For ELISA Sample Preparation

• 1. Hemolysis should be avoided as much as possible when collecting blood samples.

• 2. After collecting anticoagulated whole blood, gently invert the tube to ensure full anticoagulation, preventing clotting caused by partial blood not coming into contact with the anticoagulant.
• 3. It is not recommended to use commercial lysis buffers when collecting tissue and cell extract samples. Surfactants in the lysis buffer may affect the native conformation of the analyte, potentially leading to significantly reduced measurements or false negatives. Additionally, since a new solvent is introduced, potential matrix interference is uncertain, which could increase background levels and affect measurement accuracy.
• 4. Avoid using hyperlipidemic samples. High-fat samples contain lipids and are not homogeneous solutions. If applied directly, they may interfere with antigen-antibody binding, thus affecting measurement accuracy.
• 5. If the samples are to be tested within a week, store them at 2-8°C. If testing cannot be performed promptly, aliquot the samples according to the usage amount, and store them at -20°C or -80°C to avoid repeated freeze-thaw cycles. Serum, plasma, cell, and tissue extracts are typically diluted by 50% with a binding buffer.
• 6. The total protein concentration of homogenate should be at least 1 mg/mL. However, 2 mg/mL or more would be better.
• 7. The collected samples can be kept for different periods: 48 hours (2-8°C), 1 month (-20°C) or 6 months (-70°C).
• 8. The protein concentration of your lysates can be determined by a total protein assay not inhibited by detergents such as the Bicinchoninic acid (BCA) assay. The volume of each sample can also be normalized to deliver the same amount of total protein for each assay.

Cell Culture Supernatant

• Centrifuge cell culture media at 1,500 rpm and 4°C for 10 min.
• Assay immediately or aliquot supernatant and hold at -80°C (Avoid freeze/thaw cycles).

Cell Culture (Conditioned) Media

Since serum tends to contain cytokines which may produce significant background signals, we suggest the preparation of serum-free or low-serum medium samples. If it is necessary to test serum-containing medium, we also suggest running an uncultured medium blank to obtain the baseline signals which can then be subtracted from the cultured media sample data.

Cell Lysate

• Collect and rinse cells in PBS.
• Homogenize and lyse cells thoroughly in lysis buffer (e.g. Mammal Cell Protein Extraction Reagent, Boster Catalog Number: AR0103).
• Centrifuge cell lysate at approximately 10,000 x g and 4°C for 5 min.
• Assay immediately or aliquot supernatant and hold at -80°C (Avoid freeze/thaw cycles).

Guidelines on lysis buffer

• Avoid more than 0.1% SDS or other strongly denaturing detergents (In general, non-ionic detergents such as Triton X-100 or NP-40 are best. Zwitterionic detergents such as CHAPS or mild ionic detergents such as sodium deoxycholate will work).
• Do not use more than 2% v/v total detergent.
• Avoid the use of sodium azide.
• Avoid more than 10 mM reducing agents (e.g. dithiothreitol, mercaptoethanols).

Tissue Homogenate

• Rinse tissue with PBS to remove excess blood.
• Chop tissue into 1-2 mm pieces on ice in an ice-cold buffer, keep on ice for immediate homogenization or at -80°C for later use.
• Prepare the extraction buffer. It can be prepared ahead of time and stored at 4°C.
  • 100 mM Tris, pH 7.4
  • 150 mM NaCl
  • 1 mM EGTA
  • 1 mM EDTA
  • 1% Triton X-100
  • 0.5% sodium deoxycholate
• Immediately before use, the extraction buffer must be supplemented with the following to generate a complete extraction buffer.
  • Phosphatase inhibitor cocktail
  • Protease inhibitor cocktail
  • PMSF (Phenyl Methyl Sulfonyl Floride) to 1 mM
• For every 0.1 mg of tissue, add 500 µL of complete extraction buffer to the tube and homogenize.
• Rinse the blade of the homogenizer 2X with a 500 µL extraction buffer.
• Place the sample on a shaker at 4°C for 1 hour.
• Centrifuge the sample at approximately 10000 X g for 5 min.
• Assay immediately or aliquot supernatant (soluble protein extract) and hold at -80°C (Avoid freeze/thaw cycles).

Tissue (Bone)

• Extract de-mineralized bone samples in 4M Guanidine-HCl and protease inhibitors.
• Dissolve the final sample in 2M Guanidine-HCl.

Serum

• Collect whole blood into a tube without additives.
• Keep the blood at room temperature for 30 min.
• Centrifuge at 3,000 rpm and 4°C for 10 min.
• Assay immediately or aliquot supernatant (serum) and hold at -80°C (Avoid freeze/thaw cycles).

Note: Hemolysis should be avoided while collecting serum samples. Those samples that have undergone hemolysis may increase non-specifically staining in HRP-conjugated ELISA assay.

Plasma

• Collect whole blood into a tube containing anti-coagulant (Different protein may require different anti-coagulant, see datasheet for details on what anti-coagulant use).
• Centrifuge at 3,000 rpm and 4°C for 10 min.
• Assay immediately or aliquot supernatant (plasma) and hold at -80°C (Avoid freeze/thaw cycles).

Urine

• Collect urine into a sterile or disposable container (Fresh urine sample must be used immediately or saved to avoid the reproduction of bacteria which produce endogenous HRP for giving potential false-positive results).
• Centrifuge sample at 10,000 x g for 1 min.
• Assay immediately or aliquot supernatant and hold at -80°C. (Avoid freeze/thaw cycles).

Saliva

• Collect saliva using a collection device without any protein binding or filtering capabilities (e.g. Salivette).
• Centrifuge at 10,000 x g and 4°C for 2 min.
• Assay immediately or aliquot supernatant and hold at -80°C (Avoid freeze/thaw cycles).

Milk

• Centrifuge at 1500 x g and 4°C for 15 min.
• Collect the aqueous fraction and repeat 3X this process.
• Filter through a 0.2 µm filter.
• Assay immediately or aliquot supernatant and hold at -80°C (Avoid freeze/thaw cycles).