[The DS-DIF-KORINE kit to identify microorganisms corynebacterium, including C. diphtheriae].

The DS-DIF-KORINE kit make it possible to identify 20 types of corynebacteriae entered the Bergi identification guide (1997) including biovars gravis and mitis and belfanti version of diphtheria agent. The possibility to determine its toxigenic characteristics is also provided The test system is ready to be applied since it has all needed components to implement bacteriologic analysis in search of diphtheroids and diagnostics of diphtheria. The presented data demonstrate high diagnostic effectiveness of the new DS-DIF-KORINE kit which has the state registration in Roszdravnadzor and is adapted for spectrophotometer automatic pickup and included into Microbe-Automate and Microbe-2 programs.

[The actual issues of comparing the results of quantitative immune-enzyme tests].

The analysis was applied to the situation on national market of immune-enzyme diagnosticums with further quantitative reporting of results. The issues of coincidence and interpretation of results and causes of discrepancy are considered The notions of biologic ambiguity and metrological traceability are explained.

[Mapping of diagnostically important antigenic regions of major outer membrane protein (MOMP) from Chlamydia trachomatis using recombinant proteins with overlapping structures].

Amino acid sequence of the Chlamydia trachomatis major outer membrane protein (MOMP) was modeled using a series of the recombinant proteins containing six 100 aa fragments of MOMP overlapped in 30 aa. Testing of recombinant antigens in EIA showed that proteins containing MOMP fragments comprising 191-286 and 191-354 aa regions had the greatest activity in the reaction with anti- C. trachomatis positive serum samples. The data obtained allows the conclusion about the possibility of the use of presented recombinant proteins for development of diagnostic test for anti- C. trachomatis antibodies detection to be made.

[Antigenic activity of human herpes virus type 8 proteins coding orfK8.1 and orf65].

A peptide scanning of the sequences of human herpes virus type 8 (HHV-8) proteins coding orfK8.1 and orf65 was made. The antigenic activity of peptides was evaluated by ELISA. The fragment 101-213 aa of orfK8.1-encoded protein was shown to consist of two sites containing linear B-epitopes that had a weak antigenic activity: 121-145 and 177-209 aa. The protein coding orf65 comprised 2 sites at which linear B-cell epitopes 78-110 aa (a weak antigenic activity) and 142-170 aa (a strong antigenic activity) were located.

[Diagnostic advantages of the test system "DS-EIA-HBsAg-0.01" for detection of HBV surface antigen].

The new highly sensitive test system "DS-EIA-HBsAg-0.01" (Priority Certificate No. 2006129019 of August 10, 2006) in detecting hepatitis B surface antigen (HBsAg) was assessed. The sensitivity of the test was estimated using the federal standards sample HBsAg 42-28-311-06, panels' samples Boston Biomedica Inc. (West Bridgewater, Mass, USA) and ZeptoMetrix Corp. (Buffalo, NY, USA). The findings have indicated that "DS-EIA-HBsAg-0.01" is equally effective in detecting different subtypes of HBsAg during a seroconversion period earlier than alternative assays. Along with its high analytical and diagnostic sensitivity, the system shows a high diagnostic specificity.

A new artificial antigen of the hepatitis E virus.

An artificial antigen composed of 12 small antigenic regions derived from the ORF2 and ORF3 HEV proteins was designed. The gene encoding for this artificial antigen was assembled from synthetic oligonucleotides by a new method called Restriction Enzyme-Assisted Ligation (REAL). The diagnostic relevance of this second generation HEV mosaic protein (HEV MA-II) was demonstrated by testing this antigen against a panel of 142 well defined anti-HEV positive and anti-HEV negative serum samples. The data obtained in this study support the substantial diagnostic potential of this HEV mosaic antigen.

[A novel enzyme immunoassay system for the detection of HIV-1 p24 antigen with a sensitivity of 0.5 pg/ml].

A "DS-EIA-HIV-AG-Screen" enzyme immunoassay system has been devised to detect HIV-1 p24 antigen with a sensitivity of 0.5 pg/ml the use of which permits reduction of a seronegative window phase as compared with the tests showing a lower sensitivity threshold. The "DS-EIA-HIV-AG-Screen" system developed may be used to screen donor blood and to examine risk-group individuals for the early diagnosis of HIV infection.

[Development and assessment of a new test for verification of tests for HIV-infection markers].

The OOO "Research-and-Production Association "Diagnostic Systems" has developed a "DC-EIA-HIV-AT/AG-SPECTRUM" screening enzyme immunoassay system designed to detect separately antibodies to certain HIV-1 and HIV-2 proteins, as well as antigen p24. The determination of antibodies of all classes and the marker of early-stage infection antigen p24 with a high (5 pg/ml) sensitivity substantially reduces the number of void results obtained in the use of immunoblots. The developed "DC-EIA-HIV-AT/AG-SPECTRUM" plate system is an effective tool to support positive screening results and may be used at the final stage of laboratory diagnosis of HIV infection.

[Study of the diagnostic potential of various regions of tick-borne encephalitis virus glycoprotien E].

Bioinformation techniques were used to analyze envelope glycoprotein of tick-borne encephalitis virus in order to determine the potential diagnostically significant antigenic clusters. Five selected regions of the amino acid sequence of protein E were retranslated to nucleic sequences, by employing the optimum code for E. coli. The resultant DNA fragments were synthesized by polymerase chain reaction from synthetic oligonucleotides and expressed in E. coli cells. The recombinant antigen replicating the region from 296 to 414 aminoacids demonstrated the most significant antigenic activity. The findings lead to the conclusion that this recombinant protein may be considered as a candidate for the development of a diagnostic assay.

[Evaluation of diagnostic efficiency of the recombinant protein modeling immunodominant epitope V3 of envelope gp120 for immunoenzyme detection for HIV-1 infection antibodies].

The third hypervariable domain V3 of the human immunodeficiency virus type 1 gpl20 envelope glycoprotein contains neutralizing epitopes and plays an important role in the diagnosis of HIV infection . Neutralizing antibodies bind to conserved epitope with sequence GPG of V3 loop. The effect of sequence variation on the antigenic properties of the V3 epitope gp120 was studied using five synthetic peptides. The amino acid sequence of the peptide corresponding to the V3 region gp120 of HIV-1 subtype C showed the highest immunoreactivity. The DNA fragment encoding V3-C region gp120 was synthesized by polymerase chain reaction and cloned into pET41b vector. The recombinant plasmid was expressed in the E. coli cells, and recombinant protein was purified using glutathione-S sepharose affinity chromatography. The serological activity of the recombinant protein was tested using ELISA and compared to activity of similar synthetic peptide. The results of this study showed that most immunoreactive agent was the amino acid sequence of V3 region gp120 of HIV-1 subtype C. The recombinant antigen comprising this sequence was more antigenic than synthetic peptide with the same sequence. The evaluation of this antigen shows that this protein is a good candidate for the immunoassay development.

[Development of a regional external quality assessment program of the diagnosis of syphilis in the Volga Federal District].

In 2004, the authors first developed and implemented a regional external quality assessment program (REQAP) for the diagnosis of syphilis in the Volga Federal District (VFD). Its objective is to improve the quality of studies in the clinical laboratories of AIDS prevention and control centers (AIDS PCC) using a cardiolipin antigen precipitation microtest and/or enzyme immunoassay (EIA) for the diagnosis of syphilis and to compare the reproducibility of results at the laboratories. The program includes a theoretical stage--the issues concerning different aspects of the diagnosis of syphilis and the variants of replies to them; a practical stage--to solve enciphered problems, to draw up protocols; and a final stage--to sum up the results of two former stages and to organize a seminar. Twenty laboratories (16 laboratories of VFD AIDS centers and 4 laboratories of other therapeutic-and-prophylactic institutions) implemented REQAP for the diagnosis of syphilis. This resulted in the creation of a normative base regulating the techniques of diagnostic studies and appropriate test systems; revealed the sources of errors in the interpretation of the results obtained by the Lewis test; showed the correct interpretation of the results of EIA; indicated shortcoming when working with guide materials. Intralaboratory monitoring could reveal the incomplete realization of the stated sensitivity of test systems in considerable numbers of laboratories of the district, and well as no skills in statistical data processing. It is concluded that the normative base of diagnostic studies should be additionally worked over, that the laboratories should obligatorily participate in the Federal external quality assessment system, and that intralaboratory monitoring should be done. Most participants of REQAP for the diagnosis of syphilis are ready to use a precipitation microtest and EIA.

Distinguishing acute from chronic and resolved hepatitis C virus (HCV) infections by measurement of anti-HCV immunoglobulin G avidity index.

An assay to measure avidity index (AI) was developed to diagnose incident hepatitis C virus (HCV) infections. The assay demonstrated an AI value statistically significantly lower in primary HCV infections than in chronic infections. When the assay was applied to past resolved infections, the difference in AI values was not as significant as the difference between incident and chronic infections. Lower AI values obtained in past resolved infections may be directly related to lower levels of immunoglobulin G anti-HCV in past resolved infections than in either new infections or chronic infections.

[Impact of amino acid sequence variation of a determinant(s) on the antigenic properties of hepatitis B virus HBsAg].

Impact of amino acid sequence variation on the antigenic properties of the surface hepatitis B virus antigen HBsAg was studied. Eleven recombinant HBsAg variants of wild (adr, ayw2, adw2, adw4, aywl, adw2) and vaccine escape (adw2 T126S, adw2 Q129L, adw2 Q129R, adw2 T143K, adw2 Q145R, aywl Q145A) were obtained. All the recombinant antigens were tested on a panel of 43 monoclonal antibodies (MAb) specific to different HBsAg determinants. Amino acid sequence variation of the a-determinant of HBsAg was shown to significantly affect its immunological responsiveness and antigenic properties. Amino acid substitution in different positions or in the same position, but for various amino acids may differently affect these properties.

[Comparison of the diagnostic value of recombinant antigens and synthetic peptides that mimic the immunogenic epitopes of the envelop protein gp41 in the enzyme immunodetection of HIV-1 antibodies].

The study was undertaken to comparatively examine the efficiency of HIV antibody detection by enzyme immunoassay using recombinant antigens and synthetic peptides containing the most immunodominant region of the human immunodeficiency virus envelop protein gp41-cytotoxic T-lymphocytic (CTL) epitope. The application of the synthetic peptides that mimic this region has been to be inadequately effective. The recombinant proteins that contain the CTL epitope may be successfully used for the diagnosis of HIV-1, by using enzyme immunoassay as they show a higher sensitivity in detecting antibodies than do the synthetic peptises.

[Impact of the heterogenicity of amino acid sequence on the immunoreactivity of an antigenic epitopic complex localized within amino acids 1192-1456 of protein NS3 protein of hepatitis C virus].

Recombinant proteins were used to study the effect of heterogenicity of the primary structure of NS3 protein of hepatitis C (HCV) on the immunoreactivity of a complex of antigenic epitopes located within the amino acid sequences 1192-1456. Six genes encoding for the above fragment NS3 from different genotypes were collected from synthetic oligonucleotides and expressed in E. coli cells, by using the polymerase chain reaction. The homology of amino acid sequences of antigens ranged from 78.4 to 92.2%. All the antigens showed a higher coefficient of their reactivity with antibodies in the sera samples from patients infected by HCV of a respective genotype; however, there was no strong genotype-specific immunoreactivity. The findings lead to the conclusion that the primary structure of antigens has an impact on their immunoreactivity. Selection of variants of the primary structures of antigens is essential in developing a diagnostic test.

[Theoretical prediction of the antigenic epitopes of severe acute respiratory syndrome (SARS-CoV) proteins and evaluation of their diagnostic value].

Analysis of the sequence of a number of proteins of the virus causing severe acute respiratory syndrome (SARS-CoV) was used to theoretically predict antigenic epitopes. Nine sequences, encoding for the predicted epitopes in spike, nucleocapside, and membranous SARS CoV proteins, were synthesized by polymyrase chain reaction and cloned into the pGEX 4-T2 vector. The resultant plasmids were expressed in the E. coil cells and purified by glutathione-Sepharose affinity chromatography.

A new enzyme immunoassay for the detection of antibody to hepatitis E virus.

BACKGROUND AND AIM: The purpose of the present study was to develop enzyme immunoassay (EIA) for the detection of IgG anti-hepatitis E virus (HEV) activity using two new recombinant proteins as antigenic targets, and to evaluate these EIA with the aid of statistical methods. METHODS: Two proteins, a mosaic protein and pB166 containing region 452-617 aa of the ORF2 of the HEV Burma strain, were used to develop the new HEV EIA. This EIA was evaluated using several panels of serum specimens obtained from: (i) acutely HEV-infected patients; (ii) patients with non-A, non-C hepatitis; (iii) normal blood donors (NBD) from non-endemic countries; and (iv) experimentally infected chimpanzees. RESULTS: A new HEV EIA was developed using two new recombinant proteins. This assay was able to detect anti-HEV activity in all specimens from acutely HEV-infected patients. When NBD were tested, more than 15% of specimens were found to be IgG anti-HEV positive. All NBD anti-HEV-positive specimens were tested with overlapping synthetic peptides spanning the entire HEV ORF2-encoded protein. More than 90% of the anti-HEV-positive NBD specimens immunoreacted with an average of 15 synthetic peptides derived from different regions of the HEV ORF2 protein. These data suggest that the HEV EIA is at least 90% specific in detecting remote HEV infections. CONCLUSION: The new HEV EIA developed in the present study is a highly specific diagnostic assay for the detection of anti-HEV activity in serum specimens obtained from different epidemiologic settings.

Detection and characterisation of swine hepatitis E virus in New Zealand.

The objectives of the present study were to establish the presence of hepatitis E virus (HEV) in New Zealand pigs, first by testing for HEV antibody in pig herds throughout New Zealand to measure the herd prevalence, then by attempting to amplify HEV genomic sequences by PCR. Antibody was measured by two independently designed ELISA serology tests. HEV RNA fragments were amplified by RT-PCR of nucleic acid extracted from faeces of 10-12-week-old piglets using primers targeting ORF1, ORF2, and ORF2/3. PCR products were subject to phylogenetic analysis. Antibody to HEV was found throughout New Zealand pig herds as well as in the different age groups within the herds. Twenty herds from 22 tested were positive for HEV antibody (91% herd prevalence). Phylogenetic analysis of the amplified sequences placed this New Zealand strain of HEV closest to the human European strain It-1 (AF 110390) and U.S. swine strain (AF 082843) with 88% and 83% similarity respectively in ORF1. It was concluded that HEV is widely distributed in the New Zealand pig population. Phylogenetic analysis shows that this is a new HEV strain, grouping most closely with the United States/European cluster, which includes HEV strains of both human and swine origin.