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Application of fluorescent antibody in clinical diagnosis and treatment

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Autoimmune disease refers to a series of pathological immune responses produced by the body’s immune effect or immune effector molecules against one’s own tissues or cells, leading to a large class of diseases that damage their own tissues and organs. The pathogenesis is very complex and the clinical manifestations are variable. , Often involving the skin, serosa, joints, kidneys and central nervous system [1].

Immunofluorescence technology is an immunological detection method that uses fluorescent pigments to color-label the detection target. The detection objects include antigens and antibodies, etc., and have been widely promoted and used in clinical practice. The outstanding advantage of immunofluorescence technology is that after antibodies are combined with corresponding tissue components, the corresponding autoantigens and antibodies can be directly detected by observing the morphology of the tissue or cell components emitting specific fluorescence. The identification and localization of autoantibodies can also be carried out at the same time, and its simplicity, accuracy and rapidity are difficult to achieve by other methods. Therefore, this method is still widely used by scholars from various countries.

Many kidney diseases, especially glomerular diseases, have immunological mechanisms involved in most cases. Monoclonal immunoglobulins and complement-related kidney diseases that have received attention in recent years are also related to immunological factors. It is manifested as the accumulation of immunoglobulin or complement. Therefore, immunopathological staining is indispensable. At present, frozen sections are still the main method. In a few cases, paraffin-embedded tissues are also used for immunohistochemical staining or immunofluorescence detection. Since the routine staining items of renal puncture, such as IgG, IgA, IgM, C3 and C1q, have a strong immunohistochemical background in paraffin sections, direct immunofluorescence staining is currently used. The consensus of kidney biopsy experts recommends the use of frozen section immunofluorescence. Only when the immunofluorescence of frozen tissue and the electron microscope results conflict, it is recommended to use paraffin section immunofluorescence, and when the material is not ideal, use paraffin section for immunofluorescence or immunohistochemistry.

“Expert consensus on standardized diagnosis of kidney biopsy”[2]

The immunopathological examination of kidney biopsy includes immunofluorescence (IF) staining of frozen sections or immunohistochemical staining of paraffin sections. It is recommended to use frozen sections and use the direct IF method for inspection. If the specimen is stored and transported for a long time, it needs to be transported in a fluorescent specimen storage solution (Michel or Zeus solution); because the fluorescent signal is easy to attenuate or quench and cannot be stored for a long time, it should be photographed and stored in time. The thickness of the frozen section is generally 3 μm. The routine IF inspection items should include immunoglobulin (IgG, IgA, IgM), complement (C3, C1q) and light chain (κ, λ) staining. For special cases, other stains are needed, such as amyloid precursor protein (AA, etc.), fibronectin, type III collagen, type IV collagen alpha chain, hepatitis B virus antigen, hepatitis C virus antigen, etc.; For membranous nephropathy, IgG subtype and phospholipase A2 receptor (PLA2R) need to be added.

With the development of immunology, the research of skin immunopathology has become more and more extensive. Direct immunofluorescence has become an important method for the diagnosis of pemphigus, pemphigoid, herpetiform dermatitis, and lupus erythematosus. The classification and the discussion of pathogenesis are of great significance.

Pemphigus not only has IgG deposition between the epidermal cells of the blisters, but also between the epidermal cells of the normal-looking skin near the blisters, and the positive rate is 10%. In histopathology, it is necessary to find obvious spinal release or spinal release bullae before diagnosis. Therefore, when there are no obvious blisters in the clinic, it is more convenient to diagnose pemphigus by direct immunofluorescence than histopathology, and it is more reliable and timely. Therefore, direct immunofluorescence has been an important method for the diagnosis of various types of pemphigus.

Regarding the differential diagnosis of herpetiform dermatitis and lupus-like dermatitis: reports of these two recent variants, such as mixed bullous disease, polymorphic pemphigoid, childhood benign chronic bullous disease, etc., relying solely on tissue disease Physical methods are often difficult to diagnose, and immunofluorescence technology must be used to identify them. The study found that pemphigoid is deposited in the basement membrane area in a line with IgG, and there are cell-like fluorescent bodies in the epidermis. Herpetiform dermatitis has no IgG deposition, mainly in granular or linear deposition of IgA. Clinically, patients with mixed bullous disease deposit IgG and IgA in the basement membrane area at the same time. This illustrates the important role of immunofluorescence technology in the differential diagnosis of bullous disease [3].

IgG is the highest content of immunoglobulin in serum, accounting for 70~75%, and is also the most important anti-infective molecule. IgG can be divided according to the antigenic difference of its molecular backbone (γ chain) and the number and position of disulfide bonds. There are four subtypes, namely IgG1, IgG2, IgG3 and IgG4. Their contents in normal humans are about 65%, 23%, 8%, and 4% respectively. Various subtypes have their own characteristics and different functions, and most of the functions of IgG are accomplished by IgG subtypes. IgG1 is the most important subtype of IgG, which has a strong ability to activate complement and bind to phagocytes. IgG2 is dominant in immunity and response to polysaccharide antigens. IgG3 often shows higher affinity than IgG1 in the immune response to protein and polypeptide antigens. IgG4 may be the least active component of IgG subtypes, and it is mainly involved in allergic reactions [4].

Membranous nephropathy (MN) is a common pathological type in glomerulonephritis, which is characterized by diffuse immune complex deposition of glomerular basement membrane epithelial cells with thickened basement membrane. Membranous nephropathy is divided into two categories according to the etiology. Those who are clear are secondary MN, and those who are not clear are classified as idiopathic MN (IMN). In addition, the diagnosis will not be clear, and serology or renal tissue examination suspects secondary membranous Nephropathy is classified as atypical membranous nephropathy (UAMN) of unknown etiology. Membranous nephropathy caused by different etiologies has different mechanisms involved in the immune response, and its treatment options are also different, so its differential diagnosis is extremely important. However, many diseases have no obvious clinical manifestations in the early stages, and laboratory tests have become an important basis for early diagnosis [5, 6].

[1] Zhang Naizheng. Clinical Rheumatology [M]. Shanghai Science and Technology Press, 1999.

[2] Zhao Minghui, Wang Suxia, Zeng Caihong, et al. Expert consensus on standardized pathological diagnosis of renal biopsy[J]. Chinese Journal of Nephrology, 2018,34(12):941-946.

[3] It is Yuan Fu, Liu Jihe, Kong Qingying, et al. Research on skin immunopathology——The application of anti-human IgG fluorescent antibody direct method in certain skin diseases[J]. Skin Disease Control, 1984(Z1): 8-11.

[4] Wan Furong, Cheng Liying, Suo Cuiping. Research on IgG subtype detection and disease [J]. Medical Laboratory Science and Clinices, 2011, 22(006): 55-56.

[5] Kuroki A, Shibata T, Honda H, et al. Glomerular and serum IgG subclasses in diffuse proliferative lupus nephritis, membranous lupus nephritis, and idiopathic membranous nephropathy.[J]. Internal Medicine,2006,41(11):936 .

[6] Kawasaki Y, Suzuki J, Sakai N, et al. Evaluation of T helper-1/-2 balance on the basis of IgG subclasses and serum cytokines in children with glomerulonephritis[J]. American Journal of Kidney Diseases,2004, 44(1): 42-49.

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