The latest progress and development trend of autoantibody indirect immunofluorescence detection technology

Autoantibody detection is an important method for diagnosing autoimmune disease (autoimmune disease, AID). Commonly used detection methods include indirect immunofluorescence (IIF), enzyme-linked immunoassay (ELISA), western blotting, etc., with the continuous innovation of biotechnology , Auto-antibody detection has been gradually applied to autoantibody detection by technologies such as magnetic bead particle chemiluminescence, immune microarray, and flow immunofluorescence, which are automated and with higher detection accuracy. However, the indirect immunofluorescence method has always been the most widely used serological detection technique because of its comprehensiveness in detecting antigens. Indirect immunofluorescence method is the specific autoantibody in the patient’s serum binds to the antigen in the cell and tissue matrix, and then fluorescein-labeled anti-human IgG antibody binds to the antigen-antibody complex, which emits a characteristic green color under a fluorescent microscope Fluorescence is detected. Realizing the full automation of IIF and gradually realizing the standardization of the whole process on this basis is the future development trend.

Overview of autoimmune diseases

Autoimmune diseases are diseases caused by the reduction or destruction of immune tolerance of the immune system to its own components due to some reasons, causing autoantibodies or/and sensitized lymphocytes to damage their own organs and tissues, which are manifested in the functions of the corresponding tissues and organs obstacle. There are many types of AID. According to the tissues or organs involved in the injury, it can be divided into organ-specific autoimmune diseases (limited to a specific organ, such as autoimmune hepatitis) and systemic autoimmune diseases (lesions are found in multiple organs and connective Tissues, such as systemic lupus erythematosus). In recent years, due to the improvement of human understanding of AID, combined with the accumulation of clinical experience and the advancement of laboratory diagnostic technology, the detection rate of AID has increased significantly. Epidemiological studies have shown that the prevalence of AID worldwide has reached 3 %~5%. AID is very heterogeneous, and the clinical manifestations of patients are sometimes atypical or even without any symptoms and signs, especially in the early stages of the disease, which makes the early diagnosis of AID disease difficult. Currently, the diagnosis of AID mainly relies on laboratory tests, among which autoantibody testing is the most clinically used auxiliary diagnostic tool.

The detection of autoantibodies is an important basis for clinical diagnosis of AID, and each AID is accompanied by a characteristic autoantibody profile. The determination of autoantibodies is helpful for the diagnosis of AID, and has important clinical significance for judging the activity of the disease, observing the treatment effect, and guiding clinical medication. In addition, some autoantibodies rise several years before the onset of disease symptoms, which can play a role in disease prediction.

Indirect immunofluorescence detection method for autoantibodies

Autoantibody detection methods are based on antigen-antibody reactions. Due to the different antigen-coated carriers and forms, the antigen-antibody binding reaction labeling methods are different, and the detection methods for autoantibodies are very abundant. Commonly used are indirect immunofluorescence (IIF), enzyme-linked immunoassay (ELISA), western blotting, and Immunodiffusion methods, among which some of the detection methods have low sensitivity and specificity, and most detection methods are manual operations. There are individual differences in the manual operation process, and the test results are greatly affected by subjective factors. The same item and the same test method often appear. The test results between the two laboratories are different. The clinician will be diagnosed during the patient’s transfer. Bring confusion. In order to meet the needs of the specificity and accuracy of clinical autoantibody detection, magnetic bead particle chemiluminescence, immunomicroarray, flow immunofluorescence and other technologies are gradually applied to autoantibody detection.

Despite the continuous development of biochemical technology, new detection technologies continue to emerge and become increasingly mature, but it still cannot shake the important position of indirect immunofluorescence in the detection of autoantibodies. Indirect immunofluorescence is not only a very advanced and extremely reliable serological detection technology in the past, and even today, it has been widely used in laboratory diagnosis of AID, and its importance is recognized by rigorous laboratory workers. For autoantibody detection, indirect immunofluorescence should be the first choice as the ideal screening experiment. The American College of Rheumatology (ACR) clearly pointed out: The IIF method using human laryngeal carcinoma epithelial cells (HEp-2) cells as the matrix is the reference method for detecting antinuclear antibodies (ANA). The accuracy of the results of this method not only depends on the quality of the fluorescent slides and the accuracy of the operation process, but more importantly, laboratory professionals use the fluorescent microscope to interpret the results of the fluorescent slides. Since laboratory staff are prone to misjudgment or missed judgment on the fluorescence karyotype of the sample, and the judgment of the titer is easy to judge high or weak, the only way to accurately judge the titer of manual experiments is the gradient dilution method, which is not only a waste Time, reagents, and very few clinical applications. Therefore, to realize the automatic indirect immunofluorescence detection of autoantibodies (integration of slide processing and result interpretation (positive and negative, titer, karyotype)) and gradually realize the standardization of the whole process on this basis is the dream of inspection workers It is also the future development trend, and a lot of research is also moving in this direction.

Fully automatic indirect immunofluorescence detection platform

It is gratifying that a fully automated instrument for indirect immunofluorescence detection of autoantibodies has appeared. This is the automatic fluorescence immunoassay detection technology platform (HELIOS), which was introduced by Guangzhou Kangrun from Germany AESKU in 2014 In China, HELIOS efficiently integrates slide bar code readers, sample bar code readers, LED microscopes, high-definition cameras and other sophisticated devices, combined with digital processing software, can realize the front-end staining and back-end image collection and result interpretation of autoantibody indirect immunofluorescence experiments ( The automatic processing of negative and positive, titer, karyotype) simplifies the experimental process. Through the automation of the whole process of experimental operation and result interpretation, all experimental processes are recorded and stored for complete retrospection, and difficult results can be confirmed by experts through remote and supplementary consultations; and the use of this system between different laboratories can achieve stable indoor results and no results between rooms At the same time, the automatic fluorescence immunoassay analyzer adopts the international traditional titer, and the titer refers to the United States CDC/CAP international standard. This is a significant progress for the standardization of indirect immunofluorescence.

On the basis of the efficiency and accuracy of fluorescence experiments, due to the reduction of manual result intervention, especially titer interpretation, it can reduce the error of manual judgment of experimental results before and after the patient, and can participate in the comparison of results of related laboratories, which is in line with the state The requirement to improve the mutual recognition of the results of different laboratories also greatly reduces the medical expenses of patients and improves the clinical recognition of the results. Participate in the discussion of network experimental results through the built-in module. Under the condition of stable experimental results, the efficiency of internal staff training and mutual learning and support with advanced hospitals are greatly improved.

In China, the results of a multi-center study on the standardization of indirect immunofluorescence for antinuclear antibodies (ANA) in Guangdong Province, led by the Southern Hospital of Southern Medical University, led by 8 large units in Guangdong Province, showed that the fully automatic fluorescence immunoassay instrument can replace manual operations and improve experiments. The consistency of the test results between the rooms, and the results are more reliable, and the interpretation standards are more objective, which helps to recognize the results between the rooms.

All in all, laboratory testing, especially autoantibody testing, is essential for the diagnosis and treatment of AID. Automated and standardized detection of autoantibody detection is the goal pursued by laboratory workers. It standardizes experimental detection procedures, provides standardized detection paths, and conducts intelligent analysis of detection results to provide clinicians with accurate and reliable diagnosis.