WO2000016061A2 - Method for diagnosing the active stage of a cytomegalovirus infection in human substrates - Google Patents

Abstract

The present invention pertains to the field of virology and relates to an express diagnosis for viral infections. This invention essentially relates to a method for diagnosing the active stage of cytomegalovirus infection in a patient, wherein said method involves carrying out a sorption of the specific antigen in solid phase, adding the serum to be analysed, carrying out an incubation using a conjugate of immunologic ferments as well as a substrate and estimating the results. The antigen consists of the recombinant, main, precursor and non-structured protein of the cytomegalovirus, and the active stage of the infection is detected upon the occurrence of antibodies against said cytomegalovirus.

Description

\UΟ 00/16061 «ΡСΤ Κυ99/00246

Method for diagnosing the active stage of human cytomegalovirus infection.

Description. The method relates to the field of medicine, in particular, virology and is a method of express diagnostics of viral infections.

Cytomegalovirus (CMV) infection in humans, as stated, occurs with mild symptoms, however, is accompanied by complications of varying severity. This virus is one of the main pathogens for high-risk groups with various immunodeficiencies, including AIDS, recipients of organ and tissue transplants, when reactivation of latent cytomegalovirus, often leading to irreversible clinical pathologies of the human body. In addition to this, the infection refers to a tepathogenic gastrointestinal tract that is capable of initiating various pathologies pregnancy, often leading to serious abnormalities in new

born.

This infection is characterized by a chronic course and belongs to the latent category, and therefore its diagnosis is clinically difficult. The best-known method in this regard at present is the method of isolating virus from clinical samples of sensitive tissue. culture of the Kani Shge aηά (In Russian. Metaphysics 1985, 21: 217-221).

The disadvantage of this method is the length of the analysis (up to 72 hours) and the ambiguity of the interpretation of the results. \ΥΟ 00/16061 2 PСΤЛШ99/00246

The last time in the diagnosis of this infection is the method of determining the specific nucleotide Consistently, the genomic dosage of the vius in the clinical trial, based on the lymphase chain reaction (PCR) (Οetteger SZ, Βiιϊοηе 01, δsytοg SΜ, Mau YΑ. ϋlеϊеύοη οГсуτοте§а1ονϊшзт иπηе ιϊοт ηеννЪοгηз ьу fromϊη§ ροϊutegaze саϊη geasύοη. I. ΙηΤесΙ. ϋϊz. 1988, 158: 1177-1 184).

The disadvantage of this method is the detection of both active and latent forms of infection. This problem of human cytomegalovirus diagnostics is key due to the persistent state of CMV in the body. At present, the most adequate methodological approach in this regard is considered to be the reverse-transcribed polymerase chain reaction (RT-PCR), which detects the expression of viral genes, in particular the main pre-CMV gene, in the form of its specific information ΡΗΚ (Νа§а1а Ν. ег аϊ ϋеϊесіοη οιΗшηаη SΜU tΚΝΑ ь ΚΤ-ΡСΚ. δаρροгоο Меά. 62 (4) 1993, 193-200). However, this method has not found much use in healthcare due to its high cost.

Due to the latent course of CMV infection, its serologic diagnosis is complicated. Thus, to determine active infection, the method of detecting virus-specific antibodies of class IgM is used. However, this method has its own limitations, associated primarily with the fact that up to now it remains an old thing about detectable presence of antibodies of this class for recurrent infections (Kaη§gο, Η.Ο., Βοοοt, ΙС, Βakϊg, T.M., Τгугοгη, Υ., δиϊегΙаηе, 5.. ϋеϊесѿη οП§Μ аηύьοсϋез а§аϊηз1: суϊοте§а1ονϊгi5: сοтρагϊзοη ο νο gasϋοϊttig.οazzauz, eηζute- IkesΙ ϊttiηοozοgеηΙ azzau aηά ϊttshyuιΤigesseηΙ aηϋbοάу Ιezι. Ι.Μеά. νϊгοϊ. 14:73-80,1984). \νθ 00/16061 3 ΡСΤ/Κυ99/00246

The closest in technical essence to the proposed invention is a method for diagnosing CMV infection in humans, which includes the adsorption of a specific antigen onto a solid phase, the introduction of test serum, incubation with an immunoenzyme conjugate and a substrate with by the following evaluation of the results (patent KΚΙΙ Μ> 2028156, class 6 Α61K 39/245, 02/09/95). The disadvantage of this method is that the special immunosorbent is a nuclear fraction of cells obtained 48-72 hours after their infection with cytomegalovirus. Proteins of this type are "early" virus-specific polypeptides that are products of the expression of early (beta) genes of the CMV genome and constitute a mixture of both non-structural and structural antigens of the virus. Thus, specific antibodies of such a mixture can be detected both in the active stage of infection and during its latent course.

The objective of the present invention is to develop a method for diagnosing CMV infection that allows for rapid and adequate differentiation between the choronic and active forms of cytomegaly, which eliminates

reading relevance for timely blocking of the infectious process, as well as for predicting the development of this disease. A method for diagnosing the active stage of human cytomegalovirus infection is proposed, which includes the adsorption of a specific antigen to the target phase, the introduction of test serum, incubation with an enzyme-linked immunoconjugate and a substrate, followed by an assessment of the results, and in The recombinant main protein of the cytomegalovirus is used as an antigen, and when antibodies to it are detected, the active stage of the infection is detected. The recombinant main protein is mainly used. \νθ 00/16061 ΡСΤ/ΚШ9/00246

The primary non-structural protein of human cytomegalovirus, cloned and expressed in the E. coli protein-synthesizing system.

Example 1.

(The experiment used samples of serum from 3 groups of patients, 20 people each.

The first group: chronically infected with cytomegalovirus outside the stage of depletion, in the serum of cats were found common specific

1§0-antibodies.

The second group: patients with a diagnosis of active cytomegaly, established by both laboratory methods and the presence of a clinical picture.

Group 3: patients in remission after treatment for active form of cytomegalovirus.

The recombinant main protein of the polystyrene tablets is assembled on the solid phase of the polystyrene tablets. The main protein is the non-structural protein CMB, which is an analogue of the viral protein with a molecular weight of 72 kD. This protein, which has not been previously used to diagnose CMV infection, is known as an expression product of the IE gene region, which includes the nucleotide sequence between positions 0.66 and 0.77 on the physical map of the long unique sequence of the genome.

TsΜΒ (Υοзыыгο Τзіззϊ аηе Τакаο Νο§аtϊ-δаΙаke, ϋιιϊегеηήаϊ eχρgezzϊοη οϊte πшϊοг ϊtteάϊаϊе eagϊу §еηе οιΤshtaη suτοte§а1ονϊшз, I. οГΟеη νϊгο1ο§у, 1990, 71.

1 15-124). Detection of specific antibodies to a given antigen by means of enzyme-linked immunosorbent assay (ELISA) occurs only at the active stage of the infection process and allows its differentiation from the latent course of infection. 00/16061 ₅ ΡСΤ/Κυ99/00246

After washing the plates with a special buffer, a sample of human serum or blood plasma is added to the antigen on the solid phase. Specific antibodies present in the sample must bind to the viral antigen. To identify a specific antigen-antibody bond, an antigen (human) conjugate with the enzyme Cren's peroxidase is used. After adding the substrate and dye, the antigen-antibody-conjugate complex, through a specific interaction, produces a color reaction determined by the spectrophotometry method at a wavelength of 492 nm.

To obtain more correct results, a polypeptide systematized in E. coli cells, isogenic to the virus-specific antigen, and not containing antigenic determinants of human cyanide megaloriferous was used as a control antigen.

Table 1 (Results of a comparative analysis of the proposed method for detecting IgM antibodies and the one known from patent KII No. 2028156).

General Ig0 antibodies to human cytomegalovirus are anamnestic antibodies synthesized after a repeated encounter with the virus and persisting in the body for life. Class I antibodies are very specific antibodies, synthetically produced \νθ 00/16061 5 ΡСΤ/ΚШ9/00246

CMV infections in the active stage.

Specific Ig0 antibodies detected by the method described in the patent

Zh2028156, characterizes both active and chronic forms of infection.

Specific Ig0 antibodies determined by the proposed method do not migrate in the body and are detected only in the active stage of the infectious process.

In all examined individuals, common Ig0 antibodies were detected in 3 groups.

In the first group of examined individuals chronically infected with CMV, antibodies were detected in 10% of individuals using the method described in patent No. 2028156. At the same time, neither IgM antibodies nor Ig0 antibodies were detected using the proposed method.

In the second group of examined individuals diagnosed with active cytomegaly, IgM antibodies were detected in 75% of patients, and according to the method according to patent G°2028156, Ig0 antibodies were detected in 60% of patients. At that time, the detection of Ig0 antibodies by the proposed method was 95% of those examined.

In the third group of individuals examined after treatment for cytomegalovirus and in the remission stage, IgM antibodies were detected in 20% of cases, according to patent No. 2028156

Ig0 antibodies were detected in 20% of patients, while the detection of Ig0 antibodies using the proposed method was 10%.

The data presented in Table 1 indicate a high specificity of detection of the active stage of CMV infection when using the recombinant major precursor non-structural protein CMV as an immunosorbent in IFA. As can be seen from the data presented in Table 1, the detection rate of IgM antibodies in active CMB infection is lower than that of Ig0 antibodies to the main pre-existing antigen. This fact can be explained by the fact that, as noted, \νθ 00/16061 7 PСΤ/ΚШ9/00246

above, the formation of specific antibodies of this class is limited in the recurrent form of infection.

Thus, this approach allows for rapid and adequate differentiation between the chromatin and active forms of cytomegaly.

In addition, it should be noted that this approach provides an advantage over the method for detecting class IgM antibodies due to the difference in the dynamics of the formation of Ig0 antibodies to non-structural viral antigens and IgM antibodies to structural antigens synthesized on a more Late stages of the infection process.

Claims

\νθ 00/16061 8 ΡСГ7КШ9/00246 Φορmula of invention 1. A method for diagnosing the active stage of human cytomegalovirus infection, including the adsorption of a specific antigen onto a solid phase, the introduction of test serum, incubation with an enzyme immunoconjugate and a substrate, followed by evaluation of the results, characterized by the fact that the recombinant main precursor non-structural protein of cytomegalovirus is used as an antigen, and when antibodies to it are detected, the active stage of infection is destroyed. 2. The method according to claim 1, characterized in that it uses a recombinant basic prepared non-structural protein of human cytomegalovirus, cloned and expressed in the protein-synthesizing system E.coli.