How to properly perform ELISA

How to properly perform ELISA

I. Collection and preservation of clinical specimens The most commonly used clinical specimens for ELISA are serum (plasma). Sometimes, for specific purposes, saliva, cerebrospinal fluid, urine, feces, etc. are also used. At present, the markers used in the clinical use of serum samples generally include antigens and antibodies of infectious pathogens, tumor markers, hormones, special proteins, cytokines and therapeutic drugs. For the collection of serum samples for the determination of hormones and therapeutic drugs, it should be noted that the collection time or even the body position may have an impact on the measurement results. If cortisone is between 4 and 6 in the morning, there will be a peak: growth hormone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) are released in a paroxysmal manner. Therefore, when measuring such hormones It is necessary to take several blood samples at closely spaced intervals, with the median value as the measured value. Another example is when the renin activity is significantly increased from the supine position to the standing position. Another example is the detection of therapeutic drugs, which should be selected according to the pharmacokinetics of the optimal time after taking the drug. The collection of serum samples for the detection of antigens and antibodies, tumor markers and specialty proteins of infectious pathogens has no time or positional effects, but the following aspects should be considered in terms of handling and preservation:
(1) Care should be taken to avoid severe hemolysis. Hemoglobin contains a heme group, which has a peroxide-like activity. Therefore, in an ELISA assay using HRP as a labeling enzyme, if the hemoglobin concentration in a serum sample is high, it is easily adsorbed during the incubation. In the solid phase, it reacts with the HRP substrate added later to develop color.
(2) In the collection of samples and serum separation, attention should be paid to avoid bacterial contamination. In the case of bacterial growth, some enzymes secreted by the bacteria may decompose proteins such as antigens and antibodies; secondly, endogenous enzymes of some bacteria such as The beta-galactosidase of E. coli itself produces non-specific interference with assays labeled with the corresponding enzymes.
(3) If the serum sample is isolated by aseptic operation, it can be stored at 2-8 °C for one week. If it is operated by bacteria, it is recommended to freeze it. Long-term storage of the sample should be below -70 °C.
(4) Frozen specimens of serum should be taken care of to avoid repeated freezing and thawing caused by power outages. The mechanical shear force generated by repeated freezing and thawing of the specimen will destroy the molecules such as proteins in the specimen, causing false negative results. In addition, the mixing of freeze-thaw specimens should also be noted, do not violently shake, and repeatedly invert and mix.
(5) If the specimen is turbid or floc due to bacterial contamination during storage, the supernatant should be taken after centrifugation and sedimentation.
Second, the reagents are prepared in the clinical laboratory, and the preparation of the reagents is generally not very noticeable. The usual practice is to take the reagents out of the refrigerator and use them during the experiment, ignoring the fact that this practice may affect the insufficient incubation time. The immediate consequence of the problem is the false negative of the detection of some weakly positive specimens. Therefore, the most important thing in the preparation of the reagent in the ELISA assay is that the kit is taken out of the refrigerator before the start of the experiment, and after being allowed to stand at room temperature for more than 20 minutes, the assay is performed to make the kit before use and at room temperature. balance. The purpose of this is mainly to enable the temperature in the reaction micropores to reach the required height relatively quickly in the subsequent incubation reaction step to meet the measurement requirements. Secondly, the washing liquid in the current commercial ELISA kit needs to be diluted and prepared in the laboratory when used, so the distilled or deionized water used for dilution should be of a quality. In addition, when the kit uses OPD as a substrate, the substrate solution should be temporarily prepared before the reaction develops color.
3. Adding serum samples and reagents In the current ELISA commercial kits, the addition of serum samples is almost the only step in the medical education network to add samples using a micropipette. The key point that must be paid attention to when using the micro-sampler is that the sample should not be too fast. Avoid adding it to the upper part of the hole wall, and do not spill or create bubbles. The loading is too fast and the accuracy and uniformity of the micro-dosing cannot be guaranteed. The non-coated area applied to the upper part of the pore wall is liable to cause non-specific adsorption. Splashing can contaminate adjacent holes. When bubbles appear, there is a difference in the interface of the reaction solution. The addition of reagents is basically added from the dropper bottle in the domestic kit. In addition to paying attention to the angle of the drop, the speed of the drop is also very important. The drop is too fast, and it is easy to repeat the drop or add to the two. The phenomenon between the holes, so that non-specific adsorption occurs in the non-coated areas in the holes, causing non-specific color development. Therefore, sometimes a specimen is tested positive with the same kit, and the next measurement is negative, which is often caused by the above-mentioned errors in loading and reagents.
4. Incubation incubation is one of the most critical factors influencing the success or failure of an assay in an ELISA assay. ELISA as a solid phase immunoassay, the antigen-antibody binding reaction is carried out on a solid phase. In order to completely bind the antigen or antibody in the liquid phase to a specific antibody or antigen on the solid phase, it must react under certain temperature conditions. time. The time required for incubation is inversely proportional to temperature, ie 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 to 8 ° C.
This step of incubation is the most problematic step in clinical ELISA assays. Usually, the current incubation time of the domestic ELISA commercial kit is 37 ° C for 30 minutes to 1 hour, and the imported ELISA kit is usually 37 ° C for 1 to 2 hours to have a complete combination. Below 1 hour, it may affect the determination. Lower limit. Therefore, regarding the incubation, the following points must be noted in the actual measurement operation:
Make sure there is enough reaction time at the set temperature. Generally, after the sample and / or reagents are added, when the microplate is taken from the room temperature to the water bath or the incubator, the temperature in the well rises from room temperature to 37 ° C, which takes a certain time, especially at room temperature. In the low and non-water bath state, this heating time may be longer, but in the clinical laboratory, very few people pay attention to this problem, usually when the microplate is placed in the incubator, it starts to time, so it is very It is easy to cause the problem that the incubation time is not enough in the actual measurement, and the weak positive sample cannot be detected. There was once a blood station in the south who raised a problem. In the winter of each year, there was always more than one month. In the indoor quality control of HBsAg measurement, the measurement was provided by the clinical testing center of the Ministry of Health. When ng/ml is weakly positive, it is always undetectable. I don't know why? This may be related to the low temperature in the southern winter. At this time, the incubation time at 37 °C after the microplate was transferred to the incubator was insufficient, so that the weak positive samples were negative. Therefore, in order to ensure sufficient incubation time at 37 ° C, the clinical laboratory can determine for themselves how long it takes for the microplate in the different seasons (different room temperature) to reach 37 ° C after taking the temperature from room temperature to the incubator. So as to properly extend the placement time of the slats in the incubator. The specific method is to use a small thermometer to place the plate hole reaction solution in the medical education network for measurement and observation.

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