Processing of Uncommon Samples in ELISA

Previously we introduced the common samples preparation methods including the preparation of blood (serum, plasma), tissue homogenates, cell lysates, etc. Now we’ll present preparation methods of uncommon samples, such as sputum, bronchoalveolar lavage fluid, saliva, milk, hydrothorax and ascite, semen, etc.

1. Sputum 

Weight the viscous part of sputum, mix the sputum with 0.1 % DTT (dithiothreitol) (two fold volume) and shake for 5 minutes at 37°C. Add PBS (two fold volume) and shake again for another 15-20 minutes. After Filtering through a 150 mesh wire net, remove particulates by centrifugation of approximately 1,000×g for 10 minutes. Collect the supernatant and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use.

2. Bronchoalveolar Lavage Fluid

Centrifuge at 1,000 × g for 20 minutes. Collect the supernatant and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

3. Saliva 

Collect samples with a collection device or equivalent. and centrifuge at 1,000× g for 15 minutes at 2-8℃. Collect the supernatant and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

Note: saliva should be collected during starvation rather than within half an hour after meals, because the saliva is rich in salivary amylase after meals.

4.Red cell Lysates

1) Blood is centrifuged at 1,000 × g for 20 minutes. Discard the supernatant and collect cells.

2) Wash cells three times with cold PBS (pH 7.0-7.2).

3) Resuspend cells in cold PBS and freeze at -20℃. Then thaw and mix gently. Freeze and thaw repeatedly for three times.

4) Centrifuge at 5,000×g for 10 minutes at 2-8℃ to remove cellular debris.

5) Collect the supernatant and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

5. Milk

Centrifuge samples for 15 minutes at 10,000×g at 2 - 8℃. Collect the aqueous fraction and centrifuge twice more for a total of 3 cycles. Assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

6. Semen

Collect semen in sterile containers and liquefy it at room temperature or in water bath at 37 ℃. Then centrifuge at 4000 rpm for 10 minutes after fully liquefying to separate seminal plasma for test.

7. Hydrothorax and ascite

In order to avoid clotting, cell degeneration, bacterial destruction and autolysis during samples collection, EDTA-K2 anticoagulant should be used for routine and cytological examination of hydrothorax and ascites, and heparin anticoagulant should be used for biochemical test. Centrifuge at 2500 rpm for 5 minutes and collect the supernatant and assay immediately or store samples in aliquot at -20℃ or -80℃ for later use. Avoid repeated freeze/thaw cycles.

In addition to samples from animal, plant samples also need to be processed before assaying. Two kinds of preparation methods for plant samples are shown below for reference.

Extraction of Chemical Molecules from Plant Samples

1) Weigh 1g fresh or preserved sample (fixed with 100% methanol and placed in the refrigerator at -10 ℃ until assay). Mince the materials to small pieces and homogenize them in cold 100% methanol for 5 minutes.

2) Shake or stir the homogenate at 4 ℃ for 24 hours and then filter.

3) Shake the residue with 2ml methanol solution for 1h. Repeat twice and mix the filtrate.

4) Evaporate the filtrate on a rotary evaporator under reduced pressure at 36 ~ 38 ℃ until the final volume of the solution is 2ml.

5) Add 1ml petroleum ether to extract part of the pigment, discard the petroleum ether layer and take the lower methanol liquid layer.

6) Continue to concentrate the solution to aqueous solution. The aqueous solution contains IAA, ABA, GA, CTK, etc.

Protein Extraction from Plant Samples

1) Prepare the extraction solution and put it on ice. The volume of the extract solution is determined by the weight of the sample (For example, add 3.5mL extraction solution in 1g plant sample. This ratio can be adjusted according to different sample types).

2) Put plant samples in a mortar and grind it with liquid nitrogen. Then add it to the extract solution and leave it on ice for 3-4 hours.

3) Centrifuge at 8000 rpm for 40 minutes at 4℃ or 11100 rpm for 20 minutes at 4℃.

4) Collect the supernatant for the test.

The accuracy and reliability of ELISA test depends on high-quality reagents, precise instrument and correct experimental operation. Correct operation is not only the experimental operation, but also the samples preparation.

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