How to prepare samples for ELISA

ELISA assay is a procedure that is important in helping to quantify that amount of a target that is present within a given sample. It is vital to prepare the samples correctly, this will be fundamental in determining the success of the ELISA test.

Once the samples have been prepared, they can be aliquoted and frozen down for longer term storage. The following storage conditions are recommended: 2-8°C for 5 days, -20°C for 6 months and -80°C for 2 years. It is also advised to avoid repeated freeze-thaw cycles when retrieving samples from storage, that is why it is better to store the samples in smaller aliquots.

There are many different samples that can be used for ELISA methods. Some of the most common samples will be discussed below and general instructions will be provided on how best to prepare each of these. However, we would recommend that the specific instructions provided with each kit are followed since there might be slight variation in the protocol from one kit to another.

Different sample types

Blood sample

Serum: This does not contain any blood cells or clotting factors; it is like plasma without the fibrinogens.
(a). Allow the whole blood sample to be collected using a serum separator tube by either allowing it to stand for 2 hours at room temperature or overnight between 2-8°C. It is important to note that this is a natural blood coagulation method. Also, this clotting process will slow down if the temperature is lowered.
(b). Sample is then centrifuged for 20 mins at 1000 x g.
(c). Supernatant is transferred into a clean tube.
(d). Can analyse immediately or can freeze down (-20°C or -80°C) for later use.

Plasma (EDTA, Citric Acid, Heparin): These samples require anticoagulation, and this will depend on the target analyte being used.
(a). Collect the plasma in a tube that contains the required anticoagulant and mix gently.
(b). Sample is centrifuged for 15 minutes at 1000 x g at 2-8°C within 30 mins from when the sample was collected.
(c). Supernatant is transferred into a clean tube.
(d). Can analyse immediately or can freeze down (-20°C or -80°C) for later use.

EDTA: Functions by chelating free Ca2+ in the blood, thereby preventing coagulation. Anticoagulant working concentration for EDTA is 10ml blood consisting of 12mg EDTA.
– Advantages: Minimum influence on leukocyte and erythrocyte morphology.
– Disadvantages: Can interfere with platelet aggregation, therefore not recommended for platelet functional detection and coagulation assays.
Citrate (Sodium Citrate): Functions by chelating free Ca2+ in the blood, thereby preventing coagulation. The anticoagulant working concentration of citrate is 0.25% (ratio of 1:16 between anticoagulant and blood when using 4% anticoagulant).
– Advantages: Ideal for majority of cases since it is unable to affect coagulation factors.
– Disadvantages: It is a weak anticoagulant, displaying low solubility in blood
Heparin: Can lead to enhanced anticoagulant effect by binding to antithrombin III, this results in activating and increasing the activity of antithrombin III. Whereas the inactive form of the serine protease is prevented from forming thrombin and blood coagulation. Pure 10mg heparin is found to display anticoagulant effect present in 65-125 ml of blood.
– Advantages: It displays a strong anticoagulant effect, hard to homolyse, thermotolerant and does not alter blood cell volume.
– Disadvantages: Can result in white blood cells (leucocyte) aggregation, indicating that the anticoagulant effect will only last over a short period of time.

Tissue sample

Nearly all of tissue samples are found to be processed into tissue homogenates. The protocol used will largely be dictated by the extracted tissue that is going to be used as a sample. There may be a need to include additional protein inhibitors to minimise or prevent any further degradation of the analyte.
(a). The tissue must be dissected using clean tools, it must then be placed on ice quickly to prevent any potential degradation of proteinase.
(b). Wash the tissue using pre-cooling 0.01M PBS buffer (pH 7.4), this should remove any residual blood that may be present.
(c). Then weigh the tissue before it is homogenised.
(d). Centrifuge the processed homogenate at 5000 x g for 5 minutes.
(e). Transfer the supernatant into a clean tube.
(f). Can analyse immediately or can freeze down (-20°C or -80°C) for later use.

Some tissue samples have been found to display false positive results, this is due the endogenous biotin interacting with the avidin and streptavidin. Examples of these tissues include kidney, liver, pancreas, and brain tissues.

Cell sample

Cell Lysates: First the cells need to be lysed so that they can be used for ELISA analysis. The lysis step can either be chemical process using reagents such as Triton X-100, sodium deoxycholate, RIPA, SDS, DTT and urea or it can by a physical process such as using repeated freeze thaw cycles or ultrasonication. In general, physical procedures are the better option since the use chemicals could have a downstream effect on the ELISA results.
(a). The adherent cells are detached into a suspension using a solution of trypsin, they are then centrifuged, and the supernatant is collected into a clean tube. Cells that already in suspension, can be centrifuged and the supernatant collected.
(b). Cells are added to pre-cooling PBS and washed three times.
(c). Cells are then lysed using either chemical or physical methods. For example: 0.5ml of RIPA lysis buffer can be added, ultrasound applied four times or freeze-thaw samples four times.
(d). Apply centrifuge at 1500 x g for 10 minutes to remove cellular debris that may be present.
(e). Transfer the supernatant into a clean tube.
(f). Can analyse immediately or can freeze down (-20°C or -80°C) for later use.

Cell Culture Supernatant: This sample type is highly recommended when investigated a secreted protein as the target analyte.
(a). Centrifuge 1000 x g for 20 minutes, this will remove any precipitates that may be present.
(b). Transfer the supernatant into a clean tube.
(c). Can analyse immediately or can freeze down (-20°C or -80°C) for later use.

Saliva, milk and urine sample

These samples are becoming more popular since they are much less invasion collection procedure when compared to blood samples. These are frequently used for detecting large number of anti-microbial agents, analytes that have been secreted and many other biological enzymes.
(a). Sample is collected in a sterile centrifuge tube.
(b). It is then centrifuged at 10,000 x g at 2-8°C for 2 minutes or at 2000-3000 rpm for 20 minutes.
(c). Transfer the supernatant into a clean tube.
(d). Can analyse immediately or can freeze down (-20°C or -80°C) for later use.

Sputum sample

Sputum can be described as a mixture of mucus and saliva that is predominately present in the respiratory tract. It is normally expelled from the lungs through coughing. There are many pathogenic bacteria that may be present in the sputum that could be of particular interest.
(a). The viscous portion of the sputum is weighed.
(b). Add equal amounts of 0.1% DTT (functions in dissolving the sputum) to the weighed sputum and mix at 35°C for 5 minutes.
(c). Add equal amount of PBS to the mixture (1/2 dilution), continue to mix for further 15-20 minutes.
(d). Use a wire mesh to filter the mixture.
(e). Centrifuge at 1500 rpm for 10 minutes.
(f). Transfer the supernatant into a clean tube.
(g). Can analyse immediately or can freeze down (-20°C or -80°C) for later use.

Cerebrospinal fluid, ascites and bronchoalveolar lavage fluid (BALF) sample

(a). After collecting the sample, centrifuge at 1,2000 rpm for 20 minutes.
(b). Check to make sure there is no precipitation, need to re-centrifuge if precipitation occurs.
(c). Transfer the supernatant into a clean tube.
(d). Can analyse immediately or can freeze down (-20°C or -80°C) for later use.

Semen sample

(a). Semen sample should be collected in a sterile container.
(b). Place the container in a water bath either at room temperature or at 37°C. This will allow the fibrinolytic enzymes to make the semen thin and liquefied.
(c). Centrifuge liquefied semen for 10 minutes at 4000 rpm, this is to separate the seminal plasma.
(d). Transfer the supernatant into a clean tube.
(e). Can analyse immediately or can freeze down (-20°C or -80°C) for later use.

Stool sample

(a). Greater that 50mg of dry stool sample is collected. It is important to note that diluted stool samples are harder to process and are much more difficult to detect since the accuracy is reduced.
(b). Wash stool sample three times using PBS.
(c). Following centrifugation step, re-suspend with PBS (1g stool to 9ml PBS ratio). Can use an ultrasound to pulverise the solution.
(d). Centrifuge at 5000 x g for 10 minutes.
(e). Transfer the supernatant into a clean tube.
(f). Can analyse immediately or can freeze down (-20°C or -80°C) for later use.

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