TECHNOLOGY

1. Sample Processing methods in special circumstances

Regarding high-fat samples：High-fat samples contain fat and are not homogeneous solutions,  direct loading will affect the binding of antigen and antibody, resulting in inaccurate measurement results.It is recommended to centrifuge at a high speed of 5000×g first, let it stand for 10 minutes, take the clearer liquid in the middle layer, and then centrifuge it at a high speed (≥5000×g) to take the clear liquid in the middle and lower layers for detection.

 

Regarding hemolyzed samples：After the sample is hemolyzed, the endogenous HRP enzyme and the HRP-like properties of hemoglobin will cause uncontrollable nonspecific coloring in ELISA, affecting the accuracy of the test results.If the sample is slightly hemolyzed, the sample can be diluted and tested to reduce its impact or centrifuged to obtain the supernatant if the concentration range allows. If hemolysis is severe, it is recommended to re-collect samples.

 

2. Sample Processing methods that require crushing

Cell extract solution：Adherent cells were gently washed with cold 1× PBS, then digested with trypsin and collected by centrifugation at 1000×g for 5 minutes; suspended cells can be collected directly by centrifugation.Washing the collected cells three times with cold 1×PBS. Resuspend each 10⁶ cells in 150-200ul 1×PBS (it is recommended to add protease inhibitors to PBS; if the content is very low, reduce the volume of PBS) and break the cells by repeated freezing and thawing or ultrasound. Centrifuge the extract at 2-8℃, 1500×g for 10 minutes, and take the supernatant for detection.

 

Tissue homogenate: Rinse the tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood, weigh and cut the tissue into pieces.Mixing the chopped tissue with the corresponding volume of PBS (generally at a weight-to-volume ratio of 1:9, for example, 1 g of tissue sample corresponds to 9 mL of PBS. The specific volume can be adjusted appropriately according to experimental needs, and keep records.It is recommended to add protease inhibitors to PBS) and add it to a glass homogenizer and grind it thoroughly on ice. In order to further lyse tissue cells, the homogenate can be repeatedly frozen and thawed or ultrasonically disrupted. Finally, the homogenate is centrifuged at 2-8°C, 5000×g for 5-10 minutes, and the supernatant is taken for detection.

 

Stool samples: Add PBS buffer (0.01M, pH=7.4; 0.05M EDTA can be added optionally) to the feces at a ratio of 9mL/g, shake on ice for 15 minutes, and then centrifuge at 2-8°C, 5000×g for 5-10 minutes, and collect the supernatant for detection.

 

Paw tissue samples: To anesthetize the animal, it is necessary to remove the stratum corneum such as the nails, and only take the skin and soft tissue under the nails, and process this part of the tissue in the same way as tissue homogenization.

 

PMSF Reagents: Stored at 10 mg/mL, its effective concentration is 17~174 μg/mL, prepared according to the effective concentration. Tissue homogenate needs to strictly follow the 1:9 ratio, which requires 17~177 microliters of PMSF. If the cell extract is resuspended in 150-200ul PBS, 3-35ul of PMSF is required. Here it is recommended to give it in the amount of 150ul/1.5ul or 200ul/2.0ul for easy experimental operation.

 

3. Treatment of samples similar to cell supernatants

Urine: Collect urine samples, usually the middle of morning urine, and add them to sterile tubes; Samples were centrifuged at 2-8 ° C, 1000×g, for 15-20 min to remove impurities and cell debris. Take the supernatant for detection.

 

Saliva: Fasting 1 hour in advance and gargle to keep the mouth clean. Do not drink water or brush your teeth half an hour before sampling. At 2-8℃, centrifuge at 4000×g for 10 minutes to remove impurities, and take the supernatant to detect. A fresh saliva sample is recommended.

 

Bone marrow fluid: It is recommended to use anticoagulant tube for anticoagulation and rest, centrifuge at 2-8℃, 1000×g for 30min for 15 min, and take the supernatant for detection. It is recommended that the bone marrow fluid be treated and tested directly after extraction. If it cannot be detected immediately, the supernatant after centrifugation should be frozen. Bone marrow is an important hematopoietic tissue, and the untreated samples will have hemolysis after direct freezing and thawing, so ELISA detection is not applicable.

 

Alveolar lavage fluid: 10% chloral hydrate solution, abdominal anesthesia; open the abdominal cavity, take another 10mL syringe to collect blood from the inferior vena cava, and drain as much blood as possible. Lung lavage on the left lung on the ice. 5mL of normal saline at 37℃ was taken and irrigated three times: 2mL, 1.5mL and 1.5mL. The recovery rate is more than 70%; The alveolar lavage solution was centrifuged at 4℃, 1000×g, for 10min, and the supernatant was detected.

 

Joint fluid: Use a sterile syringe and needle to puncture the joint cavity and gently extract the joint fluid. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected.

 

Ascites: Puncture the abdominal cavity with a sterile syringe and needle to extract ascites. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected.

Cerebrospinal fluid: The subject is anesthetized, the spinal cord cavity or ventricle is punctured with a sterile needle, and the cerebrospinal fluid is slowly extracted. Collect in sterile containers. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected.

Nasal lavage solution: Use PBS(0.01M,pH=7.4) to inject the solution into the nasal cavity through a sterile syringe. The nasal lavage solution is recovered through the nasal cavity and collected in a sterile container. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected.

 

Sputum: The viscous part of sputum is weighed, and 0.1% DTT(dithithreitol) is added to double the amount of sputum. The main function of DTT is to dissolve mucus, blow with a straw repeatedly, oscillate with a whirlpool for 15S, and oscillate in a 37℃ constant temperature water bath for 5 minutes. Add PBS(0.01M,pH=7.4) twice the amount of sputum and continue oscillating for 15-20 minutes, filter with 150 mesh, centrifuge at 1500 rpm for 10 minutes, and take the supernatant for detection.

 

Prostatic fluid: There are two sampling methods for the preceding adenous fluid, transrectal massage method and prostate puncture method. The transrectal massage method first anesthetize the sample object, the operator wears sterile gloves, sterilizes the area around the anus, and then enters the rectum through the anus. Through the rectum, the operator can reach the prostate gland. Gently massage the prostate with your fingertips to promote the secretion of prostatic fluid. Massage can be pressed along both sides of the prostate toward the midline to help release prostate fluid. During the massage, the prostate fluid flows out naturally and is collected through sterile cotton swabs, tubes or needles. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected. The subject is first anesthetized and fluid is drawn directly from the secretory gland of the prostate using a sterile puncture needle. The puncture site is usually the lower pole of the prostate or another easily accessible area. The prostatic fluid obtained from the puncture was transferred to a sterile container. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected. After sampling, hemostasis should be applied to the puncture site and appropriate care should be given to the subject.

Semen: An abstinence period of 2-7 days should be maintained before semen collection to ensure semen quality and quantity are within the standard range. Semen should be collected directly in sterile containers. Normal semen is a thick jelly outside the body, after injection, it needs to be liquefied in room temperature or 37℃ water bath to make the prostate secreted by fibrinolytic enzyme to become thin. After the semen was completely liquefied, the semen was centrifuged at 4000rpm for 10 minutes to separate the seminal plasma for detection.

 

Follicular fluid: The subject was anesthetized, the location of follicles in the ovaries was determined with an ultrasound probe, and mature follicles suitable for collection were selected. The follicle is punctured with a thin needle, the tip of which is inserted into the follicle; Fluid from the follicle is sucked through a syringe or vacuum suction tube. Each mature follicle usually has a certain amount of follicular fluid. The fluid drawn from the follicle is transferred into a sterile tube. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected.

 

Vaginal secretions: Use appropriate instruments (such as cotton swabs, sampling sticks) to collect secretions from the subject (need to pay attention to the sampling time) and put them into a sterile tube. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected.

 

Milk: Collect milk by manually massaging the breast and squeezing the nipple with your hand. Pay attention to hand hygiene and avoid infection during operation. An electric or manual breast pump can also be used, usually for prolonged collection or for cases where the secretion volume is large. The breast pump must be clean and sterile. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected. Please pay attention to the physiological state of the subject and the sampling time!

 

Aqueous humor: A sterile syringe is used to puncture the anterior chamber of the eyeball of the subject to collect aqueous humor. Aqueous humor samples were collected in sterile containers. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected.

 

Tear fluid: It is usually collected by stimulating the lacrimal gland to secrete tear fluid. The common method is to use irritating eye drops, such as saline, sodium chloride, etc., to gently drop into the eye, or to use irritating substances (such as light eye massage) to promote tear gland secretion. Use a straw to collect tears directly from the corner of the eye and collect them in a sterile container. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected.

 

Bronchial fluid: The subject is anesthetized, and the bronchoscope is inserted through the patient's nasal cavity or mouth. The bronchoscope is inserted along the airway into the lungs until it reaches the small airway at the end of the lungs. A certain amount of saline (usually 10-30mL) is slowly injected into a part of the lungs (such as the bronchi, alveolar area). This flushes out the alveoli and bronchus in the area, bringing their cells, microbes, secretions and other substances back into the airway. The perfused fluid is sucked back into the bronchoscope, and the collected fluid is bronchial fluid. In order to obtain more bronchial fluid, multiple infusions and recycling can be performed. Bronchial fluid was collected in a sterile container. The collected biological fluids were centrifuged at 4℃, 1000×g, for 20 minutes, and the supernatant was detected.