Sample Processing Methods of ELISA

Serum  

Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g, 2-8°C. Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles. Centrifuge the sample again after thawing before the assay.

Plasma  

Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g, 2-8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles. Centrifuge the sample again after thawing before the assay.

Cell Culture Supernates  

Remove particulates by centrifugation for 15 minutes at 1000 x g, 2-8°C and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw cycles.

Urine  

Collect urine using a metabolic cage. Remove any particulates by centrifugation for 15 minutes at 1000x g, 2-8°C and assay immediately or aliquot and store samples at -20°C or -80°C .Avoid repeated freeze-thaw cycles. Centrifuge again before assaying to remove any additional precipitates that may appear after storage.

Tissue Homogenates  

100mg tissue was rinsed with 1X PBS, homogenized in 1 mL of 1X PBS and stored overnight at -20°C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g, 2-8°C. The supernate was assayed and removed immediately. Alternatively, aliquot and store samples at -20°C or -80°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

Cell lysates  

Remove media and rinse cells once with ice-cold PBS (pH 7.0-7.4). Scrape cells off the plate and transfer to an appropriate tube. Dilute cell suspension with 1xPBS (pH 7.0-7.4), until cell concentration reached 100 million/ml. Then store overnight at -20°C. After two freeze-thaw cycles to break up the cell membranes, the cell lysates were centrifuged for 5 minutes at 5000 g, 2-8°C. Collect the supernatant. Cell lysates should be assayed immediately or aliquotted and stored at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.

Saliva  

Remove particulates by centrifugation for 15 minutes at 10,000x g at 2-8°C and assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.

Breast Milk  

Centrifuge for 15 minutes at 10,000x g at 2-8°C. Collect the aqueous fraction and centrifuge twice more for a total of 3 cycles. Assay immediately or aliquot and store at ≤-20°C. Avoid repeated freeze-thaw cycles.

Pleural effusion  

Remove particulates by centrifugation for 15 minutes at 10,000x g at 2-8°C and assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.

Bronchoalveolar lavage fluid  

Remove particulates by centrifugation for 15 minutes at 10,000x g at 2-8°C and assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.

lysate for RBC  

Centrifuge 100ul blood sample at 2000rpm for 5min at 2-8°C to collect the RBC. Remove supernate and add 10ml ice-cold PBS (pH 7.0-7.4). Then store overnight at -20°C. After three freeze-thaw cycles to break up the cell membranes, the cell lysates were centrifuged for 10 minutes at 10000rpm, 2-8°C. Collect the supernatant. RBC lysates should be assayed immediately after 5000-fold dilution or aliquotted and stored at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles 

Cerebrospinal fluid (CSF)  

Remove particulates by centrifugation for 15 minutes at 10,000x g at 2-8°C and assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.

Ascitic fluid  

Remove particulates by centrifugation for 15 minutes at 10,000x g at 2-8°C and assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.

Solid samples (feces)

Prepare with Sample Diluent (1:10). This can be achieved by adding 0.1g solid sample to 1ml of Sample Diluent. centrifuge and collect the supernates for assay.

Plant tissue  

1g plant tissue was rinsed with 1X PBS, homogenized in 1 mL of 1X PBS with a homogenizer, or with a ultrasonic disrupter until there is no significant tissue mass. Stored overnight at -20°C. After two freeze-thaw cycles were performed to break the cell wall/membranes, the homogenates were centrifuged for 10 minutes at 3000 x g, 2-8°C. The supernate was assayed and removed immediately. Alternatively, aliquot and store samples at -20°C or -80°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles.