Processing of Common Samples in Elisa (Enzyme Linked Immunosorbent Assay) (2)

Elisa is a rapid, sensitive, accurate and reliable quantitative analysis method; sample preparation plays a pivotal role in a successful Elisa experiment. Collecting time, processing and storage all have influences on Elisa. Last time we introduce the common samples preparation including blood (serum, plasma), tissue homogenates, cell lysate and cell culture supernatant. This time we’ll present unusual samples such as skin, urine, feces, bronchoalveolar lavage fluid, saliva, cerebrospinal fluid, pleural effusion, prostatic fluid, semen, vaginal secretions, etc. for the reference.

1. Sputum

Sputum must be treated before assaying according to the following directions. Mix the sputum with 0.1 % DTT (dithiothreitol) (two fold volume) and shake for 5 minutes at 37°C.

Add PBS (two-fold volume) to shake again for another 15-20 minutes. After Filtering through a 150 mesh wire net, remove particulates by centrifugation for 10 minutes at approximately 1500 rpm.

2. Feces

Try to collect dry stool, please avoid watery stool as it is difficult to prepare and it will influence the accuracy of detection. The weight is better to be over 50 mg, washing three times with PBS(final fecal mass: PBS Volume = 1: 9), Centrifuging 5000 x g for 10 minutes after ultrasonic pulverization (or crushed), get the supernatant for detection.

3. Saliva

Collecting sample in centrifuge tube, then freeze sample at -70°C for 1 hour. Thaw it on ice, and centrifuge at 2,000 × g for 10 minutes. Transfer clarified supernatant to clean tube for use in the assay.

4. Seminal

Collecting semen in sterile containers, normal sperm in vitro after injection was thick jelly, then it needs to liquefy by fibrinolytic enzymes at room temperature or 37 degree water bath. Then centrifuging at 4000 rpm for 10 min to seperate seminal plasma for detection.

5. Skin tissue

On the day of the assay, the biopsies were allows to thaw and 20-50 mg of wet ti ssue was homogenized with the aid of a Polytron homogenizer in ice-cold PBS contai ning a protease-inhibitor cocktail. The homogenates were centrifuged for 20 minutes at 10,000 x g to remove debris and insoluble material.

6. milk

The milk sample should be centrifuged at 12000g/min with 30min at 4°C, after remove the suspended lipid, and the deposit, then the samples could be used in detection or stored for later use.

7. Cerebrospinal Fluid, Bronchoalveolar Lavage Fluid

After collecting, centrifuge samples at 1000 × g for 20 min and get the supernatants.

The processing of sample is to collect target proteins, as proteins are generally easy to be denaturated, degraded, so the process should be as mild as possible. The storage of samples is also very important, especially be assured not to go freeze/ thaw cycles. And samples could be split charging. The storage time should be less than one week if at 4°C, should not exceed 1 month if at -20°C , -80°C should not exceed 2 months. Before assaying, bring samples to room temperature, and heating samples is forbidden. The processing of samples is the first step in a successful experiment. Above all, this a sketch on how to proceess common samples for the reference.

For more information, please visit http://www.cloud-clone.us/.