• ELISA Sample Preparation

ELISA Sample Preparation

Preparing samples for ELISA

ELISA (Enzyme-Linked Immunosorbent Assay) is a technique used to quantify a target of interest in a sample. Common samples include blood (serum and plasma), tissue homogenates, cell lysates and cell culture supernatants. Depending on the target analyte, other samples may be tested such as skin, urine, feces, bronchoalveolar lavage fluid, pleural fluid, saliva, cerebrospinal fluid, prostatic fluid, semen, vaginal secretions and more. The result obtained by ELISA can be influenced by how samples have been collected, processed and stored. This page gives a general guideline on preparing and processing samples for ELISA.

• Blood
  • Serum
  • Plasma
• Tissue Homogenates
• Cells
• Cell Culture Supernatants
• Cell Lysates
• Sputum
• Feces
• Saliva
• Seminal Fluid (Semen)
• Skin Tissue
• Milk
• Cerebrospinal Fluid (CSF), Bronchoalveolar Lavage Fluid (BAL) and Urine

Blood

Whole blood samples contain many components (for further information, please see our composition of blood page). The main components are the blood cells (red blood cells, white blood cells and platelets) and the liquid component of blood. Whole blood can be centrifuged, allowing the blood cells to be separated from the blood liquid. Depending on how the blood is prepared, the resulting liquid will be serum or plasma.

Serum

Serum is a component of blood that does not contain blood cells or clotting factors. It is essentially the same as plasma, but without fibrinogens. Blood that is coagulated (clotted) yields serum without fibrinogens, though some clotting factors may remain.

Suggested Protocol:

1. Collect the serum using a serum separator tube and and allow to stand for 1-2 h at room temperature or overnight at 4°C.
   Note: Blood samples will clot naturally when left to stand; the lower the temperature the slower the clotting process.
2. Centrifuge for 20 min at 1000 × g.
3. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

Plasma

Plasma is a component of blood that makes up the extracellular matrix surrounding blood cells (red blood cells, white blood cells and platelets). Blood that is anticoagulated (not clotted) yields plasma, which includes fibrinogens and clotting factors. If the target analyte of interest is a coagulating factor, it is recommended to use plasma as a sample type rather than serum.

Plasma samples require anticoagulation. The choice of anticoagulant can depend on the target analyte of interest. Usually, 2% EDTA, 1% heparin or 3.8% sodium citrate are used. Hemolysis should be avoided as this can release protease enzymes, which could result in degradation of the target analyte present in the samples. High lipid concentrations can lead to hemolysis, so this should also be avoided.

Suggested Protocol:

1. Collect the plasma in a tube containing the desired anticoagulant.
2. Centrifuge for 15 min at 1000 × g at 2-8°C within 30 min of collection.
3. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

• Sodium Citrate
  

  Ca²⁺ present in the blood contributes to coagulation through calcium-dependent clotting pathways. Sodium citrate chelates free Ca²⁺ ions, inhibiting these pathways and therefore preventing coagulation.

Advantages: Suitable in most cases, does not affect coagulation factors.
Disadvantages: Weak anticoagulant effect, poor solubility.
Recommended Usage: Sodium Citrate (109 mmol/L) : Blood ratio of 1:9; or Sodium Citrate (106 mmol/L) : Blood ratio of 1:4.

• EDTA
  

  Ca²⁺ present in the blood contributes to coagulation through calcium-dependent clotting pathways. Ethylenediaminetetraacetic acid (EDTA) chelates free Ca²⁺ ions, which are no longer free and therefore coagulation is prevented.

Advantages: Suitable in most cases, does not affect red blood cells or white blood cells.
Disadvantages: Affects platelet aggregation, not suitable for detection of coagulation or platelet factors.
Recommended Usage: EDTA (15 g/L) : Blood ratio of 1:10

• Heparin
  

  Antithrombin III is a protein that inhibits several enzymes, mainly serine proteases, involved in the coagulation pathway. Heparin binds to antithrombin III, which activates it and causes antithrombin III activity to increase, and therefore enhances the anticoagulant effect.

  Advantages: Strong anticoagulant effect, does not alter blood cell volume, stable at high temperature, hard to hemolyse.
  Disadvantages: Causes leukocyte (white blood cell) aggregation, anticoagulant effect only lasts for a short while.
  Recommended Usage: Heparin (1 g/L) : Blood ratio of 1:10

Tissue Homogenates

The protocol for tissue homogenates will depend on the exact tissue that is used as the sample. The addition of protease inhibitors may be required to prevent the analyte of interest from being degraded. Some tissue samples such as liver, kidney, brain tissue may cause false positive results, as endogenous biotin will interact with avidin and streptavidin.

Suggested Protocol:

1. Rinse tissue in ice-cold PBS to remove excess blood. Weigh the tissue before homogenisation.
2. Finely mince the tissue and homogenise with a tissue homogeniser on ice in PBS and sonicate the cell suspension.
3. Centrifuge for 5 min at 5000 × g to remove any precipitates.
4. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

Cells

Depending on where the target analyte is expressed, cell samples may be suitable for assaying. Many factors can influence the detection result, including number of cells (more than 10⁶ cells may have a detrimental effect), pH, salt composition and concentration and so on.

Cell Culture Supernatants

Cell culture supernatants are a recommended sample type if the target analyte is a secreted protein.

Suggested Protocol:

1. Centrifuge for 20 min at 1000 × g to remove any precipitates.
2. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

Cell Lysates

Cells must be lysed before they can be assayed in ELISA. Cell lysis can be induced by physical (such as ultrasonication or freeze-thaw cycles) or chemical means (such as RIPA, Triton X-100, NP-40, SDS, sodium deoxycholate, β-Mercaptoethanol, DTT, urea). Generally, physical methods are preferred over chemical methods, as chemical detergents can influence the results obtained by ELISA. A concentration of 1% Triton X-100 and 1% NP-40 is recommended as this is sufficient for cell lysis whilst having no or little observable effect on ELISA absorbance values.

Suggested Protocol:

1. Detach adherent cells with trypsin, then centrifuge and collect the supernatant. For suspended cells, centrifuge and collect the supernatant.
2. Wash the cells three times in ice-cold PBS, and resuspend in PBS.
3. Lyse the cells by subjecting them to ultrasound four times. Alternatively, freeze-thaw the sample (-20°C to room temperature) three times.
4. Centrifuge for 10 min at 1500 × g at 2-8°C to remove any cellular debris.
5. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

Sputum

Sputum is a mixture of saliva and mucus found in the respiratory tract. It is expelled during coughing. Pathogenic bacteria may be present in sputum, which may be of interest for analysis by ELISA.

Suggested Protocol:

1. Measure the volume of the sputum sample. Add an equal volume of 0.1% dithiothreitol (DTT) to the sputum sample and mix for 5 min at 37°C.
2. Add an equal volume of PBS to the mixture (i.e. dilute 1/2) and mix for 15-20 min.
3. Filter the mixture, then centrifuge for 10 min at 1500 × g.
4. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

Feces

Fecal samples may contain pathogenic bacteria or high concentrations of an endogenous analyte, either of which can indicate disease. Dry feces samples are recommended; liquid stool samples can be difficult to prepare and can influence the detection result by ELISA.

Suggested Protocol:

1. Add PBS to at least 50 mg dry stool.
2. Wash three times with PBS, then resuspend to a fecal mass: PBS volume of 1:9.
3. Centrifuge for 10 min at 5000 × g.
4. Analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

Saliva

Saliva samples are suitable for detection of some enzymes, antimicrobial agents, and other secreted analytes.

Suggested Protocol:

1. Collect the saliva in a centrifuge tube, then freeze at -70°C for 1 h.
2. Thaw the sample on ice, then centrifuge for 10 min at 2000 × g.
3. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

Seminal Fluid

Suggested Protocol:

1. Collect the semen in a sterile container. If the sample is viscous, add fibrinolytic enzymes and leave at room temperature or at 37°C (using a water bath).
2. Centrifuge for 10 min at 4000 × g to separate the seminal plasma.
3. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

Skin Tissue

Suggested Protocol:

1. If samples are frozen, allow to thaw. Homogenise 20-50 mg wet skin tissue in ice-cold PBS containing a protease inhibitor cocktail.
2. Centrifuge for 20 min at 10,000 × g to remove debris and insoluble material.
3. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

Milk

Suggested Protocol:

1. Centrifuge for 30 min at 12,000 × g at 4°C to remove insoluble material and lipids.
2. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.

Cerebrospinal Fluid, Bronchoalveolar Lavage Fluid and Urine

Suggested Protocol:

1. Collect sample in a sterile tube and centrifuge for 20 min at 1000 × g. If precipitation occurs, centrifuge again.
2. Transfer the supernatant into a clean tube and analyse immediately or store at 4°C (up to one week), -20°C (up to one month) or -80°C (up to two months). Avoid freeze-thaw cycles. Bring samples to room temperature before carrying out the assay.