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Indirect immunofluorescence detection of autoantibodies

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The standard technique for autoantibody diagnosis is indirect immunofluorescence, which is characterized by strong specificity and obvious signal intensity contrast between positive and negative samples. Microscopic observation can accurately determine the distribution of fluorescence in tissues or cells. The position of the autoantigen determines the typical fluorescence pattern of its corresponding antibody, and the staining of all areas not related to this typical pattern is considered non-specific staining. Even with occasional strong non-specific staining, weak specific signals can be recognized. The indirect immunofluorescence method does not require complicated and time-consuming chemical preparation procedures. For experiments such as enzyme-linked immunosorbent assay, to obtain high specificity, the antigen must be extracted and purified and adsorbed on a solid carrier. If the antigen is not pure, The relevant antibody will have a false positive result.

The indirect immunofluorescence method retains the complete antigen spectrum of the original matrix, so a large number of antibodies can be detected simultaneously. Obtain higher detection efficiency. When antibodies against several different antigens need to be tested simultaneously on a biological substrate (such as anti-nuclear antigen antibodies), or when it is difficult or complicated to prepare antigens for each experiment one by one, immunofluorescence should be selected. In addition, the application of immunofluorescence can also detect some unknown antibodies.

When there is no fluorescence microscope, try to use enzyme-labeled secondary antibody instead of fluorescently labeled antibody, but the quality of the detection will be significantly reduced. Because in the immunofluorescence method, the indicator staining is produced by the antibody directly binding to the cell antigen, while in the immunoenzymatic method, the staining is diffusely surrounding the area around the antigen. Therefore, the immunoenzymatic method is not always able to distinguish the small differences in antigen distribution like the immunofluorescence method. Moreover, when using enzyme staining, many autoantibodies cannot be distinguished or cannot be detected at all. Pure photometric evaluation using only immunoenzyme staining of nuclear preparations is also not advisable, because this inevitably leads to some autoantibodies being missed.

Laboratory workers need to undergo certain professional training and continuous practice before they can be competent for the work of reading under the fluorescence microscope. The result judgment under the microscope is a manifestation of comprehensive knowledge. So far, there is no instrument that can replace manual reading.

(1) Selection of experimental substrate and dilution of serum

  1. The choice of experimental substrate The effect of indirect immunofluorescence on the determination of autoantibodies depends to a large extent on the quality of the substrate (including the quality of the substrate itself and the quality of preparation). These experimental substrates are usually processed with animal or human cells or tissues through a series of methods, such as the cultivation, adsorption and fixation of Hep-2 cells, the preparation of frozen tissue sections, and patches. Many domestic laboratories are still making their own experimental substrates, which have become increasingly unsuitable for the needs of experimental standardization, standardization, and modernization. It is recommended that commercial kits with advanced production technology and strict quality control measures should be selected as much as possible to ensure experimental results. Accurate and reliable. To detect different autoantibodies, experimental matrices from different sources (different tissues of different animals) should be selected.
  2. Dilution of serum   Generally speaking, a sample to be tested, to determine one or more autoantibodies, usually need to determine an initial dilution. If the test result of the dilution is negative, the antibody or multiple antibodies are considered to be insignificant (no clinical value), and there is usually no need to dilute the specimen for further testing. If the test result of the dilution is positive, it is considered that the antibody is obviously present (may have clinical significance), and the specimen can be further diluted to obtain the titer of the antibody. Therefore, it is very important to determine the appropriate initial dilution of the specimen to be tested. Since the initial dilution of the serum to be tested is obtained in combination with clinical statistics, and the methodologies used by different laboratories are different, the initial dilution of the serum may be different in different laboratories. In addition, in order to avoid the occurrence of prozone phenomenon, sometimes suspicious negative specimens need to be further diluted for determination.

(2) Standardized titration plate technology for experimental operation

The standard procedure of the experiment is to drop the sample and label the antibody onto the sample plate, and then cover the bio-sheet slide in the groove on the surface of the sample plate. At this time, all the bio-sheets on the slide are in contact with the droplets, and the reaction starts at the same time. Because the liquid is confined to a closed space, the traditional “wet box” is no longer needed, and a large number of specimens can be incubated at the same time under consistent reaction conditions. The following takes indirect immunofluorescence as an example to introduce the operation process of titration plate technology:

  1. Prepare and check the sample plate: observe whether the reaction area is hydrophilic but the surrounding area is hydrophobic. If not, wipe it clean with a wet tissue, then take out the slide from the kit, and wait until it reaches room temperature before opening the package. Be careful not to touch the biological flakes, use a pen to number and mark.
  2. Diluted serum “According to the user’s experimental design, use PBS-Tween buffer to dilute the serum. Each experiment requires positive and negative controls, and mix well before use.
  3. Add sample   drop 25ul diluted serum into each reaction area of ​​the sample plate in order to avoid bubbles. After dripping all the specimens to be tested, start the incubation.
  4. In the first step of incubation, cover the slide with the substrate side down in the recess of the sample plate, and the reaction starts. Incubate for 30 minutes at room temperature.
  5. Rinse Use a beaker with PBS-Tween buffer solution to rinse the slides for 1 second (note that the water flow is not too rapid, do not directly face the substrate), and then immediately immerse it in a small cup containing PBS-Tween buffer solution, and soak for at least 1 minute.
  6. Add 20ul of fluorescein isothiocyanate-labeled anti-human globulin to the reaction area of ​​a clean sample plate, and the incubation can be continued after complete addition. Fluorescein isothiocyanate-labeled anti-human globulin should be mixed before use and diluted with PBS-Tween buffer.
  7. In the second step of incubation, remove a slide from the PBSTween buffer, dry the back and edges with absorbent paper within 5 seconds, and immediately cover it on the sample plate, taking care not to wipe the reaction area. Take care to avoid direct sunlight on the slides and incubate at room temperature for 30 minutes.
  8. Rinse the slide with PBS-Tween buffer in a beaker for 1 second (the water flow should not be too rapid, do not directly face the substrate), and then immediately immerse it in a small cup containing PBS-Tween buffer for at least 1 minute .
  9. Mount the slides and add glycerol/PBS to the coverslip, about 10ul per reaction zone. Take out a slide from the pBS-Tween buffer, dry the back and four sides with paper, taking care not to wipe the whale in the reaction zone. Place the cover glass gently on the slide.
  10. Judgment result: Observe fluorescence under a fluorescence microscope.

(3) Quality control and precautions for antibody testing

l. Quality control The quality control of autoantibody detection is to control and evaluate the presence or absence and concentration of autoantibodies by various means, which is conducive to obtaining the same comparable results from the same specimen within and between laboratories. An important indicator to measure the level of laboratory testing. however. The quality control of autoantibody testing is not an easy task. Because different laboratories use different antigen substrates, different or different antigen fixation methods, different serum dilution methods, and different operators’ ability to recognize different fluorescence patterns, all of which can affect the quality of autoantibody detection. When the laboratory is conducting clinical testing, it should test the positive control and negative control at the same time to monitor the accuracy of the results. There is an urgent need for the establishment of specialized academic institutions in China, with reference to international standards, in line with international standards, establishing their own local standards, and carrying out quality control of autoantibody testing.

  1. Further confirmation of experimental results (two-stage determination)” indirect immunofluorescence method, provides an ideal way to screen autoantibodies. In many cases, only using this method can provide sufficient diagnostic information for the clinic, without further experiments. In the detection of autoantibodies, the indirect immunofluorescence method is called “level 1 determination”. If it is necessary to make further monospecific differentiation of autoantibodies, other methods can be applied, such as enzyme immunoassay, convection immunoelectrophoresis and immunodiffusion method, etc. These experiments are called “level 2 determination. The enzyme immunoassay method used The antigen must be purified. Many antigens are not easy to be purified, and the exact antigen of many autoantibodies is still unknown. Therefore, currently only a small part of antibodies detected with HEp-2 cell columns and a small part of tissue antibodies are further monospecific Detection. However, the final distinction of antibodies requires the use of purified antigen as the experimental matrix. The “level 2” determination alone cannot meet the needs of autoantibody detection. Especially for the determination of antinuclear antibodies, the “level 2” determination alone will Some autoantibodies are missed, so immunofluorescence must be used for detection at the same time.
  2. Experiment report and explanation

(1) Negative: When the entire detection system (including various quality controls) is normal, when the sample to be tested is at the appropriate initial dilution, the experimental matrix has no obvious fluorescent staining, or there is no identifiable fluorescent model , Said the serum sample to be tested negative for some autoantibody.

(2) Positive: When the entire detection system (including various quality controls) is normal, when the serum sample to be tested is at a suitable initial dilution, it will produce a specific fluorescence that is significantly higher than that of the negative control, and there are clear and identifiable Fluorescence mode, it is said that a certain antibody to be tested in the serum sample to be tested is positive.

Note that sometimes in the application report, the result of the autoantibody applied for testing is negative, while other autoantibodies are found to be positive. In this case, if the detected autoantibody has obvious clinical significance, the result should be reported. If the autoantibody has no clear clinical significance, it may not be reported to avoid confusion. However, if clinicians and laboratory workers are cooperating on research, they can report or communicate separately.

(3) Immunofluorescence mode; for positive results, the fluorescence mode should be reported to initially provide the specificity of the target antigen against which autoantibodies are directed, and provide information for further “level 2” testing.

(4) Titer: Titer is defined as the highest dilution of the specific fluorescent reaction that can be observed compared with the negative serum of the same dilution. The test results should report the titer to provide dynamic information for the clinic, because autoantibody-positive patients need to be tested multiple times during the diagnosis and treatment process, and the titer of some autoantibodies is closely related to the condition.

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