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ELISA (Enzyme-Linked Immunosorbent Assay) is a fast, sensitive, accurate, and reliable quantitative analysis method. Sample processing plays a pivotal role in the success of ELISA experiments. The types of samples tested using ELISA include common ones such as blood (serum, plasma), tissue homogenate, cell lysate, cell culture supernatant, as well as uncommon ones like skin tissue, urine, feces, bronchoalveolar lavage fluid, saliva, cerebrospinal fluid, pleural effusion, prostatic fluid, semen, vaginal secretions, etc. The collection time, processing method, and preservation of these samples can all affect the results of ELISA experiments. Below is a summary of the processing method for the uncommon ELISA semen sample, which we hope will be helpful to you.

1. Semen：

Collect semen in a sterile container. Normal semen, when ejaculated, appears as a thick, gelatinous substance. After ejaculation, it needs to be liquefied at room temperature or in a 37-degree water bath under the action of fibrinolytic enzymes secreted by the prostate, becoming thinner. After the semen is completely liquefied, centrifuge it at 4000 rpm for 10 minutes to separate the seminal plasma for testing.

2. Samples of alveolar lavage fluid and cerebrospinal fluid:

Samples were collected and centrifuged at 1000g for 20 minutes. After centrifugation, supernatant was collected and specimens were stored at -20 ° C to avoid repeated freezing and thawing.

3. Sputum sample

1) Select the more viscous part of sputum and weigh it, adding 0.1% DTT(dithithreitol) twice the amount of sputum. The main function of DTT is to dissolve mucus, blow repeatedly with a straw, oscillate 15s with a whirlpool, and oscillate in a 37 degree constant temperature water bath for 5 minutes;

2) Add PBS buffer twice the amount of sputum and continue oscillating for 15-20 minutes, filter with 150 mesh, and centrifuge at 1500 rpm for 10 minutes to absorb the supernatant for detection.

4. Saliva sample

Saliva is generally not easy to collect within half an hour after a meal, because the saliva that has just eaten is rich in salivary amylase, and the best time to collect is on an empty stomach.

Samples were collected in a centrifuge tube and frozen at -70 ° C for 1 hour. After melting the sample on the ice, centrifuge 2000×g for 10 minutes and take the supernatant test.

5. Milk sample

The milk was centrifuged at 4 degrees and 12000g for 30 minutes to remove the upper fat and the lower precipitate, and the middle layer sample was taken for testing.

6. Urine sample

Collect with sterile tube. Centrifuge for about 20 minutes (2000-3000 RPM). Collect supernatant carefully. If precipitate forms during storage, centrifuge again.

7. Fecal Samples

Try to collect dry feces. Excessively watery feces can be difficult to process and may reduce the accuracy of detection. The collected weight should be greater than 50 mg. Wash the feces three times with PBS (final feces mass to PBS volume ratio of 1:9). After ultrasonic crushing (or mashing), centrifuge at 5000×g for 10 minutes and take the supernatant for detection.

8. Cell Samples

According to the location of the target, cell lysate or cell culture supernatant can be selected as the sample. It should be noted that because there are many interfering factors in this type of sample, such as cell state, cell number (greater than 10^6), ph(about 7), salt ion concentration in the medium, sampling time, etc., there may be undetectable conditions.

If the target is secretory and mainly extracellular, the cell culture supernatant can be selected as the sample; if the target is mainly intracellular, the cell lysate is recommended as the sample type. Cell culture supernatant treatment method is relatively simple, take the cell culture supernatant, 1000×g centrifuge for 20 minutes, take the supernatant can be detected.

Cell lysate:

1) The adherent cells need to be digested with pancreatic enzymes first, and the cells can be collected by centrifugation (suspended cells can be collected directly by centrifugation);

2) Wash the collected cells with cold PBS for 3 times;

3) Cell lysis by physical method (can be broken by ultrasound first, and then repeatedly frozen and thawed) :

⇒ ultrasonic crushing: Take an appropriate amount of PBS to suspend the cells, and use ultrasonic wave of a certain power to treat the cell suspension, so that the cells crack sharply;

⇒ repeated freeze-thaw: Freeze the cells to be broken below -20 ° C and melt at room temperature for 3 times to make the cells swell and break;

4) Centrifuge the specimen at 2-8 ° C 1500×g for 10 minutes and collect the supernatant for later use.

In general, physical methods such as repeated freeze-thaw, homogenization, etc. will be better than chemical methods, because chemical methods introduce acids or bases, which may have an impact on the ELISA experiment. We also performed experiments to evaluate the effects of chemical extracts and detergents such as RIPA, TritonX-100, NP-40 and SDS, sodium deoxycholate, β-mercaptoethanol, DTT and urea on ELISA tests. According to our quality inspection results, high concentration of detergent will affect the final result of ELISA test. However, its effect is reduced by the reduction of detergent concentration. Compared with other chemically cracked buffer liquid phases, 1% Triton X-100 and 1% NP-40 have less effect on ELISA detection. If chemical extraction methods must be used, these two detergents are recommended to extract target proteins. Chemical cell lysis solution is used to decompose cells. If the detergent component will interfere with the antigen-antibody reaction and affect the detection effect, dialysis and ultrafiltration are recommended to remove the detergent component.

The process of handling samples is the process of collecting target proteins. Since proteins are prone to denaturation and degradation, this process should be as gentle as possible. Storage after sample processing is also crucial, and special attention should be paid to avoiding repeated freezing and thawing. After sample processing, they can be packaged and sealed for storage. Storage at 4 degrees Celsius should be less than 1 week, -20 degrees Celsius should not exceed 1 month, and -80 degrees Celsius should not exceed 2 months. Before using the specimens, they should be slowly equilibrated to room temperature without heating to melt. Sample processing is the first step to successful experiments. The above is a brief description of the special sample processing method for Elisa, for researchers' reference. 

9. Plant Samples

Preparation of Plant Tissue Homogenate

1. Take tissue blocks (0.1g to 0.5g, with a minimum of 5 to 10mg) and rinse them in ice-cold PBS. Dry them with filter paper, accurately weigh them, and place them into a 5ml homogenization tube.

2. Add 9 times the volume of homogenization medium to the homogenization tube at a ratio of weight (g) to volume (ml) of 1:9. Quickly cut the tissue blocks into smaller pieces using ophthalmic scissors while keeping the tube in an ice-water bath.

3. Homogenization methods: Manual homogenization and mechanical homogenization.

① Manual homogenization: Hold the homogenization tube with your left hand and insert the lower end into a container filled with ice-water mixture. With your right hand, insert the pestle vertically into the tube and rotate it up and down for dozens of times (6 to 8 minutes) to fully grind the tissue into a 10% homogenate.

② Mechanical homogenization: Use a tissue grinder at 10,000 to 15,000 rpm to grind the tissue up and down to make a 10% tissue homogenate. An internal cutting tissue homogenizer can also be used (homogenize for 10 seconds each time, with a 30-second interval, repeating 3 to 5 times, all performed in ice-water. The homogenization time can be appropriately extended). Observe under a microscope:

4. Centrifuge the prepared 10% homogenate using a conventional centrifuge or a low-temperature low-speed centrifuge at approximately 3,000 rpm for 10 to 15 minutes. Collect the supernatant for measurement.

II. Homogenization Medium

Generally, 0.05 mol/L Tris-HCl, pH 7.4 phosphate-buffered saline (PBS) is used. Customers can set the concentration according to the sample and measurement indicators to maintain an isosmotic environment for the sample.

III. Sample Storage:

If the plant tissue sample is not immediately measured, it can be frozen at low temperatures. The lower the temperature, the better. Without repeated freeze-thaw cycles, it can be stored for three months at temperatures below -20℃ and for six months at temperatures below -70℃.