Antibody response in salmonids against the 70 kDa serine protease of Aeromonas salmonicida studied by a monoclonal antibody-based ELISA.

An antigen-capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies (mAb) was set up and evaluated for selective detection of salmonid antibody responses to the antigen P1, which is a weakly immunogenic exoprotease of typical Aeromonas salmonicida. This new assay permits a specific determination of anti-protease-antibodies, without antigen purification. Serum antibodies induced by the strongly immunogenic lipopolysaccharide could reliably be discriminated from anti-P1-antibodies. Antibody titres of 45 experimental antisera recorded by cELISA were moderately correlated with titres determined by routinely used indirect ELISA (iELISA) by detecting partially different antibody populations (r=0.753). Substitutions of immunoreactants and confirmatory immunoblotting strongly suggest that the mAb-based assay selectively recognises antibodies directed to epitopes of native protease. A conjugate of inhibited protease and cationized bovine serum albumin (cBSA) was found to engender a significant anti-protease-response in three salmonid species (P<0.05), whereas the unconjugated antigen and Apoject 1-Fural were proved to be ineffective. Recorded specific antibody titres were as high as 1:381,400, indicating a considerable enhanced immunogenicity of cBSA-conjugated P1 and high assay sensitivity. The established cELISA offers a promising approach to further improvement of monitoring fish humoral immune response to surface accessible epitopes of the immunosuppressive exoprotease, P1, and to scrutinize its protective significance.

A-protein from achromogenic atypical Aeromonas salmonicida: molecular cloning, expression, purification, and characterization.

Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the protein in Escherichia coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein. Recombinant A-protein was compared by biochemical, immunological, and molecular methods with the A-protein isolated from atypical A. salmonicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type when examined by SDS-PAGE and gel filtration chromatography. The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western blot techniques. All forms of A-protein were found to activate the secretion of tumor necrosis factor alpha from murine macrophage. To date, this represents the first large-scale production of biologically active recombinant A-protein.

Generation and preliminary characterization of monoclonal antibodies directed to glycerophospholipid:cholesterol acyltransferase (GCAT) native epitopes of Aeromonas salmonicida.

Four monoclonal antibodies (MAbs) directed to native glycerophospholipid:cholesterol acyltransferase (GCAT) epitopes of Aeromonas salmonicida were isolated using an esterase capture assay. The molecular mass of this MAb-defined antigen was estimated to be 26 kDa in SDS-PAGE. Three different epitope specificities of these MAbs were demonstrated. It was shown that all 4 MAbs recognize GCAT in culture filtrates of the strain MT004 excluding the simultaneous trapping of other components. None of the MAbs react with the denatured GCAT in Western blots.

Protein-water interaction energies as predictor for antigenic determinants.

Goodford's GRIN/GRID method is used for the prediction of antigenic determinants of lysozyme by calculation of protein-water interaction energies. The comparison of the regions of high interaction energy with experimentally determined contact surfaces in antigen-antibody complexes and epitopes obtained by cross-reactivity measurements shows a noteworthy agreement. The model is proposed to enlarge the basis of theoretical models for epitope prediction. It may contribute to the increase of the prediction value when applied together with other methods.

[Coupling of bovine gamma globulin to carbonochloridate-activated cellulose beads, CNBr-activated Sepharose CL-4B and NaIO4-oxidized Sepharose 6B and use of the coupled products for immunoaffinity chromatography]. / Kopplung von Rindergammaglobulin an Chlorameisensäureester-aktivierte Perlcellulose, CNBr-aktivierte Sepharose CL-4B und NaIO4-oxidierte Sepharose 6B sowie Einsatz der Kopplungsprodukte zur Immunaffinitätschromatographie.

For coupling 25 mg of bovine IgG (BGG) were given to 5 ml volumes of packed bead cellulose activated by 5-norbornene-2.3-dicarboximido carbonochloridate and CNBr-activated Sepharose CL-4B, respectively. Thus, BGG-immunosorbents were obtained with 4.6 to 4.9 mg BGG/ml matrix. 5 ml volumes of packed NaIO4-oxidized Sepharose 6B coupled 27% from 25 mg of BGG, only. In this case, immunosorbents with 1.35 mg BGG/ml matrix were produced. All BGG-immunosorbents were chemically relatively stable. The use of these immunosorbents for affinity chromatography results in the isolation of one milligram of pure rabbit anti-BGG antibodies by means of about 4.6 mg of BGG coupled to the cellulose or the Sepharose-CL-4B matrices. On the other side, only 3.4 mg of BGG coupled to the NaIO4-activated Sepharose 6B were necessary in order to isolate one milligram of antibodies in an immunoelectrophoretically pure state.

Comparative studies on the structure of biliary immunoglobulins of some avian species. I. Physico-chemical properties of biliary immunoglobulins of chicken, turkey, duck and goose.

In pooled bile of chicken, turkey, duck and goose immunoglobulins (Igs)+ were found in relatively high amounts between 4.5 and 15.0 mg/ml (corresponding to 28 to 36% of the total protein contents). All biliary Ig fractions studied possess (H2L2)n structure estimated by SDS-PAGE. In chicken and turkey L chains are assumed to be non-covalently linked to the polymeric H chains. In duck and goose L chains are completely linked to H chains. The Mr of H chains are rather similar: 64 kDa for the Igs of chicken and turkey bile and 67 kDa for the biliary Igs of duck and goose. There are slight differences in the electrophoretic mobility: Chicken and turkey Igs are beta 1/alpha 2-globulins and the corresponding Ig fractions from duck and goose are beta 2-globulins.

Comparative studies on the structure of biliary immunoglobulins of some avian species. II. Antigenic properties of the biliary immunoglobulins of chicken, turkey, duck and goose.

Studies on the immunological cross-reactivity of the Igs+ from the bile of chicken and turkey (order Galliformes) as well as duck and goose (order Anseriformes) to Igs with antigenic known properties carried out by a series of RIA. Various class-specific anti-Ig sera produced in rabbits, guinea-pigs and mice were used. The following results were obtained: (1) A high extent of antigenic relationship between the chicken and turkey biliary Igs was demonstrated by a radiobinding system consisting of 125I-turkey biliary Ig and class-specific rabbit anti-turkey biliary Ig antibodies. The biliary Igs of duck and goose did not inhibit this antigen binding system. (2) The duck and goose biliary Igs were able to inhibit the binding of 125I-carp IgM to Fc-fragment-specific rabbit anti-carp IgM antibodies and of 125I-human IgM to Fc-fragment-specific guinea-pig anti-human IgM antibodies, respectively. (3) None of the biliary Igs show cross-reactive properties common with human and porcine IgA. That was detected by inhibition of the radio-binding system from 125I-human IgA and alpha chain-specific mouse anti-human IgA antibodies. Therefore, the conclusion was made that in anseriform birds IgM-like Igs are the secretory Igs, while in galiform birds biliary Igs with special antigenic properties occur.

[Production and characterization of monoclonal antibodies to the secretory components of human IgA]. / Herstellung und Charakterisierung von monoklonalen Antikörpern gegen die sekretorische Komponente des humanen IgA.

The monoclonal antibodies BL-HSC/1-3 are characterized by means of indirect radioimmunotechnique with regard to their binding pattern to a set of human proteins. The results suggest a binding specificity of the three monoclonals for A-determinants of the human secretory component. However, we could not point out a different binding pattern to the immunoglobulin-bound and the free molecule, respectively. By use of appropriate immunoassays, the monoclonal antibody BL-HSC/3 is the most suitable one for the detection of the secretory component and secretory IgA in body fluids. The protein band labeled by BL-HSC/3 in the immunoblotting analysis corresponds to a molar mass of 79,000 D according to the data known for the human secretory component.

Sepharose gels as antigenic matrices for specific binding of anti-galactosyl antibodies.

From 100 ml of pooled normal serum taken from different species (man, cow, rabbit, chicken, duck, carp) 0.5 to 4.0 mg of high and low molecular weight anti-galactosyl antibodies could be isolated by affinity-chromatography on non-ligand-attached Sepharose 4B. The antibodies bound to the agarose matrix were eluted with 0.5 M D-galactose containing buffers. Using species-specific anti-immunoglobulin sera in immunoelectrophoresis, the immunoglobulin character of the protein fractions prepared was analyzed. In addition, the H and L chain structure of the anti-galactosyl antibodies was checked by SDS-PAGE.

On the theoretical prediction of protein antigenic determinants from amino acid sequences.

The applicability range of a modified version of the Hopp-Woods model for the determination of protein antigenic determinants is estimated by comparing experimental data based on monoclonal and polyclonal antibodies. The results indicate a close correlation between the hydrophilicity of protein regions and their antigenicity. The efficiency of various hydrophilicity/hydrophobicity scales for the amino acid side chains is tested. A comment on the recognition factor concept of Fraga is given.

Evolution of low molecular weight immunoglobulins. V. Degree of antigenic relationship between the 7S immunoglobulins of mammals, birds, and lower vertebrates to the turkey IgY.

There are close antigenic relations between the turkey IgY--the dominant 7S Ig of serum--and the corresponding IgY-like Igs of chicken, duck, goose, tortoise, and frog as well as serum or colostral IgA of man and swine. The RIA-systems used for the determination of immunological cross-reactivity among the Ig types consisted of 125-I-labelled turkey IgY and class-specific rabbit anti-turkey IgY antibodies. The results stand in a good agreement with our findings demonstrating strong antigenic relationships between various serum or colostral Igs of IgY and IgA types, respectively, to the chicken IgY. Therefore, the IgY-like Igs seem to be the precursors for the IgA class of mammals.

[Evolution of the immunoglobulins]. / Zur Evolution der Immunglobuline.

Review. All vertebrates, so far tested, possess immunoglobulins of IgM class similar to the IgM of mammals in structure and function. On the phylogenetic level of anuran amphibians a low-molecular weight 7S immunglobulin type appears occurring in the representatives of reptiles and birds, too. These 7S immunoglobulins (earlier called IgY-like immunoglobulins) show physico-chemical and antigenic properties common to the mammalian IgA. Therefore, they can be considered the precursors of mammalian IgA. The immunoglobulins of IgG, IgD, and IgE classes represent phylogenetically modern molecules appearing during the evolution on the level of mammals. Besides, some special developments can be observed amongst the immunoglobulins.

Evolution of low molecular weight immunoglobulins--IV. IgY-like immunoglobulins of birds, reptiles and amphibians, precursors of mammalian IgA.

Radioimmunochemical studies on the comparison of the immunological cross-reactivity between the 7-S Igs of birds, reptiles and amphibians (IgY-like Igs) and the IgA of mammals (man and pig) using 125I-chicken IgY and anti-chicken IgY(Fc) or anti-chicken IgY(H) antibodies from rabbits and carp for the detection led to the conclusion that there are close antigenic relationships between them. Therefore, the IgY-like Igs seem to be the precursors for the IgA class of mammals. From that, we give a phylogenetic tree of Igs in accordance with the evolutionary development of vertebrates.

Radioimmunochemical studies on 7.8S and 5.7S duck immunoglobulins in comparison with Fab and Fc fragments of chicken IgY.

In ducks two forms of low molecular weight Ig's +), 7.8S and 5.7S, have been described. To compare their antigenicity with Fab and Fc fragments of chicken IgY a double-antibody RIA technique was used in which the binding of 125I-chicken IgY or 125I-chicken IgY (Fc) to rabbit and carp anti-chicken IgY (Fab) and anti-chicken IgY (Fc) antibodies, respectively, was inhibited by duck 7.8S and 5.7S Ig's. Chicken IgY and duck 7.8S Ig ( IgY -like protein) are highly cross-reactive with respect to their Fab as well as Fc part determinants. On the other hand, the duck 5.7S Ig shows nearly identical determinants to the Fab fragments of 7.8S Ig, but a Fc part is lacking. Therefore, we conclude that the 5.7S Ig molecule in ducks doesn't represent a separate Ig class. It consists of the same principal type of heavy chains as the 7.8S Ig, but the H chain of the 5.7S Ig lacks very likely the last two homologous constant regions.

Evolution of low molecular weight immunoglobulins--III. The immunoglobulin of chicken bile--not an IgA.

Studies on comparison of antigenic relationships between the immunoglobulin of chicken bile--the so-called chicken IgA--and human, porcine, as well as murine IgA were performed by a series of double-antibody and solid-phase RIA systems using H chain-specific anti-chicken biliary Ig sera and alpha chain-specific anti-human IgA sera produced in rabbits and in carp. It was not possible to demonstrate a significant immunological cross-reactivity of the Ig of chicken bile to the Ig's of IgA class. Besides, we could not find an Ig type similar to this chicken biliary Ig in pooled normal sera or in the crude Ig fractions precipitated by (NH4)2SO4 of several vertebrate species (man, swine, buzzard, duck, goose, kestrel, kite, three tortoise species, frog, carp and perch); or in the bile of duck, goose, two snake species and frog. Only pooled chicken normal serum contained this biliary Ig type. Accordingly, the chicken biliary Ig can no longer be considered a precursor of mammalian IgA. This Ig of chicken bile represents a unique Ig class of birds and is to be called IgB (dominant biliary Ig of some birds).

[Structural and functional characteristics of IgG1- and IgG2-antibody subclasses in the guinea pig. 2. Production and testing of specific antisera against IgG1 and IgG2-antibody subclasses and against Fc-, Fc'- and Fab-papain fragments of IgG2-antibodies in guinea pigs]. / Untersuchungen über strukturelle und funktionelle Eigenschaften von IgG1- und IgG2-Antikörpern des Meerschweinchens. 2. Herstellung und Testung spezifischer Antiseren gegen IgG1- und IgG2-Antikörper-subklassen sowei gegen Fc-, Fc'- und Fab-Papainfragmente der IgG2-Antikörper von Meerschweinchen.

Rabbit antisera against specifically purified guinea pig IgG1 and IgG2 antibodies and against several papain-fragments of IgG2 were produced. These antisera were made specific by absorption with purified immunoglobulin of the other subclass and assayed for monospecificity by two immunological tests. In the macrophage rosette inhibition test, only the anti-IgG2 and the anti-Fab (IgG2) sera were effective. Inhibition of the rosette formation could be obtained at dilutions up to 1:100 or higher. Attempts to induce passive cutaneous anaphylaxis in the guinea pig skin after sensibilization with homocytotropic antibodies were evidently positive only with rabbit anti-guinea pig IgG1 serum. Anti-IgG2 and anti-IgG2 Fab sera revealed a positive reaction in higher concentrated forms (1:2 or 1:5 diluted) only.