Immunocytochemical analysis of a monoclonal antibody specific for rainbow trout (Oncorhynchus mykiss) granulocytes and thrombocytes.

A monoclonal antibody against rainbow trout peripheral blood leucocytes was selected for its lack of reactivity with rainbow trout immunoglobulin. Its reactivity with leucocytes from peripheral blood, head kidney and spleen was analysed by flow cytometry and electron microscopy, and compared with that of monoclonal antibodies directed against rainbow trout immunoglobulin, which reacted with B cells, B lymphoblasts and plasma cells. The antibody reacted with 5-20% of the peripheral blood leucocytes, 8-9% of head kidney leucocytes and 5-7% of spleen leucocytes. Electron microscopical immunocytochemistry revealed that the antibody reacted strongly with granulocytes and weakly with thrombocytes, and not with erythrocytes, lymphocytes, monocytes or macrophages. The antibody has possible applications in the identification and isolation of rainbow trout leucocytes, either alone or in combination with other monoclonal antibodies.

Complete coding sequence of rainbow trout Mhc II beta chain.

Several cDNA clones comprising the entire coding sequence of the rainbow trout (Oncorhynchus mykiss) major histocompatibility comlex (Mhc) class II B gene have been isolated from different sources. A single B gene appears to be transcribed in the rainbow trout and it encodes a 247 amino acid long polypeptide, which is of similar size to mammalian, avian, and amphibian and other teleost beta chains. The amino acid sequence identity to mammalian, amphibian, and avian class II beta chains is only about 30%. Despite the low similarity, a striking pattern of conservation is observed, both in the putative peptide-binding domain and in the Ig-like domain. Most of the conserved residues are located in the Ig-like domain and in the transmembrane segment. The majority of polymorphic residues reside in the beta 1 domain, with the greatest variability found in the amino-terminal half of the domain. The sequence data are compatible with a rather limited polymorphism of a single, expressed Mhc class II B gene.

Affinity purification of antigen-specific serum immunoglobulin from the European eel (Anguilla anguilla).

Immunization of specimens of the European eel with a hapten-carrier conjugate resulted in a significant rise in anti-hapten titres. Antigen-specific immunoglobulin was purified on a matrix onto which the hapten-carrier conjugate had been immobilized. Rabbit antisera raised against the product of the affinity purification recognized two molecular moieties. The first was eel immunoglobulin as inferred by the polypeptide composition, i.e. disulphide-linked heavy and light chains of 72 kDa and 25 kDa respectively. The second was a 110-kDa protein. A 800-kDa and a 400-kDa molecular form of eel immunoglobulin were disclosed by a combination of gel filtration and immunoelectrophoretic detection. The latter was the more abundant serum form. The 110-kDa protein was found non-covalently associated with the 400-kDa immunoglobulin. Both molecular forms of immunoglobulin from immune sera exhibited antigenic specificity and reactivity.

Characterization of a macaque recombinant monoclonal antibody that binds to a CD4-induced epitope and neutralizes simian immunodeficiency virus.

A potent neutralizing Fab fragment from a long-term survivor of simian immunodeficiency virus (SIVsm) infection was used to construct a recombinant macaque immunoglobulin G1kappa (IgG1kappa) molecule, designated IgG1-201. A Chinese hamster ovary cell line expressing IgG1-201 was derived by stable transfection and optimized for antibody secretion by methotrexate selection and dihydrofolate reductase gene amplification. IgG1-201 effectively neutralized the homologous, molecularly cloned SIVsmH4 virus but had no activity against the heterologous SIVmac251/BK28 virus. The previously characterized, neutralization-resistant SIVsmE543-3 virus was also not neutralized by IgG1-201. Binding to SIVsmH4 gp120 was enhanced in the presence of recombinant soluble CD4, suggesting that IgG1-201 bound a CD4-induced epitope. IgG1-201 immunoprecipitated the SIVsmH4 but not the SIVsmE543-3 envelope despite a close relationship between these two clones. Immunoprecipitation of a panel of SIVsmH4/SIVsmE543-3 chimeric viruses tentatively assigned the neutralization epitope to the third constant domain, immediately C terminal to the V3 loop. These findings suggest the presence of at least one CD4-induced neutralization epitope on SIV, as is the case with human immunodeficiency virus type 1.

MHC class II beta-chain expression in the rainbow trout.

A cDNA clone corresponding to the MHC class II beta-chain of the rainbow trout (Oncorhynchus mykiss) has been isolated and used in restriction fragment length polymorphism (RFLP) studies in a family of full siblings of rainbow trout. A very simple RFLP pattern was detected, suggesting segregation of a homozygote AA genotype and a heterozygote AB genotype. The MHC class II beta-chain of the rainbow trout seems to be transcribed in the same type of cells as class II genes of higher vertebrates even though the cDNA clone recognizes atypical messenger sizes of 2.2 kb and 3.6 kb in the analysed family. Surprisingly the transcripts seem to be allele-specific for the assigned genotypes.

Identification by phage display and characterization of two neutralizing chimpanzee monoclonal antibodies to the hepatitis E virus capsid protein.

Two monoclonal antibodies (MAbs) against the ORF2 protein of the SAR-55 strain of hepatitis E virus (HEV) were isolated by phage display from a cDNA library of chimpanzee (Pan troglodytes) gamma1/kappa antibody genes. Both MAbs, HEV#4 and HEV#31, bound to reduced, denatured open reading frame 2 (ORF2) protein in a Western blot, suggesting that they recognize linear epitopes. The affinities (equilibrium dissociation constants, K(d)) for the SAR-55 ORF2 protein were 1.7 nM for HEV#4 and 5.4 nM for HEV#31. The two MAbs also reacted in an enzyme-linked immunosorbent assay with recombinant ORF2 protein from a heterologous HEV, the Meng strain. Each MAb blocked the subsequent binding of the other MAb to homologous ORF2 protein in indirect competition assays, suggesting that they recognize the same or overlapping epitopes. Radioimmunoprecipitation assays suggested that at least part of the linear epitope(s) recognized by the two MAbs is located between amino acids 578 and 607. MAbs were mixed with homologous HEV in vitro and then inoculated into rhesus monkeys (Macaca mulatta) to determine their neutralizing ability. Whereas all control animals developed hepatitis (elevated liver enzyme levels in serum) and seroconverted to HEV, those receiving an inoculum incubated with either HEV#4 or HEV#31 were not infected. Therefore, each MAb neutralized the SAR-55 strain of HEV in vitro.

Simian immunodeficiency virus (SIV) envelope-specific Fabs with high-level homologous neutralizing activity: recovery from a long-term-nonprogressor SIV-infected macaque.

An antibody phage display library was constructed from RNA extracted from lymph node cells of a simian immunodeficiency virus (SIV)-infected long-term-nonprogressor macaque. Seven gp120-reactive Fabs were obtained by selection of the library against SIV monomeric gp120. Although each of the Fabs was unique in sequence, there were two distinct groups based on epitope recognition, neutralizing activity in vitro, and molecular analysis. Group 1 Fabs did not neutralize SIV and bound to a linear epitope in the V3 loop of the SIV envelope. In contrast, two of the group 2 Fabs neutralized homologous, neutralization-sensitive SIVsm isolates with high efficiency but failed to neutralize heterologous SIVmac isolates. Based on competition enzyme-linked immunosorbent assays with mouse monoclonal antibodies of known specificity, these Fabs reacted with a conformational epitope that includes domains V3 and V4 of the SIV envelope. These neutralizing and nonneutralizing Fabs provide valuable standardized and renewable reagents for studying the role of antibody in preventing or modifying SIV infection in vivo.