ELISA Protocol-Elisa Experiment Standard Operating Method (Part II)

Step 3: Add Sample

The specimen is serum: it is best to store the blood naturally for 1-2 hours, and then centrifuge at 3000 rpm for 15 minutes; the specimen is plasma: a blood specimen collection tube containing anticoagulant must be used, and the blood collection tube must be inverted immediately after blood collection Mix for 5-10 times. After standing for a period of time, centrifuge at 3000 rpm for 15 minutes; if it is tested within a few days, it can be placed in a refrigerator at 2-8°C, and if it is to be stored, it should be placed in a low-temperature refrigerator at -20°C.
Put the sample into the incubator in time after adding the sample.
After adding the enzyme reagent, wipe the surface of the microtiter plate with absorbent paper to dry.
If AT or other automatic sample addition is used, it is best to choose FAME or other post-processing instruments to add enzyme reagents.
When there are many specimens, please operate in batches.
The addition of serum samples is almost the only step to add samples using a micropipette. The key points that must be paid attention to when using the micropipette to add samples are: do not add the sample too fast, avoid adding to the upper part of the hole wall, and do not splash or generate bubbles. The sample addition is too fast, and the accuracy and uniformity of the micro sample addition cannot be guaranteed. The non-coated area added to the upper part of the hole wall can easily lead to non-specific adsorption. Spills can contaminate adjacent holes. When bubbles appear, the interface of the reaction liquid is different.

Step 4: Incubate

Incubation is the most critical factor affecting the success or failure of the ELISA assay. ELISA is a solid-phase immunoassay. The antigen-antibody binding reaction is carried out on the solid phase. To make the antigen or antibody in the liquid phase completely bind to the specific antibody or antigen on the solid phase, it must be reacted under certain temperature conditions. time. The time required for incubation is inversely proportional to the temperature, that is, the higher the temperature, the shorter the time required. The most commonly used incubation temperatures are 37°C and room temperature, followed by 43°C and 2-8°C. Strictly control the incubation time according to the operating instructions;
Attach a cover or cover during incubation to prevent the specimen or diluent from evaporating and adsorbing on the wall of the hole.

Step 5: Wash the Board

Make sure that the lotion fills the holes and the washing needle is unblocked. After washing the plate, it is best to gently pat dry on absorbent paper (choose a clean, no or less dusty absorbent material); when there are too many reaction plates, arrange them reasonably, or use more Several plate washer to avoid long waiting time.

Step 6: Color Development

The color developer should be prepared before use as far as possible, and the expired color developer should not be used. The light blue TMB color developer is not used; when adding samples, the color developer should not flow out.

Step 7: Termination

Avoid generating bubbles when adding stop solution, which may lead to false positive results.

Step 8: Read the Board

During the entire operation, ensure that the ELISA plate does not touch hypochlorous acid; realize the automation of ELISA testing standards as much as possible, and effectively improve the quality of testing.

In actual operation, in addition to selecting high-quality reagents, you must strictly follow the operating steps, and at the same time perform indoor quality control and inter-room quality evaluation, and test each specimen with a rigorous work style to ensure the quality of the test.

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