ELISA test must see; ELISA kit use operation points

ELISA test must see; ELISA kit use operation points

Enzyme-linked immunosorbent assay (ELISA) is widely used in clinical practice because of its simple operation, high sensitivity, good specificity, and economical safety. However, due to its complicated operation steps, it generally includes reagent preparation, sample collection, sample loading, and temperature. Breeding, washing, color development, colorimetry, result determination, etc., any of the improper operations will have a great impact on the test results. Therefore, it is necessary to strengthen the overall quality control of the ELISA test to ensure the accuracy and reliability of the results.

1 reagent

Quality reagents are the basis for quality assurance. The reagents taken from the refrigerator at 4 °C must be allowed to stand at room temperature for 20 to 30 minutes before being activated. Otherwise, the formation of the hydration layer may affect the original concentration of the reagent and the uniform distribution of the solute molecules in the reagent. Unused reagents and controls should be sealed and returned to the refrigerator for storage. According to the characteristics of protein molecular distribution changes during the freezing process, the reagent should be fully mixed before it is officially activated. The distilled water, deionized water, and self-prepared buffer must be adjusted for pH and ionic strength. Different batches of reagents are difficult to ensure the same quality during the manufacturing process. Therefore, it is necessary to select and order long batch of reagents, and to ensure the storage conditions. Strict implementation of this standard can avoid the reagent batch number change and rebuild the quality control system and re-evaluate the reagents. The complex process and the stability of the results.

2 specimens

The patient specimen may contain immunological substances that interfere with the test, resulting in false positive or false negative results. The interference factors can generally be divided into two categories, namely endogenous and exogenous interference factors. Endogenous interfering factors generally include rheumatoid factor, complement, high concentration of non-specific immunoglobulin, heterophilic antibody and some autoantibodies, etc., avoiding endogenous interference factors mainly by selecting appropriate reagents; exogenous interference Factors include hemolysis of specimens, contamination of specimens by bacteria, excessive storage of specimens, and coagulation of specimens. Care should be taken to avoid severe hemolysis in the specimen. The hemoglobin in the specimen contains the heme gene, which has peroxide-like activity. Therefore, in the ELISA assay using horseradish peroxidase (HRP) as a marker, it is easy to be warm. During the incubation process, it is adsorbed to the solid phase to react with the added substrate to develop color. Specimens are stored for too long at low temperatures, and IgG can be polymerized into multimers, which can lead to deeper backgrounds and even false positive results in indirect ELISA assays. Normally, serum samples can be stored at 2 to 8 ° C for 1 week, and the cryopreservation time will be longer. Serum samples should be fully centrifuged, otherwise false positive results may result from non-specific adsorption of fibrinogen to the micropores. Cryopreservation of specimens avoids repeated freezing and thawing. Repeated freezing and thawing of specimens may cause damage to proteins and other molecules in the specimen due to the mechanical shearing force generated by the specimens, causing the antibody titer to decrease and causing false negative results. The collection and separation of specimens should be careful to avoid bacterial contamination. In addition, specimens will be unevenly distributed due to partial concentration of proteins during cryopreservation, so the reconstituted specimens must be mixed, but do not violently oscillate and repeatedly reverse.

3 operational factors

3.1 Loading

The sampler is a relatively sophisticated tool that can cause inaccurate sample loading due to mechanical wear or blood sticking in the rod during prolonged use, so the dispenser must be maintained and calibrated periodically. Each time you add different specimens, you need to replace the nozzle to avoid cross-contamination; the loading speed should not be too fast, so as not to add the specimen to the upper part of the hole wall, and be careful not to splash or generate bubbles. Pay attention to the operation time difference when loading. The impact on the results, especially on the results of competition law testing, is even greater. Therefore, it is recommended to use a multi-channel dispenser when adding the enzyme conjugate and substrate, or to add the sample at the same time to reduce the effect of the operation time difference on the results. In addition, some items need to be diluted before the test to reduce the non-specific reaction. The way to dilute is very important, the best way is to mix with the oscillator, you can not change the mixing method.

3.2 Incubation

3.2.1 Temperature of incubation: The temperature used for breeding is 43, 37 ° C, room temperature or 4 ° C. 37 ° C is the incubation temperature commonly used in the laboratory, and is also the optimum reaction temperature for most antigen-antibody binding. The antigen-antibody reaction generally reaches a peak at 37 ° C for 1 to 2 h. To accelerate the reaction, the reaction temperature can be increased within a certain range and continuously oscillated during the reaction to increase the probability of antigen-antibody contact. The antigen-antibody reaction is more thorough at 4 ° C, and the resulting product is more and more stable, but it is generally not used in ELISA assays because the time required is too long.

3.2.2 Ways of incubation: The incubation method often uses the thermostat method, the microwave irradiation method and the water bath method. Among them, the water bath method can better solve the difference in absorbance (ie, "edge effect") between the surrounding hole and the central hole due to uneven heat. The ELISA plate can be placed in a water bath, and the bottom of the plate should be placed against the water surface to quickly balance the temperature. To avoid evaporation, the plate hole can be covered with plastic sealing paper. If a thermostat is used, the ELISA plate should be placed in a wet box. The wet box should be made of a material with good heat transfer (such as metal), a wet gauze at the bottom of the box, and finally the ELISA plate should be placed on the wet gauze. Each reaction plate cannot be placed in an overlapping process during the incubation process to prevent unevenness in temperature diffusion.

3.3 Washing

Washing is a major feature of ELISA unlike homogeneous immunological detection techniques. Washing is not a reaction step throughout the ELISA reaction, but it is critical. The purpose is to remove components unrelated to the reaction in the reaction system, including unbound enzyme conjugates and interfering substances that are non-specifically adsorbed to the solid support during the reaction. The washing solution is usually a buffer containing a certain concentration of Tween 20, which is a non-ionic detergent containing both a hydrophilic group and a hydrophobic group. The mechanism of action in washing is by means of its hydrophobic group. Hydrophobic interactions are passively adsorbed on the hydrophobic groups of the protein molecules on the solid phase of the polystyrene to form hydrophobic bonds, thereby weakening the adsorption of the protein molecules and the solid phase. At the same time, under the combination of the hydrophilic group and the water molecules in the liquid phase, the protein molecules are promoted to leave the solid phase and enter the liquid phase, and finally eluted. Attention must be paid to the concentration of non-ionic detergents. The most common washing solution is 0.05% Tween20, a pH-neutral phosphate buffer. If the Tween20 in the washing solution exceeds 0.2%, it can be coated on a solid phase. The antigen-antibody desorption affects the lower limit of the assay. Washing can be done by washing machine or manual washing. Manual washing is divided into immersion and running water washing. Regardless of the method of washing the plate, the inside of the hole should be avoided when injecting liquid into the plate, otherwise the washing liquid will be difficult to enter the hole and the washing effect will be affected. When washing the plate with a washing machine, ensure that each liquid aspirating needle on the washing machine can be uniformly inserted into the bottom of the plate hole to completely absorb the washing liquid. It is required that the residual liquid after washing the plate is less than 2 μL, that is, the artificial gusset pad The paper is not wet and soaks for more than 45 s.

3.4 color development

In most cases, HRP and alkaline phosphatase (ALP) are used in ELISA products. The substrate catalyzed by HRP is hydrogen peroxide, and the coloring hydrogen donors participating in the reaction include o-phenylenediamine (OPD), o-toluidine (OT) and tetramethylbenzidine (TMB). Among them, OPD is hardly soluble in water, and it is easy to deteriorate. It is prepared before use. The reaction process should be protected from light and OPD has certain toxicity. The reaction product of TMB is blue, with clear contrast, stable nature and no toxicity to human body. If you choose a variety of acidic stop solutions, the blue color will turn yellow. When the TMB is used as a substrate, the detection wavelength of the product is 450 nm, and ALP is used as a label. The substrate is generally p-nitrophenyl phosphate (pNPP), and the product is yellow p-nitrophenol with an absorption wavelength of 405 nm. In order to ensure the stability of the results, it must be strictly in accordance with the temperature and time specified in the kit instructions, and the temperature and time should not be changed arbitrarily.

3.5 colorimetric

The results of ELISA color detection must be detected by a microplate reader, and the results cannot be judged by the naked eye. Because the color perception of different individuals is different, especially for the detection items of hepatitis B virus e antibody and hepatitis B virus core antibody, it is difficult for the naked eye to judge. . The microplate reader should be measured at two wavelengths, including two absorption peak wavelengths that are sensitive and insensitive to color products, and non-sensitive wavelengths are used to remove interference due to scratches, fingerprints, background, etc. on the container. The maximum absorption wavelength of TMB is 450 nm, and the non-sensitive wavelength is 630 nm. Generally, there is no blank hole, otherwise the phenomenon that the absorbance value is negative. After adding the stop solution, the color should be colorimetrically as soon as possible, otherwise the absorbance value of the sample will gradually decrease, and the deeper the color development, the faster the color change, and the color should be measured within 2 min.

3.6 Quality control of testing instruments should be strengthened

For example, the quantitative pipette should be calibrated regularly; the washing machine should dump the waste tank in time, replenish the washing liquid, run the rinsing procedure (double steaming) after starting up, check the system liquid system (with or without water leakage), check the liquid absorption and injection. Liquid speed, shutdown operation flushing procedure (double steaming), cleaning suction needle and water tank, the instrument appearance is clean; the microplate reader should regularly check whether the filter is qualified, whether the light source is stable, whether the mechanical system is faulty, keep the appearance clean, and Regularly ask the manufacturer's engineers for maintenance and repair; the water bath and the refrigerator that stores the reagents should monitor the temperature every day, and have records.

4 Determination and reporting of results

4.1 Determination of results

The result is judged strictly according to the positive judgment value (CO) provided by the kit. While the manufacturer provides the reagents, the CO values ​​listed in the manual are based on a series of scientific experiments and statistical studies, and should not be changed at will. Quantitative determination requires the preparation of a standard curve, and the results are calculated from the standard curve.

4.2 Gray area settings

Usually, the ELISA results are reported as “negative” or “positive”. There is a clear dividing line between the two, which is the so-called positive judgment value, ie, the CO value. The absorbance value (A) for the sample is higher than CO. The value is judged to be positive, and the opposite is judged to be negative. However, in practice, the population with the sample A value near the CO value often appears, that is, the "grey area" detected by the ELISA. In the domestic ELISA kit for detection of infectious pathogen antigens and antibodies, the setting of “grey area” is not involved, and the CO value alone is used to determine the presence or absence of infection, especially for screening blood donors. In the actual work, the author found that patients with results near the CO value have poor reproducibility and are also likely to cause medical disputes. Therefore, patients whose test results are located in the “grey area” can be confirmed by confirmatory test or follow-up test.

4.3 Review of specimens

The manual operation process of ELISA is complicated and has many influencing factors. Even if the quality control in the room is controlled, the results may be different due to the difference between the holes. Therefore, the development of the review system and the increase of the review can ensure the accuracy of the results, such as the hepatitis B. Review the results of the semi-" gray area" specimens and rare patterns. The hospital strictly implemented the review system in actual work and achieved good results.

5 quality control

5.1 Indoor Quality Control (IQC)

To ensure the accuracy and reliability of the test results, IQC must be done. First, the environment, equipment and hardware facilities of the laboratory must be in good condition. Secondly, the reagents and measurement methods should be idealized. In addition, the laboratory technicians must After good training. The clinical significance of the ELISA qualitative test is whether the pathogen is detected, regardless of the amount of detection. Therefore, quality control should ensure the sensitivity of the test. At present, there is no uniform standard for the quality control rules of domestic laboratory qualitative tests. More units use the “immediate method” and combine the method of LeveyJennings quality control chart. In addition to the negative control of the kit attached to the kit, at least 2 indoor controls should be selected, one of which is a weakly positive control close to the CO value, and the S/CO value should be 2 to 4. The other is a negative control. Carefully analyze the cause of IQC out of control each time, regularly compare the mean value and coefficient of variation of each test, find out the change of the detection ability of the kit in time, and take appropriate measures to improve the reliability of the test.

5.2 Inter-room quality assessment (EQA)

EQA is a retrospective evaluation that primarily controls the accuracy of laboratory results. The main point is to ensure that the same patient's specimens are treated like inter-room quality. It must be emphasized that EQA results must be analyzed together with IQC to find out the causes, improve the deficiencies in the work, and guide the laboratory to select the appropriate kit.

In summary, the ELISA detection operation steps are complicated, and there may be many factors affecting the measurement results, which are distributed in each step of the measurement operation. Therefore, it is necessary to strengthen the quality control of each link in order to obtain accurate and reliable detection results.

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