Immunoenzyme analysis of adenovirus hexon. Determination of antibodies from hyperimmunized chickens

We have elaborated three systems of enzyme-linked immunosorbent assay (ELISA) for detection of chicken IgG antibodies specific for hexon antigens of three immunologically distinct adenovirus groups: those of mammalian adenoviruses (Mastadenovira), typical avian adenoviruses (Aviadenovira) and of egg-drop syndrome-76 (EDS-76) virus. In each system the antibodies against respective hexons were specifically detected. In mammalian adenovirus hexons the ELISA detects primarily the type-specific (epsilon) and genus-specific (alpha) antigenic determinants. The time course of anti-hexon antibodies content was followed during immunization. The level of anti-hexon antibodies in egg yolk reflects adequately their content in blood serum. The technique is suitable for serological diagnosis of chicken adenoviral infections as well as for characterization of egg-yolk antibodies obtained by preparative hyperimmunization of hens.     

Antigenic composition of adenovirus hexons]

Quantitative relations between the group-specific and the type-specific components of the hexons of adenovirus type 2 and 5 were studied by means of FITC-conjugated Fab-fragments of antibodies directed against type 2 and type 5 hexons. From the sedimentation constant of the complexes of hexons and Fab in the region of excess of Fab we conclude that there are at least 20 determinants on the hexon. Half of these are type-specific and the others are group-specific. Both components of the type 2 hexon consist of equal parts of carbodiimide sensitive and carbodiimide resistent determinants.     

Structural and immunochemical analysis of the products of proteolysis of simian adenovirus hexons

Hexon capsomers of simian adenovirus sim16 (SA7) and of human adenoviruses h5 (Ad5) and h6 (Ad6) were proteolytically digested and the resulting products studied by SDS-polyacrylamide gel electrophoresis and by radioimmunoprecipitation analysis. The trypsinolysis of native SA7 hexon leads to a stable molecular "core" containing 4-5 fragment species of 10 to 65 kDa and resembling the intact capsomer in quarternary structure (trimer). Similar cores but consisting of smaller fragments (less than 40 kDa) were obtained after chymotryptic digestion of native SA7, Ad5 and Ad6 hexons. The chymotryptic hexon fragments were also held together in pseudotrimeric structures. The similarity of proteolytic hexon fragment patterns between different primate adenoviral hexons suggested a homology to exist in localisation of the exposed tryptic and chymotryptic cleavage sites in their respective hexon polypeptide chains. Papain caused a complete hydrolysis of native SA7 hexon (trimer) yielding small peptides, but at first stage of digestion a stable papain hexon core containing small fragments (less than 10 kDa) was observed. The tryptic SA7 hexon cores in native state retained their antigenicity in reactions with homo- and heterologous antibodies, but after core denaturation the resulting fragments had no antigenic activity of native capsomer. In contrast to the data previously published, chymotryptic cores of SA7, Ad5 and Ad6 hexons not only reacted with respective homologous antibodies but also retained (at least in part) cross-reactive antigenic determinants. The questions of formation and stability of native adenoviral hexon conformation are discussed as well as the possible nature of hexon antigenic determinants.     

Hexagonal crystalline arrays of adenovirus type 1 hexons

Separated, highly purified and concentrated adenovirus type 1 soluble hexon capsomers were crystallized by dialysis against 0.5 M acetate buffer. The crystallization process was followed electron microscopically. In the early phase of the crystallization, groups of a few hexons began to appear, then the two-dimensional crystal lattices grew gradually to a size of 1-2 micron. Simultaneously three-dimensional crystals of tetrahedral and prismatic shapes developed. The hexons in the two-dimensional crystal lattice formed regulator dense arrays corresponding to the hexagonal packing. Analysis of the crystal structure revealed 15-20% local irregularity (short range disorder) and about 10% deviation in the values of the lattice constant if determined from three different directions. The average lattice constant values showed considerable differences in different preparations. Angles formed by non-parallel hexon rows deviated by a few degrees from the regular hexagonal order. Consequently, the position of the hexons in dense two-dimensional crystals was found slightly skew and irregular, although each unit stayed within a certain distance as compared to its equilibrium position defined theoretically in the network. Dislocations were frequently found to disturb the regular arrays. The extra hexon row developing between two rows deverted them from their original direction. At these sites the crystal lattice slanted and the dense array of the hexons loosened. High resolution electron microscopy revealed fine linking structures between the hexons. In several cases the aggregated hexons failed to show a ring-like appearance, they were situated in lying--profile--position and the hexon-building polypeptide fibres became visible. The diameters of the hexons and the distance between them were measured in three directions and the size of the hexon-building polypeptides was determined as well.     

Immunoenzyme analysis for determining class G and M antibodies to HBsAg

A scheme of the purification of hepatitis B virus surface antigen (HBsAg) as applied to the enzyme immunoassay (EIA) for the detection of antibodies to HBsAg is described. An indirect EIA technique for the detection of IgG and IgM antibodies to HBsAg has been developed and the diagnostic assay system based on the use of immunoreagents and solid-phase carriers produced in the USSR has been obtained. The sensitivity of the indirect EIA technique in the detection of IgG antibodies to HBsAg exceeds that of double immunodiffusion in gel used for this purpose 2,500- to 5,000-fold. The study has shown the possibility of using the indirect EIA technique for the detection of antibodies to HBsAg, both free and bound in immune complexes, of detecting antibodies to HBsAg in patients with acute and chronic viral hepatitis B, as well as of simultaneous detection of IgG and IgM antibodies to HBsAg without pseudonegative results.     

Immunoenzyme assay of human cartilaginous proteoglycans and their antibodies

An ELISA has been developed to measure human cartilaginous proteoglycans (PG) and their auto-antibodies. The assay is described step by step: successively the nature of the microtiter wells, the concentration of the PG to be coated, the optimal dilution of the antiserum, the length of various incubations and their respective temperature, are described. Standard curve obtained with purified PG is linear in a logit-log representation. The sensitivity of the assay is 2 ng/tube. Finally, biological fluids-sérum and synovial fluid-show a good parallelism with PG.     

Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic,molecular modeling,and sequence-based methods

A major impediment to the use of adenovirus as a gene therapy vector and for vaccine applications is the host immune response to adenovirus hexon-the major protein component of the icosahedral capsid. A solution may lie in novel vectors with modified or chimeric hexons designed to evade the immune response. To facilitate this approach, we have distinguished the portion of hexon that all serotypes have in common from the hypervariable regions that are responsible for capsid diversity and type-specific immunogenicity. The common hexon core-conserved because it forms the viral capsid-sets boundaries to the regions where modifications can be made to produce nonnative hexons. The core has been defined from the large and diverse set of known hexon sequences by an accurate alignment based on the newly refined crystal structures of human adenovirus types 2 (Ad2) and Ad5 hexon. Comparison of the two hexon models, which are the most accurate so far, reveals that over 90% of the residues in each have three-dimensional positions that closely match. Structures for more distant hexons were predicted by building molecular models of human Ad4, chimpanzee adenovirus (AdC68), and fowl adenovirus 1 (FAV1 or CELO). The five structures were then used to guide the alignment of the 40 full-length (>900 residues) hexon sequences in public databases. Distance- and parsimony-based phylogenetic trees are consistent and reveal evolutionary relationships between adenovirus types that parallel those of their animal hosts. The combination of crystallography, molecular modeling, and phylogenetic analysis defines a conserved molecular core that can serve as the armature for the directed design of novel hexons.     

Immunoenzyme analysis with visual demonstration for detecting antibodies to the tick-borne encephalitis virus

Good prospects for the use of enzyme immunoassay (EIA) with the simple visual indication of results have been shown with the detection of specific antibodies to tick-borne encephalitis virus in blood serum used as an example. When compared with such highly sensitive method as radioimmunoassay, visual EIA is inferior in both sensitivity and selectivity, but its special advantage is that it requires no instrument for evaluating the result.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Inhibition of a plasminogen activator from oncogenic virus-transformed mouse cells by rabbit antibodies against the enzyme

Antisera were raised in rabbits against an electrophoretically pure 48 000 dalton plasminogen activator from mouse cells transformed by an oncogenic virus. The IgG fraction of the antisera inhibited 48 000 dalton mouse plasminogen activators from a variety of sources (neoplastic and nonneoplastic), a 29 00) dalton plasminogen activator from mouse urine and a 48 000 dalton plasminogen activator from rat urine. No inhibition was observed of a 75 000 dalton plasminogen activator extracted from mouse lung, of mouse plasmin or of plasminogen activators from human urine and from oncogenic-virus transformed chicken cells. The IgG antibodies were stronger and more specific inhibitors of the 48 000 dalton mouse plasminogen activator than any previously tested compounds.     

Antigenic determinants of adenovirus capsids. II. Homogeneity of hexons, and accessibility of their determinants, in the virion.

We have tested the two principal theories which explain the previous finding that small amounts of type-specific antibody to the adenovirus hexon can neutralize infectivity, whereas even large amounts of cross-reactive antibody do not. a) It has been suggested that the type-specific determinants are especially prominent in the virion. We have therefore measured the capacity of whole virus to bind appropriate antibodies, using a sensitive radioimmunoprecipitation (RIP) system. In fact, virions bound type-specific and cross-reactive antibodies impartially. Moreover, they bound both much less effectively than did free hexon or disrupted virus, suggesting that many of each kind of determinant are inaccessible in virions. b) It has been suggested that the type-specific determinants are confined to those hexons located next to the pentons, and that they are the targets for neutralizing antibody. We have therefore studied the antigenicity of peripentonal and nonamer hexons isolated from virions, and found that each possessed both kinds of determinants. Furthermore, these were present in the same proportion as in hexons purified from the soluble antigens in infected cells ("free hexons"). We concluded that the mechanism of neutralization by antibody is complicated, and that the type-specific determinants exposed on the virion must play a crucial role.     

Immunoenzyme analysis method for detecting anti-Pseudomonas aeruginosa antibodies in persons inoculated with pyoimmunogen

The possibility of using the indirect ELISA techniques for evaluating the level of the post-vaccinal production of humoral antibodies in donors immunized with Pyoimmunogen. P. aeruginosa vaccine, has been studied. The specificity and high resolution of this test system, based on the immobilization of the antigens of the vaccinal preparation on a solid-phase carrier, have been demonstrated. A rational method for the evaluation of specific antibody titers with due regard to the spectrophotometric data indicating the results of the reaction and the degree of the dilution of the serum under test has been proposed.     

Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of 125I-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for 125I-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. 125I-M110 and 125I-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL (ID50 = 0.44 nM) was comparable to that of 125I-oPRL by unlabeled oPRL (ID50 = 0.35 nM), while 125I-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82. These findings indicate that monoclonal antibodies can be readily prepared from partially purified PRL receptors from rabbit mammary gland; two antibodies (M110 and A82) are hormone binding site specific while the other (A917) binds a domain partially but not entirely distinct from the hormone binding site, and that all three antibodies have strong species specificity.     

Homophilic binding of mouse monoclonal antibodies against GD3 ganglioside.

R24, a mouse IgG3 mAb against GD3 ganglioside, was shown to bind to itself in a homophilic manner. This was demonstrated by augmented binding of 125I-labeled R24 to the cell surface of GD3+ cells by unlabeled R24 and by direct binding of biotinylated R24 to R24 adsorbed on solid phase. Although homophilic binding was evident when R24 was bound to solid phase, R24-R24 aggregates could not be detected in solution under otherwise identical conditions. R24 bound to four other mAb (two IgG3, one IgG2a, one IgM) directed against GD3 but did not bind to a panel of 21 other mAb including other IgG3 mAb and mAb directed against non-GD3 ganglioside. Evidence implicating the GD3-binding site of R24 in homophilic binding included the following observations: 1) F(ab')2 fragments of R24 could bind to R24, 2) an antiidiotypic mAb against the GD3-binding site of R24 inhibited R24 homophilic binding, 3) an IgM anti-GD3 mAb also demonstrated homophilic binding to R24, and 4) homophilic binding was a function of immunoreactivity and avidity for GD3. R24 variants with 40-fold lower avidity for GD3 demonstrated a similar decrease in homophilic binding. Inasmuch as R24 bound to R24 F(ab')2 fragments and specifically to anti-GD3 mAb, it appeared that the target for homophilic binding was an epitope within the V region of anti-GD3 mAb. It is likely that homophilic interactions result in increased affinity of R24 for GD3 through increased effective valency of antibody-Ag complexes.     

Isotype-specific rabbit antibodies against chinchilla immunoglobulins G, M, and A.

Chinchillas have become a preferred animal model for studying otitis media, and are also useful in studying insulin release, gastrin physiology, intestinal infection, and hepatocellular pathophysiology. Immunopathologic studies in the model, however, have been limited by absence of specific antibody reagents against chinchilla immunoglobulins. We describe a method for preparing isotype-specific rabbit antibodies against the heavy-chain components of chinchilla immunoglobulins G, M, and A. Chromatographic techniques were used to isolate chinchilla immunoglobulins from serum and breast milk; heavy-chain fractions were isolated and used as antigens to produce isotype-specific antibodies in New Zealand White rabbits. Enzyme-linked immunosorbent assay of these antisera disclosed anti-light chain cross-reactivity, which was removed by affinity chromatography. The isolation and affinity purification techniques were highly reproducible. The availability of these reagents should greatly enhance the utility of the chinchilla in modeling human disease.