Usually we can use it forELISAThere are various types of samples for testing, such as serum, plasma, urine, cell culture supernatant, or tissue homogenate, and the processing methods for different types of testing samples are different. Proper handling of samples is a guaranteeELISAThe steps for the correctness and accuracy of detection are briefly introduced below, along with the processing methods for different types of samples.

1、 serum

Serum is the most commonly usedELISAThe type of sample being tested is also relatively easy to process.

Collect blood samples using test tubes or centrifuge tubes that are free of pyrogen and endotoxin, and leave the test tubes or centrifuge tubes at room temperature2Hour or4Overnight at ℃ to allow serum to precipitate. (Tilting the test tube or centrifuge tube to increase the cross-sectional area of the liquid surface can facilitate greater precipitation of serum.)4℃1000×gcentrifugation20minuteCarefully collect the supernatant. Suggest dividing the serum into multiple portions,-20℃ or-80Store at ℃ to avoid repeated freezing and thawing.

Hemolysis should be avoided during the blood collection process, as red blood cell lysis releases substances with peroxidase activityHRPFor markingELISAThere may be non-specific color development during testing, which can lead to inaccurate detection. At the same time, bacterial contamination should also be avoided, as bacteria may contain endogenous substances inside their bodiesHRPThis leads to false positives in the detection.

2、 plasma

Collect blood samples using blood collection tubes or centrifuge tubes containing anticoagulants, and after sample collection30mininside4℃ 1000×gcentrifugation15minTake the supernatant, which is plasma. Divide the supernatant into multiple portions,-20℃ or-80Store at ℃ to avoid repeated freezing and thawing. Avoid using specimens with hemolysis or hyperlipidemia.

The commonly used anticoagulants areEDTAHeparin sodium and sodium citrate, etc., should also be carefully read from the kit instructions during testing to check if the kit has special requirements for anticoagulants.

3、 Cell culture supernatant

Take the cell culture supernatant and transfer it to a centrifuge tube,1000×gcentrifugation20minRemove cell debris and impurities, take the supernatant,-20℃ or-80Store at ℃ to avoid repeated freezing and thawing.

4、 cell lysate

1） Suck out the culture medium from the culture plate, digest the cells with trypsin, and add an appropriate amount of culture medium to blow the cells off the culture plate. Suspended cells can be omitted.

2） Collect cell suspension,1000×gcentrifugation10minDiscard the culture medium and use pre cooledPBSrinse3Next time.

3） Add an appropriate amount of pre coolingPBSOr resuspend the cells in cell lysate (with protease inhibitor added before use). usually6The cell count required for one well of an orifice plate150~250μL PBSResurrected.

4） Place the sample in-20℃ or-80Freeze the sample at ℃, then thaw it at room temperature and repeat the process several times to fully lyse the cells. The sample can also be subjected to ultrasonic fragmentation to achieve the purpose of cracking.

5） 4℃10000×gcentrifugation10minRemove cell debris and take the supernatant,-20℃ or-80Store at ℃ to avoid repeated freezing and thawing.

5Tissue homogenate solution

1） Use organizational samplesPBS （0.01M, PH 7.4）Rinse off residual blood or impurities on the surface of the tissue.

2） Weigh the tissue block, record it, and cut it into pieces. The fragments should be as small as possible to facilitate more thorough homogenization

3） Add the organization to the pre cooled system in a certain proportionPBS(Add protease inhibitor before use) homogenize, and place on ice or in an ice bath during homogenization. (Usually based on organizational weight:PBSvolume=1:9The proportion of homogenate, for example1gCorresponding organizational samples9mLofPBSThe specific volume can be adjusted appropriately according to experimental needs. When calculating the sample concentration after detection, it should be multiplied by the corresponding dilution factor

4） Suck the homogenate into a centrifuge tube,4℃5000×gcentrifugation5~10minTake the supernatant,-20℃ or-80Store at ℃ to avoid repeated freezing and thawing.

6、 Urine, saliva, and other liquid biological samples

1000×gcentrifugation20minTake the supernatant for detection.

Overall, becauseELISAOnly soluble protein content can be detected, so it should be ensured that all samples are clear liquids, and sediment or suspension should be removed by centrifugation.

To ensure the accuracy of the detection, it is saved in-20℃ or-80The sample at ℃1~6Testing within one month;4Samples stored at ℃ should be1Conduct testing within the week.

In addition, it should be ensured that the sample does not containNaN₃BecauseNaN₃Will inhibitHRPThe activity leads to false negative results.