[The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").

[Influence of physiological state of Escherichia coli cells on the expression of soluble protein--recombinant analog of glycoprotein G of Herpes simplex virus of type 2].

It is shown that the recombinant protein GST-HSV2gG, containing the immunodominant regions of glycoprotein G of HSV-2 is accumulated in the form of inclusion bodies or in soluble form in the Escherichia coli BL21 (DE3) cells. The ratio between protein fractions varied depending on the physiological state of cells before biosynthesis. The kinetic parameters of bacterial populations were determined by mathematical modeling of growth curves based on the Verhulst logistic function. It was established that the induction of biosynthesis in the growth acceleration phase (at OD600 = 0.3) with 0.1 mM IPTG gives the maximum yield of soluble protein (26.75 mg/l or 17.6 mg/g biomass). The target protein was purified using the immobilized metal ion affinity and affinity chromatography technologies. Antigenic activity of the soluble form of recombinant protein GST-HSV2gG, was significantly (three times) higher than that of the protein purified from inclusion bodies (p < 0.05) and was comparable with the activity of the commercial analog (p > 0.05), that allows using this product in the immunosorbent test kits for diagnosis of IgG to HSV-2.

[Purification and properties of the hexon antigen of canine adenovirus serotype CAV-1]. / Ochistka i svoistva geksonobogo antigena adenovirusa sobak serotipa CAV-1.

The main antigen and immunogen of canine adenovirus type 1 (CAV-1) has been purified to near homogeneity from cultural fluid of a CAV-1-infected primary cell culture by hydrophobic and anion-exchange chromatography. The hexon native form (trimer) was shown to be resistant against denaturation by SDS under conditions of SDS-PAGE performed without heating the samples. The monomer chain of the CAV-1 hexon was apparently identical in terms of electrophoretic mobility with that of the previously sequenced BAV-3 hexon polypeptide (103 kDA). In blot enzyme immunoassay only native trimers of CAV-1 hexon were detected by cross-specific polyclonal and monoclonal anti-hexon antibodies.

Comparison of adenoviral hexon polypeptides (monomers) and of native hexons (trimers) by SDS-polyacrylamide gel electrophoresis.

Purified hexons of 27 serotypes of human, simian, bovine and avian adenoviruses were analysed by SDS-PAGE. The apparent molecular weights of hexon polypeptides calculated by comparison with 5 non-hexon and 3 sequenced hexon polypeptide markers ranged from 98 kDa (for bovine adenovirus Ad bos7) to 118 kDa (for simian adenovirus Ad sim13; SV36). A stability of native hexon capsomers (trimers) in SDS at room temperature permitted us to resolve native (trimeric) hexon by SDS-PAGE and to distinguish them from denatured (monomeric) hexon polypeptides by electrophoretic mobilities. Hexon trimer bands with slow mobility in SDS-PAGE (unlike hexon monomer polypeptide bands) retained native hexon antigenicity as revealed by immunoblot analyses. Possible applications of simultaneous analyses of hexon trimers and monomers by SDS-PAGE are discussed.

Characterization of a series of monoclonal antibodies specific for bovine adenovirus serotype BAV3 hexon antigen and their use in an ELISA for detection of adenoviral hexons.

After immunization of mice with purified hexon (the main capsid antigen) of bovine adenovirus serotype BAV3 we have obtained a set of 16 individual hybridoma clones producing MAb's against BAV3 hexon. All MAb's were shown to belong to immunoglobulin G class. Specificity of the most avid MAb marked B3Hx-1 was tested on a panel of representative hexon antigens from 16 adenovirus serotypes of human and animal origin using several immunoassays. In Western blot analysis the MAb B3Hx-1 reacted only with native (trimeric) form of hexon protein and not with denaturated hexon polypeptide chains. The epitope defined by B3Hx-1 appeared stable against SDS at ambient temperature and against chloramine-promoted iodination. The specificity of the epitope was characterized as almost genus-crossreactive: it was absent from hexons of avian and of bovine subgroup 2 adenovirus serotypes and present in most hexons of bovine, canine, simian and human adenoviruses tested. Within the latter group its expression was weak or absent only for human subgenus C serotypes. Several variants of sandwich-type ELISA were developed using MAb B3Hx-1 and different polyclonal antibodies against hexons of mammalian adenoviruses. The level of hexon detection for different adenovirus serotypes varied in range 10(-9) to 10(-8) g per ml.

[Immunoenzyme analysis of adenovirus hexons. Indication and characteristics of rabbit and mouse antibodies against hexons]. / Immunofermentnyi analiz geksona adenovirusov. Indikatsiia i kharakteristika krolich'ikh i myshinykh antitel protiv geksonov.

An enzyme-linked immunosorbent assay (ELISA) was developed for analysis of rabbit and mouse IgG antibodies specific to adenoviral hexon. The anti-hexon antibodies were detected by capture with purified hexon coated onto polystyrene microtiter plates and visualizing them by respective anti-IgG horseradish peroxidase conjugates. In the sera from hyperimmunized rabbits and mice as well as in the mouse ascite fluids the ELISA procedure revealed primarily type-specific (epsilon) and genus-specific (alpha) antigenic determinants in hexon but not those of intermediate specificities.

[Stability of the structure and antigenic determinants of adenovirus type 1 native hexon to proteases]. / Ustoichivost' struktury i antigennykh determinantov nativnogo geksonaadenovirusa tipa 1 k proteazam.

Hexon capsomers of human adenovirus type 1 (h1) labeled by iodine 125 were digested in a native state (trimers) by trypsin, chymotrypsin or papain, and the resulting hydrolysates were analyzed by SDS-PAGE. In each case, a discrete and temporally stable pattern of relatively large fragments was revealed. The degree of hexon polypeptide hydrolysis was maximal for papain, intermediate for chymotrypsin and minimal for trypsin, the largest fragments in the digest being 32, 40 and 80 kD, respectively. At room temperature, all the electrophoretically discernible hexon proteolytical fragments were held together in structures resembling intact hexon trimers and could be regarded as "hexon cores", of which papain hexon cores were the most stable during SDS-PAGE. Radioimmunoprecipitation analysis revealed a complete absence of native hexon antigenicity in thermodenaturated fragments of hexon protease digests, while native trypsin, chymotrypsin and papain hexon cores could be precipitated by hexon-specific antibodies. The immunoprecipitated material contained all of the hexon fragments found in appropriate hexon cores and retained the structure of the original cores. Trypsin, chymotrypsin and papain hexon cores were shown to possess at least part of native Ad h1 hexon antigenic determinants of each of the following specificities: species-specific (epsilon), cross-reactive with hexon of human adenoviruses (h3 and h6), simian adenovirus (sim 16), bovine adenoviruses (bos 3 and bos 7) and avian adenovirus (Aviadenovirus gal 1 or CELO). Thus, the full spectrum of known hexon antigenic determinants (species-specific to intergenus-crossreactive) is at least portly stable against protease attack of native hexon capsomers.

Changes of adenovirus hexon associated with different passage history of Ad h 1.

Two descendants of the prototype strain AD71-Washington D. C. were obtained by independent passaging for at least 18 years in Kiev, and in Budapest (Ad h 1 kappa, and Ad h 1B, respectively). By restriction endonuclease mapping, the DNA was identical corresponding to the patterns of human adenovirus type 1. In spite of this, SDS-polyacrylamide gel electrophoresis revealed that the purified hexon of Ad h 1 kappa was of lower Mr than the subunit of Ad h 1B. In contrast to this, the native capsomer (hexon) of Ad h 1 kappa exhibited lower electrophoretic mobility in agarose gel electrophoresis than the native hexon of Ad h 1B. Oligopeptide mapping of the main hexon bands from SDS-polyacrylamide gels revealed the presence of unique spots among the chymotryptic oligopeptides of Ad h 1B, too. Thus, the differences in the sensitivity to proteolytic cleavage during purification seem to have a structural basis. Antigenic analysis of the native hexon capsomers was performed using polyclonal antihexon immunsera. Immunodiffusion, immunoelectrophoresis, and competitive RIA were used for comparison. The results indicate that native hexon capsomers of Ad h 1 kappa and Ad ha 1B possess antigenic differences within the type-specific regions, nevertheless, their genetic background could not be detected by the restriction endonucleases applied. It cannot be excluded that the differences were results of altered assembly of virions under different passage conditions.