The Elisa experiment has been widely used clinically due to its high sensitivity and good specificity. However, each link in the operation has a greater impact on the detection effect of the experiment. If you are not careful, it may lead to incomplete color rendering and patterning. Wait for the result.
ELISA Protocol
The following step by step analysis of the factors that may affect the results of the Elisa test operation.
Step 1: Specimen Selection and Preparation
The most commonly used clinical specimens for ELISA determination are serum (plasma), and sometimes specimens such as saliva, cerebrospinal fluid, urine, and feces are also used for specific testing purposes. At present, the clinically used serum samples to determine the markers generally include antigens and antibodies of infectious pathogens, tumor markers, hormones, special proteins, cytokines, and therapeutic drugs. For the collection of serum samples used for hormone and therapeutic drug determination, it is necessary to pay attention to the collection time or even the body position that may affect the determination results. For example, cortisone will have a peak between 4 and 6 in the morning: growth hormone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) are all released in a paroxysmal manner. Therefore, when measuring these hormones , It is necessary to take several blood samples in closely connected time intervals, and use the median value as the measured value. Another example is when changing from a lying position to a standing position, the serum renin activity will increase significantly. Another example is the detection of therapeutic drugs, the best time after taking the drug should be selected according to the pharmacokinetics for blood sampling. The collection of serum specimens used for the detection of antigens and antibodies of infectious pathogens, tumor markers, and special proteins, etc., has no time and position impact, but the following aspects should be considered in the handling and storage:
- Pay attention to avoid severe hemolysis. Hemoglobin contains a heme group, which has a peroxide-like activity. Therefore, in the ELISA assay with HRP as the labeling enzyme, if the concentration of hemoglobin in the serum sample is high, it is easy to adsorb during the incubation process. In the solid phase, it reacts with the HRP substrate added later to develop color.
- During sample collection and serum separation, attention should be paid to avoid bacterial contamination as much as possible. For the growth of bacteria, some of the secreted enzymes may decompose proteins such as antigens and antibodies; the other is for some bacterial endogenous enzymes such as The β-galactosidase of Escherichia coli itself can cause non-specific interference with the assay method labeled with the corresponding enzyme.
- If the serum specimen is separated by aseptic operation, it can be stored at 2～8℃ for one week, if it is for bacteria operation, it is recommended to freeze preservation. The long-term storage of samples should be below -70°C.
- For frozen serum specimens, care must be taken to avoid repeated freezing and thawing caused by power outages. The mechanical shearing force produced by repeated freezing and thawing of the specimen will have a destructive effect on the protein and other molecules in the specimen, thereby causing false negative results. In addition, attention should be paid to the mixing of freeze-thaw specimens. Do not vigorously shake, just invert and mix repeatedly.
- If the specimens have turbidity or flocculation caused by bacterial contamination during storage, the supernatant should be centrifuged for testing.
Step 2: Preparation of Reagents
In clinical laboratories, generally little attention is paid to the preparation of reagents. The usual practice is to take the reagents out of the refrigerator and use them during the experiment, ignoring that this method may affect the problem of insufficient incubation time later. The consequence of this is false negatives on some weakly positive specimens. Therefore, the most important thing in the preparation of reagents in the ELISA assay is to take the kit out of the refrigerator before starting the experiment and place it at room temperature for 30-60 minutes before performing the measurement, so that the kit is at room temperature before use. balance. The purpose of this is mainly to enable the temperature in the reaction micropores to reach the required height quickly in the subsequent incubation reaction step to meet the measurement requirements.
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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