High quality reagents, good instruments and proper operation are necessary to ensure the accuracy and reliability of ELISA test results.
The operation of ELISA varies with the formation of solid - phase carriers.
This article will describe the attention of the various steps of plate ELISA points (bead type, tubular type and magnetic ball ELSIA, reagents, with a special instrument used abroad both have detailed instructions, must strictly follow the procedures).
The adoption and preservation of specimens
We usually can be used for ELISA test of sample is there are many types, such as serum, plasma, urine, clear cell culture or tissue of slurry, etc., the early treatment of different types of test sample is not the same.
The correct treatment of samples is the first step to ensure the correctness and accuracy of ELISA detection. The following is a brief introduction to the treatment of different types of samples.
serum
Serum is one of the most commonly used samples for ELISA detection, and the treatment is relatively simple.
With no pyrogen, endotoxin test tube or centrifugal tube of blood samples, place the tube or the centrifugal tube at room temperature for 2 hours or 4 ℃ for the night, make serum separation.
(it is better to tilt the tube or centrifuge to increase the cross section of the liquid surface, so that the serum can be precipitated out more.)
Centrifugal 20 min 4 ℃ 1000 x g, carefully collected supernatant.
Suggest that serum dispatched more than - 20 ℃ or 80 ℃ preservation, avoid repeated freezing and thawing.
Blood should be avoided in the process of hemolysis, because red blood cells, cracking will release with peroxidase activity material, in for HRP labeled ELISA detection are nonspecific in color, and lead to the detection accuracy.
It should also avoid bacterial contamination, since the bacteria may contain endogenous HRP, leading to false positives.
The plasma
By containing anticoagulant blood vessels or centrifugal tube of blood samples, specimens collected within 30 min after 4 ℃ 1000 x g 15 min, the centrifugal supernatant or plasma.
The supernatant dispatched more than - 20 ℃ or 80 ℃ preservation, avoid repeated freezing and thawing.
Avoid using hemolytic or hyperlipidemic specimens.
The commonly used anticoagulants are EDTA, heparin sodium, sodium citrate, etc., and the test kit should be carefully read in the test kit to check whether the kit has special requirements for anticoagulants.
Cell culture is clear
In cell culture supernatant to centrifuge tube, 1000 x g centrifugal 20 min, remove debris and impurities, take supernatant, - 20 ℃ or 80 ℃ preservation, avoid repeated freezing and thawing.
Cell lysis fluid
1) the culture medium in the culture plate was absorbed, and the cells were digested with the pancreatic enzyme, and the cells were blown off the culture plate by the appropriate medium.
Suspension cells may be omitted.
2) collect cell suspension, 1000 x g centrifuge 10min, discard the culture medium, and wash three times with precooled PBS.
3) add a moderate amount of precooled PBS or cellular lysis (before using the protease inhibitor) to rehang the cells.
Normally the amount of cells in a hole of 6 holes is 150 ~ 250 mu L PBS.
4) put samples in - 20 ℃ or 80 ℃, make samples frozen, put samples thawing at room temperature, several times repeated freezing and thawing, make cells full pyrolysis.
The samples can be broken by ultrasonic to achieve the purpose of cracking.
Centrifuge for 10 min 5) 4 ℃ 10000 x g, remove debris, take supernatant, - 20 ℃ or 80 ℃ preservation, avoid repeated freezing and thawing.
homogenizate
1) wash tissue samples with PBS (0.01 M, PH 7.4) to remove blood or impurities from the surface of the tissue.
2) to weigh the tissue and cut it after recording, the broken pieces should be as small as possible, so as to make the slurry more fully.
3) mix the tissues in a certain proportion to the pre-cooled PBS (before using the protease inhibitor) to mix the slurry and place it in ice or ice bath.
(usually by tissue weight: volume = 1:9 proportion of PBS homogenate, such as 1 g tissue samples corresponding to 9 ml of PBS, specific volume can be adjusted according to the experiment, testing samples is calculated after concentration should be multiplied by the corresponding dilution multiples).
4) learn well slurry to the centrifugal tube, 4 ℃ 5000 x g centrifugal 5 ~ 10 min, take supernatant, - 20 ℃ or 80 ℃ preservation, avoid repeated freezing and thawing.
Urine, saliva and other liquid biological samples
20min for 1000 x g centrifuge.
In general, because ELISA can only detect soluble protein content, it should be ensured that all samples are clarified liquid, and the precipitation or suspension should be centrifuged.
In order to ensure the accuracy of the detection, save in - 20 ℃ or 80 ℃ test samples of the best in 1 ~ 6 months;
4 ℃ of samples should be kept within 1 week for testing.
In addition, the samples should be guaranteed not to contain NaN3, because NaN3 will inhibit the activity of HRP, leading to false negative results.
Preparation of reagents
Prepare the reagent for the test according to the instructions of the kit.
Distilled water or deionized water used in ELISA, including for washing, should be fresh and high quality.
The self-matching buffer and cleaning solution are measured by pH meter.
The test reagent removed from the refrigerator should be used after the temperature and room temperature balance, and the general room temperature balance shall not be less than 30min.
The part of the kit that is not needed in the kit should be returned to the refrigerator in time.
Add the sample
In ELISA, there are usually three additional sample steps, namely, the addition of the specimen, the enzyme binding, and the substrate.
When adding samples, add the added items to the bottom of the hole of LEISA, avoid adding to the wall of the hole, and be careful not to splash out, do not create bubbles.
The specimen is generally used to add the sample to the plate hole.
The mouthpiece should be replaced every time the specimen should be replaced to avoid cross-contamination and a disposable quantitative plastic tube (generally not recommended).
Some indicators, such as indirect method ELISA, require diluted serum, which can be diluted and diluted in the test tube.
It is also possible to add the diluent in the plate hole and then add the serum samples to it, then oscillate for 1min on the tiny vibrator.
The process of adding the liquid and substrate working fluid can be used to make the process of adding liquid to be completed in the shortest time.
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