Abbkine Recommended Sample Preparation Method for ELISA

The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. Some examples include: diagnosis of HIV infection, pregnancy tests, and measurement of cytokines or soluble receptors in cell supernatant or serum. The ELISA principle can be categorized into sandwich ELISA, competitive ELISA and indirect ELISA.

(Picture Source: https://microbenotes.com/enzyme-linked-immunosorbent-assay-elisa/ )

 

 

Abbkine supplies more than 7000+ ELISA Kits, and EliKine™ ELISA Kit is the most eye-catching part among it. We have clarified the sample preparation method for different samples as below for your convenient use.

 

A.Serum

Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4 °C before centrifugation for 20 minutes at approximately 1000×g. Assay freshly prepared serum immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.

 

B.Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2-8 °C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.

(Note: Samples should be centrifugated adequately and no hemolysis or granule was allowed)

 

C.Tissue homogenates

1.Please choose fresh samples

2.The weight of the sample is not less than 50mg, generally 1g, but it is not strictly required that each weight of the sample must reach the same level

3.The proportion of homogenate was selected to be 10%, which is equivalent to 1g of tissue and 9ml of homogenization buffer. The component of homogenization buffer: PBS(PH=7.2-7.4,concentration: 0.01mol/L)

4.Tissue pieces should be minced to small pieces which will be homogenized with a glass homogenizer on ice. (or using liquid nitrogen to milling)

5.The homogenates are then centrifugated for 15 minutes at 5000×g to collect the supernate.

 

D.Cell culture supernatants

Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.

 

E.Cell lysate

1) Digest adherent cells with trypsin, and collect cells by centrifugation (suspended cells can be directly collected by centrifugation);

2) Wash cells 3 times with cold PBS;

3) Physical method to break the cells (You can also use Ultrasonic breaking first, then freezing and thawing):

⇒ Ultrasonic breaking: Resuspend cells in propriate amount of PBS, and break cells with suitable ultrasound;

⇒ Repeat freezing and thawing: Freeze cells below -20 degrees, thaw at room temperature, repeat 3 times;

4) Centrifuge the sample at 1500 × g at 2-8 degrees for 10 minutes and collect the supernatant for later detection.

(Note: The quantity of cells shall be about 1×106)

If you need to use cell lysis buffer, please set one negative control (cell lysis buffer ) to deduct background value. Be careful not to contain protein denaturants in lysis buffer,  like SDS, it may affect the binding between antigens and antibodies. Suggested protocol as below;

Transfer the cell lysate to a centrifuge tube, centrifuge at 1,500 rpm for 10 minutes at 4 °C. Collect supernatant for detection.

 

F.Urine

Use a sterile tube to collect samples and centrifuge samples for 20 minutes at 2000×g – 3000×g. Thoroughly collect supernatants and assay immediately or store samples in aliquot at 4°C (1 week), -20°C(1 month) or -80 °C(2 month) for later use. Avoid repeated freeze/thaw cycles. If there are sediment during storage, please centrifuge again. (Note:Samples should be should be equilibrated to room temperature before use, don’t heat to dissolve.)

For the other types of sample inquiry, please email service@abbkine.com, our technical specialist will come back to you shortly.

Refer to our ELISAs at https://www.abbkine.com/product-category/assay-kit/elisa-kit/ .

 

About Abbkine Scientific Co., Ltd.

Abbkine offers services for global scientists in the fields of proteomics and cytology, and is committed to the innovation and development of various scientific reagents related to proteomics and cytology, to promote the development of fields including life science research and drug discovery. Proteomics products cover the preparation of samples (protein extraction, purification, coupling), protein quantification, antibodies and kits for protein detection. Cytology products involve cytokines (cell culture), cell status detection, cell staining, organelle extraction, cell metabolism and cytopathology reagents (kits). Abbkine relies on the product portfolio and unique marketing support as the main market strategy and product innovation mode, to help your research career!

 

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