【ELISA Sample Processing Guide】Serum / Plasma
In ELISA experiments, sample quality directly affects the accuracy of results. Even the best reagents and equipment cannot compensate for problems caused by improper sample handling.
Common issues such as protein degradation, contamination, and improper storage conditions often stem from negligence in sample processing. These seemingly minor details can lead to experiment failure or data deviation.
This guide will help you:
ü Master the correct processing procedures for different sample types
ü Avoid common sample processing errors
ü Establish standardized operating procedures
Correct sample handling is the first step to obtaining reliable ELISA results. Let us start from the basics to ensure every sample can achieve its full value.
Differences Between Serum and Plasma Components:
Serum and Plasma are samples obtained from blood through different processing methods. Due to the influence of the coagulation process, there are certain differences in their components, and these differences are reflected throughout sample preparation and storage. It is important to understand these before sample collection to choose the most appropriate sample preparation strategy.
Serum-Plasma Difference Comparison Table
| Dimension | Serum | Plasma |
|---|---|---|
| Collection Method | Supernatant sample obtained after blood naturally coagulates and is centrifuged to remove cell debris and blood clots | Sample obtained by adding anticoagulants (EDTA, heparin, sodium citrate, etc.) to blood and performing centrifugation |
| Coagulation Factors | Fibrinogen, factors II, V, VII, VIII, IX, X, etc. are consumed during coagulation and are almost absent in serum | All coagulation factors and fibrinogen are completely preserved |
| Platelet-Derived Proteins | During coagulation, after platelets are activated, the following types of cytokines are released in large quantities: Chemokines: CXCL5, CXCL12, CXCL2, etc. Inflammatory factors: IL-1β, IL-1α, IL-6, etc. Immunoregulatory factors: CD40L, sP-selectin, HMGB1 Growth factors: TGF-β1/TGF-β2, VEGF-A, FGF-2, etc. | Minimal platelet-derived proteins |
| Albumin | Similar content | Similar content |
| Globulin | Similar to plasma, but some globulins may be precipitated during the coagulation process. | Similar content |
| Metabolites | • Concentrations of amino acids, free fatty acids, acyl-carnitine, LPC, LPE, PC, SM, etc. are generally higher than in plasma (approximately 20% or more) • 104 metabolites are significantly higher in serum | • Some phospholipids (such as PI) are higher in plasma • Overall metabolite concentration is slightly lower but more stable |
| Enzyme Components | Naturally occurring enzymes in blood: • Serine proteases (such as thrombin, plasmin) • Cysteine proteases (such as trypsin inhibitor-bound enzymes) • Metalloproteinases (such as matrix metalloproteinases MMP-2, MMP-9) • Phosphatases/kinases (such as acid phosphatase, protein kinase C) • Lipases/phospholipases (such as phospholipase A2) However, during coagulation, platelets are activated and release a large amount of intracellular enzymes (such as phosphatases, esterases, proteases, phospholipases, etc.), resulting in higher enzyme content than in plasma. | Due to the absence of the coagulation process, in addition to naturally occurring enzymes in blood, the anticoagulation process also affects the activity of some enzymes. Please refer to the Serum and Plasma Enzyme Source Reference Table for specific details. |
| Cellular Debris/Peptides | Platelet activation produces a large number of peptides and low-molecular-weight fragments, with protein fragments accounting for approximately 75% in serum | Protein fragments account for approximately 25% |
| Red Blood Cells | Not included | Not included |
| White Blood Cells | Not included | Not included |
| Volume Yield | At the same hematocrit, serum yield is approximately 15-20% lower than plasma (due to clot occupying some volume) | Slightly higher yield |
| Processing Time | Need to wait for blood to fully coagulate (≥30 min) before centrifugation | Can be centrifuged immediately after blood collection |
| Sample Stability | Due to enzymes released during the coagulation process still present in the sample, and a small amount of residual white blood cells, serum composition changes over time. Long-term storage stability is poorer than plasma. | Due to the absence of coagulation process, no additional proteases are released, and anticoagulants inhibit enzyme activity, resulting in overall better long-term storage stability than serum. |
Serum Preparation
The following is the serum preparation process:
Collection - Coagulation - Centrifugation - Aliquoting - Storage
Preparation Work
| Item | Specific Requirements | Purpose |
|---|---|---|
| Blood Collection Tube | Use serum separator tubes (SST) or plain vacuum tubes without additives. The inner wall of SST contains coagulant + gel, which can automatically form a serum layer after centrifugation, facilitating aliquoting. | Unify serum production conditions and reduce errors caused by manual operation. |
| Needle Size | Commonly used 21-23G (diameter 0.6-0.8 mm) disposable needles. | Needles that are too thin can create negative pressure, leading to red blood cell rupture. |
| Tourniquet Use | Use only when necessary, for ≤1 min, then release immediately. | Prolonged tourniquet use can cause local blood stasis and increase the probability of red blood cell rupture. |
| Blood Draw Speed | Use smooth, uniform aspiration, avoid rapid or forceful aspiration. | Excessive negative pressure is the main cause of hemolysis. |
Blood Collection
Use disposable blood collection needles with Serum Separator Tube (SST) for blood collection. Throughout the process, pay attention to avoid blood sample contamination. After collecting the blood sample, gently invert 1-2 times to allow the coagulant to fully mix with the blood sample. Label and let stand.
Coagulation
| Precautions | Specific Requirements |
|---|---|
| Standing Time | Place the collected tubes at room temperature (20-25°C), horizontally, avoiding tilting. Coagulation time: 30 min-1h (SST recommended 30 min, regular tubes 45-60 min), until blood is completely coagulated with no flow. |
| Avoid Agitation | Prohibit vigorous shaking or tapping of the tube. Gently invert 1-2 times (only when using tubes with coagulant) to mix. |
| Temperature Control | Do not perform coagulation in refrigerator or high-temperature environments. Low temperature delays fibrin mesh formation, while high temperature accelerates enzyme activity, leading to protein degradation. |
Centrifugation
| Parameters | Recommended Range | Description |
|---|---|---|
| Speed | 1000-1500 × g (speed depends on centrifuge specifications) | Ensuring sufficient centrifugal force can effectively separate cells and cell debris from serum. |
| Time | 10-15 minutes | Ensure sufficient time for complete cell separation. |
| Temperature | 4°C or room temperature (20-25°C) | Low temperature helps reduce enzyme activity and minimize protein degradation. |
| Brake | Slow deceleration or no brake | Avoid sudden stopping that may disturb the separated layers. |
Tip： The serum layer should be clear and light yellow, with no cloudiness between the cellular layer and the gel layer (if using SST). If there is floating blood cells or the serum appears pink, it indicates hemolysis, which releases a large amount of endogenous HRP enzyme and can cause significant false positives. In such cases, a new blood draw is required.
Aliquoting
1. Operating Environment
Carry out the procedure in a clean biosafety cabinet or a clean bench. Wear disposable gloves and use sterile consumables to avoid contamination. Avoid additional enzymes introduced by contaminants that may cause protein degradation.
2. Aliquoting Method
Use low-binding centrifuge tubes (e.g., 0.5 mL, 1.5 mL, 2 mL) for single-use aliquoting. Avoid repeated pipetting.
Each tube should contain ≤ 80% of the tube's total volume, leaving sufficient space to prevent rupture due to expansion during freezing.
3. Addition of Inhibitors (Optional, Based on Experimental Requirements)
It is recommended to add inhibitors after centrifugation, but you must confirm in advance whether the inhibitors affect downstream experiments. Specific inhibitors can be referenced in the Reference Table for Enzyme Sources in Serum and Plasma.
Storage
| Storage Condition | Duration | Notes |
|---|---|---|
| Short-term (≤7 days) | 2-8°C | Store in refrigerator. Ensure sealed to prevent contamination and evaporation. |
| Long-term | -20°C or -80°C | Avoid repeated freeze-thaw cycles. It is recommended to aliquot in advance according to experimental needs. For -80°C storage, use specialized freezer-safe tubes. |
| Transportation | Ice pack or dry ice | Maintain low temperature during transportation to ensure sample stability. |
Plasma Preparation
The following is the plasma preparation process:
Collection - Anticoagulant Addition - Centrifugation - Aliquoting - Storage
Preparation Work
| Item | Specific Requirements | Purpose |
|---|---|---|
| Anticoagulant Type | EDTA (most common, concentration 1.5-2.0 mg/mL blood) Heparin (suitable for certain coagulation tests) Sodium Citrate (concentration 3.2% or 3.8%, ratio 1:9 with blood) | Prevent blood coagulation by chelating calcium ions (EDTA, citrate) or inhibiting thrombin activity (heparin). |
| Blood Collection Tube | Use dedicated vacuum blood collection tubes with anticoagulants. | Ensure accurate anticoagulant-to-blood ratio. |
| Needle Size | Commonly used 21-23G (diameter 0.6-0.8 mm) disposable needles. | Needles that are too thin can create negative pressure, leading to red blood cell rupture. |
| Tourniquet Use | Use only when necessary, for ≤1 min, then release immediately. | Prolonged tourniquet use can cause local blood stasis and increase the probability of red blood cell rupture. |
| Blood Draw Speed | Use smooth, uniform aspiration, avoid rapid or forceful aspiration. | Excessive negative pressure is the main cause of hemolysis. |
Blood Collection
Use disposable blood collection needles with dedicated plasma collection tubes for blood collection. Throughout the process, pay attention to avoid blood sample contamination. Immediately after blood collection, gently invert the tube 8-10 times to ensure proper mixing of anticoagulant with blood. Do not shake vigorously.
Centrifugation
| Parameters | Recommended Range | Description |
|---|---|---|
| Time | Perform centrifugation within 15-30 minutes after blood collection | Prolonged standing can lead to platelet activation and release of platelet-derived factors. |
| Speed | 1000-1500 × g (speed depends on centrifuge specifications) | Obtain platelet-poor plasma (PPP) by increasing centrifugal force or performing secondary centrifugation. |
| Time | 10-15 minutes | Ensure sufficient time for complete cell separation. |
| Temperature | 4°C or room temperature (20-25°C) | Low temperature helps reduce enzyme activity and minimize protein degradation. |
| Brake | Slow deceleration or no brake | Avoid sudden stopping that may disturb the separated layers. |
Tip: If the determination of platelet factors or coagulation factors is required, the handling time of plasma must be strictly controlled. Typically, centrifugation should be completed within 30 minutes after blood collection, and a second high-speed centrifugation is also required.
Secondary Centrifugation (Platelet-Poor Plasma, Requires Secondary Centrifugation)
| Parameters | Recommended Range | Description |
|---|---|---|
| Speed | 2000 - 3000 x g (speed depends on centrifuge specifications) | Further remove residual platelets and cell debris to obtain purer plasma (commonly referred to as Platelet-Poor Plasma (PPP)). |
| Time | 10-15 min | Sufficient to sediment platelets and cell debris, improving sample clarity. Completely sediment platelets to obtain pure plasma. |
| Temperature | 4 °C | When measuring platelet factors, to prevent mechanical stress during centrifugation from activating platelets, pre-cooled centrifuges are typically used and operations are performed at low temperature (on ice). This is to obtain platelets that are not activated in vitro for accurate measurement of endogenous platelet factor concentrations. |
| Brake | Light brake or turn off brake (depending on centrifuge) | Strong braking creates shock, resuspends blood cells, and increases the risk of hemolysis. |
Tip: The aliquoted plasma must be clear and transparent. If red turbidity appears (red blood cell rupture), it indicates sample hemolysis, and blood collection needs to be redone. Hemolysis causes hemoglobin, iron ions, and intracellular enzymes to appear in plasma, which severely interferes with experimental results.
Aliquoting
1. Operating Environment
Carry out the procedure in a clean biosafety cabinet or a clean bench. Wear disposable gloves and use sterile consumables to avoid contamination. Avoid additional enzymes introduced by contaminants that may cause protein degradation.
2. Aliquoting Method
After the first centrifugation, the blood separates into distinct layers: the clear top layer is plasma, the bottom consists of blood cells, and there may be a thin intermediate layer of white blood cells. Use RNase- and protease-free low-binding centrifuge tubes (e.g., 0.5 mL, 1.5 mL, 2 mL) for a single-step aliquoting. Avoid contacting the intermediate white cell layer and refrain from repeated aspiration. Typically, aliquot into small volumes ranging from 200 µL to 500 µL to prevent multiple freeze-thaw cycles.
Labeling: Affix labels directly onto the tube bodies, recording essential information such as the sampling time, subject ID, centrifugation parameters, and anticoagulant type.
3. Addition of Inhibitors (Optional, Based on Experimental Requirements)
It is recommended to add inhibitors after centrifugation, but you must confirm in advance whether the inhibitors affect downstream experiments. Specific inhibitors can be referenced in the Reference Table for Enzyme Sources in Serum and Plasma.
Storage
| Storage Condition | Duration | Notes |
|---|---|---|
| Short-term (≤7 days) | 2-8°C | Store in refrigerator. Ensure sealed to prevent contamination and evaporation. |
| Long-term | -20°C or -80°C | Avoid repeated freeze-thaw cycles. It is recommended to aliquot in advance according to experimental needs. For -80°C storage, use specialized freezer-safe tubes. |
| Transportation | Ice pack or dry ice | Maintain low temperature during transportation to ensure sample stability. Note: Avoid freezing EDTA plasma as this may cause cell lysis. |
Key Considerations
Common Pitfalls and Solutions
| Issue | Possible Causes | Solutions |
|---|---|---|
| Hemolysis | • Improper needle size • Excessive suction force • Vigorous mixing • Prolonged tourniquet use • Delayed centrifugation | • Use appropriate needle size (21-23G) • Maintain moderate suction force • Mix gently by inversion • Release tourniquet promptly • Centrifuge within specified time |
| Lipemia | • Recent meal intake • Certain medications • Metabolic disorders | • Fast for 8-12 hours before collection • Collect morning fasting samples • Consider using fasting plasma |
| Protein Degradation | • Prolonged standing at room temperature • Improper storage temperature • Repeated freeze-thaw cycles | • Process samples promptly after collection • Store at appropriate temperature • Avoid repeated freeze-thaw • Use protease inhibitors if necessary |
| Contamination | • Non-sterile equipment • Improper handling • Inadequate sealing | • Use sterile equipment • Follow aseptic procedures • Ensure proper sealing • Work in clean environment |
| Insufficient Volume | • Difficult blood draw • Improper collection technique • Inadequate anticoagulant ratio | • Use appropriate collection technique • Ensure proper anticoagulant-to-blood ratio • Plan for adequate sample volume |
Summary
Proper sample processing is crucial for obtaining reliable ELISA results. Key points to remember:
ü Choose the appropriate sample type (serum or plasma) based on experimental needs
ü Follow standardized operating procedures for sample collection, processing, and storage
ü Pay attention to details that may affect sample quality, such as hemolysis prevention and temperature control
ü Record all relevant information, including collection date, processing time, and storage conditions
ü Understand the differences between serum and plasma to make informed decisions for specific experimental requirements
We hope this guide helps you improve experimental success rates and obtain more reliable research results.