Method for the diagnosis of active phase cytomegalovirus infection of human

 

(57) Abstract:

The invention is intended for rapid diagnosis of active CMV infection. The method is based on identifying virousspecificakih IgG to recombinant main predannomu non-structural protein of cytomegalovirus, which represents an analogue of the viral protein with a molecular weight of 72 KD, enzyme-linked immunosorbent assay. This cluster of antibodies is detected only in case of active replication of cytomegalovirus and is a marker for differentiation of active and chronic infection. The invention provides the ability to quickly diagnose active form cytomegaly that is of exceptional relevance for timely blocking the infectious process, as well as for prediction of development of this disease. 1 C.p. f-crystals, 1 table.

The method applies to Virology, in particular, to the rapid diagnosis of viral infections.

Cytomegalovirus infection of humans usually occurs with mild symptoms, however, is accompanied by complications of varying severity. This virus is one of the main pathogens to risk groups with different the nom condition occurs reactivation of latent cytomegalovirus, often leading to irreversible clinical pathologies of the human body. In addition, CMV infection is classified as teratogenic, able to initiate various pathologies of pregnancy, often leading to serious anomalies in newborns.

This infection is characterized by a chronic form of current and belongs to the category of latent, therefore, its diagnosis is clinically difficult. The most prominent approach in this regard is currently the method for isolation of virus from clinical specimens on sensitive tissue culture (Gleaves CA, Smith TF, Schuster EA, Pearson GR. Comparison of standard tube culture and shell viral culture techniques for the detection of cytomegalovirus in clinical specimens. J. Clin Environ 1985, 21: 217-221). The disadvantage of this method is the duration of analysis (up to 72 hours) and the ambiguity of interpretation of the results.

Recently in the diagnosis of this infection is used the polymerase chain reaction (PCR), detection of specific nucleotide sequence in the genomic DNA of the virus in clinical samples (Demmler GJ, Buffone GJ, Schimbor CM, May RA. Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction. J Infect Dis 1988, 158 : 1177-1184). The disadvantage of this method is the detection of both active and latent is the CMV status in the body.

Currently, the most appropriate methodological approach in this plan is back-transcriptase polymerase chain reaction (RT-PCR), detecting the expression of viral genes, in particular the main pretannage gene CMV, in the form of specific messenger RNA (Nagata N., et al Detection of Human CMV mRNA by RT-PCR. Sapporo Med. J. 62 (4) 1993, 193-200). However, this method has not found wide application in health care practice because of the high cost and use of precision imported equipment.

In connection with the latent for CMV infection complicated and serodiagnosis. So, to determine active infection used method of identifying virousspecificakih antibodies of class IgM. However, this method has its limitations, primarily related to the fact that still remains an open question detektiruya the antibodies of this class when recurrent form of infection (Kangro, N. About., Booth, J. C., Bakir, T. M., Tryhorn, Y., Sutherland, S., Detection of lgM antibodies against cytomegalovirus: comparison of two radioimmunoassays, enzyme-linked immunosorbent assay and immunofluresc..ent antibody test. J. Med. Virol. 14:73-80, 1984).

Closest to the proposed invention is a method for the diagnosis of cytomegalovirus infection of the person, including the specific sorption of om with subsequent evaluation of the results (patent RU N 2028156, class 6 And 61 To 39/245, 09.02.95).

The disadvantage of this method is that specific immunosorbent is a nuclear cell fraction obtained within 48-72 hours after infection with cytomegalovirus. Proteins of this fraction constitutes "early" virousspecificakih polypeptides, which are the products of expression of early (beta) gene of CMV genome, and is a mix of both non-structural and structural antigens of the virus. Thus, specific antibodies such a mixture can be detected as in active stages of infection, and when it is latent within.

Eliminate these drawbacks is achieved by the fact that for the diagnosis of active phase of the infection uses recombinant main pretani non-structural protein of cytomegalovirus, similar viral protein with a molecular weight of 72 KD, which is the expression product predannih (alpha) genes viral genome. Detection of specific antibodies in ELISA to this antigen occurs only in the active phase of the infectious process and allows to differentiate it from the latent period of infection.

The method consists in the following:

Solid phase polystyrene tablets sorbed nuclear biological chemical (NBC weight of 72 KD. This protein is known as the expression product of the region IE gene comprising the nucleotide sequence between 0.66 and 0,77 positions on the physical map of the long unique sequence of the genome of CMV (Yoshihiro Tsutsui and Takako Nogami - Satake, Differential expression of the major immediate early gene of human cytomegalovirus, J. Of Gen. Virology, 1990, 71, 115-124). However, information about the use of this antigen for the diagnosis of cytomegalovirus infection was not found.

After washing tablets special buffer to the antigen on the solid phase is deposited sample is serum or blood plasma of a person. Specific antibodies present in the sample should contact a viral antigen. To identify specific communication antigen-antibody used individuai (human) conjugated with the enzyme horseradish peroxidase. After addition of the substrate and dye complex antigen-antibody-conjugate for specific interaction gives a color reaction, which is determined by spectrophotometry at a wavelength of 492 nm.

For more accurate results as the control antigen was used polypeptide synthesized in the cells of E. cloi, isogenic virousspecificakih antigen and does not contain antigenic determinant is a group of 20 patients each.

The first group is chronically infected with cytomegalovirus outside the acute stage, in the serum were detected General specific IgG-antibodies.

The second group: patients with a diagnosis of active cytomegaly established as laboratory methods and clinical picture.

The third group: patients after treatment active forms cytomegaly in a state of remission.

The results of the analysis are presented in the following table.

The data presented in table indicate high specificity detection of the active phase of CMV infection when used as immunosorbent assay ELISA main pretannage non-structural protein of CMV. As can be seen from the data presented in the table, detection of IgM antibodies with active CMV infection is lower than IgG antibodies to the main predannomu antigen. This fact can be explained by the fact that, as noted above, the formation of specific antibodies of this class is limited in recurrent form of infection.

This approach allows for rapid and adequate differentiation between chronic and active forms cytomegaly, and what is the project for development of this disease.

In addition, it should be noted that this approach provides advantages over the method of detection of antibodies of class lgM due to differences in the dynamics of the formation of IgG antibodies to non-structural viral antigens and IgM antibodies to structural antigens synthesized in the later stages of the infectious process.

1. Method for the diagnosis of active phase cytomegalovirus infection of the person, including sorption specific antigen on the solid phase, the introduction of the test serum, incubation with enzyme conjugate and substrate with subsequent evaluation of the results, characterized in that as antigens using recombinant main pretani non-structural protein of cytomegalovirus, which represents a similar protein with a molecular weight of 72 KD, and when detecting antibodies to it detects the active phase of infection.

2. The method according to p. 1, characterized in that the use of recombinant main pretani non-structural protein of human cytomegalovirus, cloned and expressed in protein-synthesizing system of E. coli.

 

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