ELISA Sample Preparation & Collection Guide

If you are using serum, collect blood using a serum separator tube (SST) and allow the sample to clot for approximately 30 minutes at room temperature. Centrifuge for 15 minutes at 1000 × g, remove the serum, and assay immediately or aliquot and store at ≤ -20 °C.

Best Practices

• Allow complete clot formation before centrifugation. 
• Avoid hemolyzed samples whenever possible. 
• Grossly lipemic or grossly icteric samples may also affect performance for certain assays. 
• Store samples in aliquots to minimize freeze-thaw cycles. 

If you are using plasma, collect blood using EDTA, heparin, or citrate as the anticoagulant. Centrifuge within 30 minutes of collection for 15 minutes at 1000 × g. Remove the plasma and assay immediately or aliquot and store at ≤ -20 °C.

Best Practices

• Process samples promptly after collection. 
• Select the anticoagulant recommended for your assay. 
• Avoid repeated freeze-thaw cycles. 
• Different anticoagulants can affect analyte measurements differently, and assays are validated for only certain plasma anticoagulants, so consult the product datasheet before selecting one. 

If your assay requires platelet-poor plasma, first prepare plasma as described above. Collect and keep samples on ice during collection and prior to centrifugation. Then perform a second centrifugation at 10,000 × g for 10 minutes at 2–8 °C to remove residual platelets before analysis.

Why this matters

Platelets can secrete cytokines and growth factors during processing and storage, potentially affecting biomarker measurements. Keeping samples cold or on ice during collection and centrifugation helps mitigate secretion prior to platelet removal.

If you are analyzing secreted proteins from cultured cells, centrifuge the culture medium at 500 × g for 5 minutes to remove cells and debris. Collect the clarified supernatant and assay immediately or aliquot and store at ≤ -20 °C.

Best Practices

• Avoid disturbing the cell pellet. 
• Analyze freshly collected samples whenever possible. 
• Store clarified supernatants in aliquots and avoid repeated freeze-thaw cycles. 

If you are using tissue homogenates, rinse tissue with PBS to remove excess blood before homogenization. Homogenize in PBS, perform freeze-thaw cycles if required to disrupt cell membranes, then centrifuge at 5000 × g for 5 minutes. Collect the clarified supernatant for analysis or store at ≤ -20 °C.

Best Practices

• Process tissues consistently between samples. 
• Keep samples cold during preparation. 
• Remove debris completely before analysis. 
• Consider adding protease inhibitors (and phosphatase inhibitors, if needed) to the PBS prior to homogenization. 

If you are measuring intracellular proteins, lyse cells using an appropriate lysis buffer and incubate on ice for approximately 30 minutes. Centrifuge at 14,000 × g for 5 minutes to remove insoluble material. Transfer the clarified lysate to a clean tube, determine total protein concentration, and assay immediately or store aliquots at ≤ -20 °C.

Best Practices

• Use a lysis buffer compatible with your ELISA. 
• Include protease inhibitors when appropriate. 
• Normalize samples based on total protein concentration. 

If you are preparing tissue lysates, rinse tissue with PBS, mince into small pieces, and homogenize thoroughly. Add an equal volume of a lysis buffer (such as RIPA buffer) containing protease inhibitors and incubate for approximately 30 minutes with gentle mixing. Centrifuge to remove insoluble material, determine protein concentration, and assay immediately or store aliquots at ≤ -20 °C.

Best Practices

• Use consistent tissue-to-buffer ratios. 
• Keep samples cold throughout preparation. 
• Quantify protein concentration before analysis. 

If you are using saliva, collect the sample into a clean collection tube and centrifuge at 10,000 × g for 5 minutes. Carefully transfer the clarified aqueous phase for immediate analysis or aliquot and store at ≤ -20 °C.

Best Practices

• Minimize mucus contamination. 
• Avoid food contamination before collection. 
• Freeze aliquots immediately if not analyzing the same day. 

If you are using urine, collect the first morning mid-stream sample into a sterile container whenever possible. Centrifuge to remove particulate matter and assay immediately or aliquot and store at ≤ -20 °C.

Best Practices

• Collect samples using sterile containers. 
• Remove particulates before analysis. 
• Record collection time if comparing multiple samples. 

If you are analyzing human milk, centrifuge samples for 15 minutes at 1000 × g at 2–8 °C. Carefully collect the aqueous fraction and repeat this clarification step a total of three times to remove lipids before analysis. Assay immediately or store aliquots at ≤ -20 °C.

Best Practices

• Thorough lipid removal improves assay consistency. 
• Avoid disturbing the fat layer during collection. 
• Store clarified samples in aliquots. 

Yes. Most ELISA samples can be frozen for later analysis. Aliquot samples before freezing to avoid repeated freeze-thaw cycles, which may reduce analyte stability.

Not always. While many ELISAs support both sample types, some assays are validated for only serum or only plasma. Always consult the product datasheet for approved sample types.

Most ELISAs are compatible with EDTA, heparin, or citrate plasma, but compatibility can vary depending on the analyte. Follow the recommendations provided for your specific assay.

Repeated freezing and thawing can degrade proteins and introduce variability, potentially affecting assay sensitivity and reproducibility.

Centrifugation removes cells, debris, and particulates that may interfere with antibody binding or increase background signal, helping ensure more accurate and reproducible results.