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.

Development of an ELISA-system for the assessment of furunculosis vaccines.

As an alternative method for challenge experiments in fish a sandwich-ELISA has been established for the detection of potentially protective antibody populations. Assay specificity depends on monoclonal antibodies (mabs) directed against different antigens of Aeromonas salmonicida, the causative agent of furunculosis. Comparable levels of salmon antibodies against LPS and the A-layer protein were recorded by ELISA and antigen binding assay.

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).

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.

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.

[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].

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.

[Production and characterization of monoclonal antibodies to the secretory components of human 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.

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).

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.

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.

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.

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.

[Evolution of the immunoglobulins].

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.

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.

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.

[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].

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.

[Extension of the rosette test in the estimation of TAA-binding cells in breast cancer and mastopathy patients].

The antigen specific rosette test was used for the evaluation of a specific sensibilization of a tumour bearer to TAA. The frequency of the rosettes formed by peripheral blood leucocytes of breast carcinoma patients with antigen-coupled particles is significantly higher than that produced by leucocytes of normal donors. Following operation normally the number of TAA specific rosettes decrease, in 4 cases with recidives we found high numbers of TAA binding cells.

Binding of non-mammalian immunoglobulins to staphylococcal protein A.

Immunoglobulins M of the chicken (about 3% of serum IgM), clawed toad (12%) and carp (13%) bind to the protein A-Sepharose. A monomeric immunoglobulin with four domain heavy chains from the chicken serum is also bound (30% of bound immunoglobulins). The protein A-Sepharose can be utilized for isolation of IgM of the clawed toad and carp directly from the serum.