[Effect of pH of Adsorption Buffers on the Number and Antigen-Binding Activity of Monoclonal Antibodies Immobilized on the Surface of Polystyrene Microplates].

The change in the concentration and antigen-binding activity of 28 monoclonal antibodies was studied after their adsorption on the surface of polystyrene microplates in buffers with different pH values (1.0, 2.8, 7.5, 9.6, and 11.9). We used 16 clones to the HIV p24 protein and 12 clones to the surface antigen of Hepatitis B Virus. The binding efficiency of adsorbed antibodies to the labeled antigen was evaluated by the slope of the linear region of the binding curve to the concentration axis. It was shown that the antigen-binding activity of six antibodies (21.5%) statistically significantly increased after adsorption at pH 2.8 and 11.9 as compared to pH 7.5 and 9.5. The maximum amount of antibodies was found to be adsorbed on the solid surface at pH 7.5. The analysis of the binding of 125I-HBs-antigen to adsorbed antibodies made it possible to evaluate the concentration of active antibodies on the polystyrene surface. It was shown that the increase in the antigen-binding activity was due to an increase in the proportion of antibodies with retained activity after adsorption at pH 2.8 and 11.9. Under these conditions, about 20% of the antibodies retained their antigen-binding activity, and 6% did so after immobilization at pH 7.5.

[The comparative study of specificity of test-systems in diagnostic of HIV-infection on categories of samples of blood serum of pregnant women].

The detection of false positive serological reactions (FPSR) on HIV-infection under screening examination of pregnant women is an actual problem of practical health care. The original observations testify that under analysis of the same samples of blood serum of pregnant women using screening immune enzyme test-systems of various manufacturers the unmatched data concerning FPSR can be obtained. The purpose of this study was to implement comparative evaluation of specificity of immune enzyme test-systems of three different manufacturers: "DS-IFA-HIV-AGAT-SCREEN" ("Diagnostic Systems"), "Genscreen Ultra HIV Ag-Ab" "Bio Rad" France) and "The CombiBest HIV-1,2 AG/AT" ("Vector-Best" Novosibirsk). The sampling of 440 samples of blood serums of pregnant women from various medical institutions of Nizhnii Novgorod was analyzed. The results of the study demonstrated that FPSR were detected in all test-systems and at that spectrum of samples differed. The identical specificity of compared test-systems amounted to 98.64%. The alternative approach to FPSR to HIV issue under screening examinations of pregnant women was proposed. The proposed mode consisted of consistent application of two test-systems of fourth generation with different format of setup of reaction.

[Comparative analysis of the immune response to DNA constructions encoding hepatitis C virus nonstructural proteins].

A promising approach to construction of antiviral vaccines consists in activation of cellular immunity with the DNA vaccines. The goal of this work was to evaluate the efficacy of genetic immunization of mice with DNA pcNS3-NS5B encoding five hepatitis C virus (HCV) nonstructural proteins: NS3, NS4A, NS4B, NS5A, and NS5B in comparison with plasmids containing genes of same individual nonstructural proteins. The DNA constructions were injected intramuscularly in DBA mice three times. The humoral immune response was assessed with ELISA; cellular immune response--in blast transformation reaction, by quantitation of CD4+ and CD8+ T cell proliferation using flow cytofluorometry, by intracellular synthesis and secretion of IFN-gamma and IL-2 in ELISpot and ELISA. It was found that the functionally active T cell response was achieved to antigens presenting NS3, NS4, NS5A, and NS5B epitopes of different HCV genotypes in response to pcNS3-NS5B plasmid and was stronger than that to plasmids carrying individual genes. A high proliferation rate of CD4+ T cells, secretion of IL-2 and IFN-gamma, induction of anti-NS3 and anti-NS5B IgG2a were demonstrated. These findings indicate that DNA construction pcNS3-NS5B is one of promising candidates for anti-HCV vaccine developing.

[The combined application of nucleotide and amino acid sequences of NS3 hepatitis C virus protein, DNA encoding granulocyte macrophage colony-stimulating factor and inhibitor of regulatory T cells induces effective immune response against hepatitis C virus].

Hepatitis C is related to the most important socially significant human infectious diseases; however, vaccine against this virus up to now has notbeen created. One of the possible components of vaccine is the nonstructural protein NS3 of hepatitis C virus (HCV), which is synthesized in the infected cells and displays protease, NTPase, and helicase enzymatic activities. The connection between the effectiveness ofT cellular response to NS3 epitopes and the spontaneous resolution of acute hepatitis C was shown. The purpose of this work was to compare the immune response of mice to the inoculation of nucleotide and amino acid sequences of HCV NS3 and their combination, to evaluate the adjuvant activity of the DNA encoding granulocyte macrophage colony-stimulating factor (GM-CSF) and the influence of regulatory T cells on the effectiveness of immune response. The maximum anti-HCV NS3 antibody level in the serum (to 1:640000) induced the recombinant protein rNS3 introduced with aluminum hydroxide. The most intensive cellular immune response was observed after the simultaneous administration of rNS3 and DNAs encoding full-size NS3 and GM-CSF. A high level of lymphocyte proliferation, accumulation of IFN-gamma-secreting cells and IFN-gamma, and IL-2 release in response to the stimulators--NS3 antigens of different composition were observed in this group of mice. It has been established that the suppression of regulatory T cells in vitro leads to the statistically significant increase in the secretion of IFN-gamma. Thus, simultaneous application of rNS3 along with the DNAs encoding full-size NS3 and GM-CSF is promising approach for development of hepatitis C vaccine. The expediency of inclusion in the vaccine composition of regulatory T cell inhibitors will be clear after special studies.

[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.

[DNA immunization with a plasmid containing gene of hepatitis C virus protein 5A (NS5A) induces the effective cellular immune response].

In spite of extensive research, no effective vaccine against hepatitis C virus (HCV) has been developed so far. DNA immunization is a potent technique of vaccine design strongly promoting the cellular arm of immune response. The genes encoding nonstructural HCV proteins (NS2-NS5B) are promising candidates for vaccine development. NS5A is a protein involved in viral pathogenesis, in the induction of immune response, and probably in viral resistance to interferon treatment. The objective of this study was to construct a DNA vaccine encoding NS5A protein and evaluate its immunogenicity. A plasmid encoding a full-size NS5A protein was produced using the pcDNA3.1 (+) vector for eukaryotic expression system. The expression of the NS5A gene was confirmed by immunoperoxidase staining of the transfected eukaryotic cells with anti-NS5A monoclonal antibodies. Triple immunization of mice with the plasmid vaccine induced a pronounced cellular immune response against abroad spectrum of NSSA epitopes as assessed by T-cell proliferation andsecretion of antiviral cytokines IFN-gamma and IL-2. In in vitro T-cell stimulation experiments, NS5A-derived antigens were modeled by synthetic peptides, recombinant proteins of various genotypes, and phages carrying exposed NS5A peptides. A novel immunomodulator Immunomax showed high adjuvant activity in DNA immunization. The data obtained indicate that the suggested DNA construct has a strong potential in the development of the gene vaccines against hepatitis C.

[Use of pH-sensitive immobilized monoclonal antibodies for optimization of immunoenzyme sandwich technique of detection of HBsAg of hepatitis B virus].

AIM: To develop highly sensitive sandwich technique for identification of surface hepatitis B virus antigen (HBsAg) in serum and analyse of possible improvement of solid phase for immunoenzyme sandwich technique of HBsAg identification through variation of pH-dependent sorption of monoclonal antibodies on the surface of immune plates. MATERIALS AND METHODS: Calibration curves for identification of HBsAg in sandwich techniques using 36 possible binary combinations of monoclonal antibodies of our panel (including high affinity antibodies to HBsAg produced by 6 hybridomas) were compared. Immobilization of antibodies on solid phase (by passive sorption) was performed at different pH values (2.8, 7.5, and 9.5). RESULTS: Analysis of panel of antibodies to HBsAg produced by 6 hybridomas revealed pH-dependent monoclonal antibodies (18C8), which immobilization at low pH values together with detecting antibodies F4F3 allowed to greatly improve sensitivity of the sandwich technique. Minimal credibly detectable concentration of HBsAg in sera of persons infected with hepatitis B virus was 0.013 - 0.017 ng/ml. Validation of sandwich technique was performed on certified panel of serum samples with various concentrations of HBsAg (different serotypes). CONCLUSION: Highly sensitive sandwich technique for detection of HBsAg was developed. It was shown that analysis of panel of monoclonal antibodies on pH-dependence could be used as simple methodical approach for optimization of immunoenzyme sandwich techniques for detection of different antigens.

[HIV-1 resistance in the Yamal region and the comparison of mutation frequency with various score values].

The amino acid sequence of the known strain HXB-2 belonging to subtype B is used as a reference strain matrix to determine HIV genotypic resistance by the sequencing technique. However, numerous studies have ascertained that HIV non-B subtypes have been widely accepted in Russia. The authors' data show that the significance of low score mutations in the Russian populations of HIV-1 is yet to be explained, unquestionably, arouses great scientific interest, and calls for further study.

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.

[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.

[The capacities of current test systems to verify early HIV infection].

The purpose of the present investigation was to comparatively evaluate the performance characteristics of the test systems designed to verify the positive results of screening survey for HIV infection, such as the solid-phase immunoassay DS-EIA-HIV-AB/AG-SPECTR (Diagnosticheskiye Sistemy (Diagnostic Systems) Research-and-Production Association, Nizhni Novgorod) and tests based on immune blotting (IB). The investigation examined 15 seroconversion panels produced by ZeptoMetrix (USA) and BBI (USA). The use of the DS-EIA-HIV-AB/AG-SPECTR test system determined 88 of the 167 seroconversion panels as HIV positive. The IB-based tests revealed only 45 of the 167 samples as positive. Consequently, the application of the DS-EIA-HIV-AB/AG-SPECTR test system is more effective than the IB-based tests in early HIV infection.

[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.

[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.

[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.

[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.

[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.

[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.

[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.