RU2028156C1 - Method of immunosorbent preparing for diagnosis of cytomegaloviral infection - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: monolayer of cell cultures is infected with cytomegalovirus, virus is incubated for 48-72 h. Then nuclear fraction containing "early" cytomegaloviral proteins is isolated from virus-infected cells. This nuclear fraction is used as an antigen for immunosorbent preparing on polystyrene 96-well plates. Method is used for diagnosis of "current" cytomegaloviral infection using enzymoimmunoassay. EFFECT: development of method for immunosorbent preparing indicated above. 2 tbl

Description

 The invention relates to medicine and for the diagnosis of cytomegalovirus (CMV) infection, which is of great importance.

 It is known that CMV - a representative of herpes viruses - is capable of causing both acute and latent disease. In 80% of people, primary infection with the virus occurs in the first 2-3 years of life, while in 95% of cases there are no clinical symptoms. However, the transmitted infection is accompanied by the development of virus-specific antibodies, which persist throughout almost the entire life of a person (the so-called "anamnestic" antibodies). During the latent state of CMV infection, virus is not released into the environment, but humoral "anamnestic" antibodies can be detected in various serological reactions.

 In case of violation of the immune status of a previously infected human CMV, the virus begins to actively replicate, and it can be detected in various biological fluids (blood, urine, saliva, etc.). In response to virus replication, specific antibodies to the so-called “early” viral proteins (proteins that develop within 24-72 hours after infection with CMV or its reactivation) begin to be synthesized in the body (Landini, Michelson, 1988). Detection of antibodies to “early” proteins indicates a “current” infection at the moment and is important for the correct administration of patients with congenital and acquired immunodeficiencies (AIDS, intrauterine infection, organ transplantation).

 To diagnose CMV infection, the virological method of isolating the pathogen in a sensitive cell culture is used, followed by identification of the isolated isolate by immunofluorescence with monoclonal antibodies (Drew, 1988; Swenson, Kaplan, 1987; Gleaves et al., 1984). The disadvantage of this method is its duration (confirmation of the diagnosis can be obtained only 20-30 days after taking a sample of blood or urine from the patient) and its complexity.

 Serological diagnosis of CMV infection is of limited use, since due to the latent nature of its course, the fact of detection of antibodies does not indicate a current infection. Even the use of various methods based on the detection of virus-specific immunoglobulins of class M in the case of a recurrent infection does not provide reliable results, because relapse is not always accompanied by the formation of antibodies of this class (Doers et al., 1985).

 Closest to the proposed method is the detection of antibodies to CMV in human sera in an enzyme-linked immunosorbent assay (Abbott CMV total AA EIA). The immunosorbent in such test systems are antigens isolated from infected cell cultures at the height of the development of the cytopathic effect.

 The disadvantage of this method is the inability to diagnose CMV infection, since this test system does not allow differentiating "anamnestic" antibodies from the antibodies synthesized in a given period of time, which are produced in response to virus infection or reactivation of a latent virus.

 The aim of this invention is to develop a method of preparing an immunosorbent for the diagnosis of "current" CMV infection using enzyme-linked immunosorbent assay (ELISA). This goal is achieved by the fact that from the nuclear fraction of cells infected with CMV, 48–72 hours after their infection, virus-specific “early” proteins are isolated that adsorb in the wells of 96-well polystyrene plates.

Example 1. The transplanted cell line of human embryo fibroblasts is infected with cytomegalovirus strain AD-169, which was obtained from the American Virus Collection and is currently maintained at the Laboratory for Diagnosing Viral Infections, Research Institute of Viral Preparations, Russian Academy of Medical Sciences. 48-72 hours after infection, the nuclear fraction of the cells is isolated. For this, virus-infected cells are separated from the glass with a trypsin solution and washed once with a phosphate-buffered solution of pH 7.4 (FSB). The cell pellet diluted in twice distilled water containing 2% fetal calf serum was incubated for 15 minutes in an ice bath. An equal volume of a 1% solution of non-ionic detergent (NP-40) is then added and the resulting mixture is homogenized in a Downes homogenizer. The nuclei are separated from the cytoplasmic fragments by centrifugation through a 22.5% Ficoll 400 solution prepared on PBS, pH 7.4, at 1800 g for 15 min at 4 ^o C. The pellet is washed once with PBS, pH 7.4, resuspended in the same buffer and treated with ultrasound (three cycles of 30 s with an interval between cycles of 1 min.). The resulting "nuclear" antigen containing the early CMV proteins is clarified by centrifugation at 8000 g for 20 minutes.

 To obtain the immunosorbent, in the wells of odd rows (A, C, E, G) of polystyrene 96-well microtiter plates, 100 μl (0.1 ml) of the viral antigen is added at a concentration of at least 1 μg per well. In the even-row wells (B, D, G, H), 100 μl of the control antigen, isolated from an uninfected cell culture, was added in the same manner as the viral antigen.

The plates covered with lids are incubated at 4-10 ^° C for 18-20 hours. After washing 3 times with distilled water containing 2% Tween-20, the plates with loaded antigens are stored at 4-10 ^° C until use. The shelf life of the immunosorbent is 6 months.

 PRI me R 2. To obtain the drug "early" proteins using the same cell culture as described in example 1, infected with strain Dov-90, isolated from the urine of a patient with CMV infection. The strain "Dov-90" is stored in the museum of the Research Institute of Viral Preparations. All stages of the preparation of the immunosorbent and its control are carried out as described in example 1.

 PRI me R 3. To prepare a solid-phase immunosorbent, the culture of diploid cells of a human lung embryo is infected with strain CMB AD-169. Cell culture is deposited in the collection of cell cultures of the State Research Institute for Control and Standardization of Medical Biological Preparations named after L.A. Tarasevich. All procedures for the preparation and control of immunosorbent are carried out in the same way as described in example 1.

 PRI me R 4. For the preparation of the preparation of the "early" CMV proteins in order to use it to obtain the immunosorbent, the diploid cell culture described in Example 3 is charged with CMV, the Dov strain. The remaining procedures for the preparation and control of the immunosorbent are carried out as described in example 1.

 Example 5. The study of sera in ELISA is carried out according to the previously described method (Voller et al., 1979) using antibodies against human immunoglobulins labeled with peroxidase. Each serum is examined at a dilution of 1: 100 with both viral and control antigens. As a result of the studies, a threshold value of optical density (OD) was established during the reaction of antibodies with viral and control antigens, delimiting positive and negative sera, which amounted to 0.2 optical units. If the difference in optical densities (AOP) during the reaction of serum with the tier and control antigens is more than 0.2 O.E., then the serum is regarded as positive, i.e. containing antibodies to the "early" CMV proteins.

 The examples of preparation of an immunosorbent using various strains of CMV and different types of cells in which this virus is capable of multiplying, as well as the results of a study on engineered immunosorbents of a panel of sera containing and not containing antibodies to “early” CMV proteins, indicate the possibility of using any available strain of CMV actively propagating in a suitable cell culture line. This is evidence of the industrial applicability of the proposed method for the preparation of immunosorbent for the rapid diagnosis of CMV infection in the ELISA test system for the detection of antibodies to CMV.

 56 individuals were examined whose serum antibodies to CMV were detected on a prototype test system. Of this group examined in 31 patients with a clinically expressed disease (pneumonia, splenomegaly, hepatitis, etc.), the cytomegalovirus infection was confirmed by the isolation of the virus from the urine or by histological detection of "giant" cells in the urine. 25 donors at the time of the examination were almost healthy.

 The results of the study of sera on the developed test system in comparison with the prototype are presented in table.2.

 The data show that in the ELISA test system of the firm "Abbott" it is impossible to differentiate "anamnestic" antibodies from antibodies synthesized in response to the current infection. Using the proposed immunosorbent, antibodies to "early" proteins were detected only in a group of patients with CMV infection confirmed by other methods.

 The results obtained indicate the effectiveness of the proposed method for producing an immunosorbent for early and rapid diagnosis of CMV infection.

 Using an immunosorbent prepared by the developed method, more than 300 sera from sick people with suspected CMV infection have been examined to date. In 36 cases, also confirmed by the isolation of the virus on a sensitive cell culture, antibodies to “early” CMV proteins were detected.

 100% coincidence of the results of virological and serological examinations was recorded.

Claims (1)

 METHOD FOR PREPARING AN IMMUNOSORBENT FOR DIAGNOSTIC OF CYTOMEGALOVIRUS INFECTION, characterized in that, in order to diagnose a "current" cytomegalovirus infection, monolayer cell cultures are infected with cytomegalovirus, virus-specific cells are used for infection 48 to 72 hours after infection, and their specific cells are used for their specificity well polystyrene 96-well plates as the target product.

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