Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism.

The gene encoding IgH δ has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated δ-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory δ transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory δ transcript resulted in two δ-H chains, which incorporated Cµ1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory δ mRNA and IgD(+) plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.

Production and characterization of monoclonal antibodies to IgM of Pacific herring (Clupea pallasii).

Pacific herring (Clupea pallasii) have a central role in the North Pacific ecosystem as a forage fish species and are natural reservoirs of several important finfish pathogens, including Viral hemorrhagic septicemia virus (VHSV). Here, we report the identification of the gene encoding the immunoglobulin mu (IgM) heavy chain, as well as the development and characterization of monoclonal antibodies (MAbs) that specifically react with Pacific herring IgM. Pacific herring immunoglobulin was purified and consisted of heavy and light chains of approximately 80 and 25 kDa. Three hybridoma clones were initially identified by ELISA as reactive with purified immunoglobulin but only one clone was able to detect an 80 kDa protein in Pacific and Atlantic herring (Clupea harengus) whole plasma by denaturing western blot. However, all three MAbs were able to precipitate an 80 kDa protein from Pacific herring and LCMS sequencing of peptide fragments derived from this protein matched the predicted amino acid sequence of the cloned, heavy chain gene. In addition, two of the MAbs stained cells within the putative lymphocyte gates for the spleen, anterior kidney and posterior kidney but were not reactive for myeloid/granulocyte gates, which is consistent with these MAbs reacting with surface IgM⁺ B-cells. To our knowledge, this is the first report of IgM-related gene sequences and anti-IgM monoclonal antibodies from any member of the family Clupeidae. The antibodies produced in this study are critical for achieving our long-term goal of conducting serological surveillance to assess pathogen exposure in natural populations of Pacific herring.

The strength of B cell interaction with antigen determines the degree of IgM polymerization.

The induction of variable disulfide polymerization of IgM in the trout (Oncorhynchus mykiss) and its effect on its half-life were examined. An association between greater Ab affinity and increased disulfide polymerization was first indicated by the observation of this increased IgM disulfide polymerization during the process of affinity maturation. A direct association between Ab affinity and disulfide polymerization was then established by the fractionation of individual sera into high- and low-affinity subpopulations, which also resulted in the partitioning of high and low degrees of disulfide polymerization. The ability of high-affinity B cells to produce more highly polymerized Abs upon Ag induction was demonstrated by in vitro Ag-driven selection. Low Ag concentrations, which elicited only high-affinity Abs, also possessed the highest degree of polymerization, whereas higher concentrations of Ag elicited a broader array of Ab affinities, yielding a lower average affinity and degree of polymerization. Half-life studies revealed that the high-affinity, highly polymerized Abs possessed longer half-lives than the lower-affinity, lightly polymerized Abs. Finally, although the affinity for Ag is associated with elevated levels of polymerization, analysis of naive Ig revealed that the degree of polymerization alone, not affinity, appears sufficient to prolong Ig half-life.

Genomic architecture and repertoire of the rainbow trout immunoglobulin light chain genes.

The genomic loci encoding the four immunoglobulin light chains (IgL1, IgL2, IgL3, and IgL4) in the Swanson trout genome assembly were annotated in order to provide a measurement of the potential IgL repertoire. IgL1 and IgL3 gene segments are co-localized on chromosomes 21, 18, 15, and 7 while IgL2 and IgL4 were found on chromosomes 13 and 17, respectively. In total, 48 constant (CL), 87 variable (VL), and 59 joining (JL) productive genes are described. Pairwise alignment of the VL segments revealed that they belong to nine different families, three of which (kappa IV, V, and VI) are described for the first time in this study. VL and CL sequences on chromosome 15 and 21 and those on chromosomes 7 and 18 clustered together in phylogenetic analysis. PCR was used to examine IgL CL and VL genes in 9 lines of rainbow trout. IgL4 in the Hot Creek and Golden trout lines was missing 42 nucleotides resulting in a loss of 14 amino acids. The sigma IV variable family was completely absent from the Swanson, Arlee, Hot Creek, and wild type lines and silenced in the Skamania line with the addition of 176 bp mini-satellite insert. Similarly, the Whale Rock, Arlee, and wild type lines were all found to encode two sigma II products, a functional 252 bp product and a larger 425 bp product that contained a 172 bp insert. Results from this study indicate that there are genomic differences in IgL repertoire between different lines of trout that could affect humoral immune responses post vaccination and during disease.

Characterization of immunoglobulin light chain utilization and variable family diversity in rainbow trout.

This study characterizes immunoglobulin light chain (IgL) expression and variable family usage in rainbow trout. IgL transcripts were generated by 5' RACE from both immune and TNP-KLH immunized fish. Phylogenetic analysis revealed that the IgL variable regions clustered into seven different families: three kappa families (two newly described in this study), three sigma families, and a single lambda family. IgL1 and IgL3 transcripts expressing identical variable regions were identified and genomic analysis revealed that the two isotypes are co-localized on chromosomes 7, 15, 18, and 21 allowing for potential rearrangement between clusters. Fish were immunized with TNP-KLH (n = 5) and percent expression of IgL1, IgL2, IgL3, and IgL4 measured by qRT-PCR from immune tissues and magnetically sorted TNP-specific lymphocyte populations. In all samples IgL1 constituted 80-95% of the transcripts. The percentage of anti-TNP specific IgL1 transcripts was measured in naïve, unsorted, and TNP-specific cell populations of TNP-KLH fish (n = 3) and found to be significantly higher in the TNP positive cell population (21%) compared to the naïve population (1%; p = 0.02) suggesting that there is a selection of TNP specific IgL sequences.

The development and evaluation of monoclonal antibodies for the detection of polycyclic aromatic hydrocarbons.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of 3- to 5-ring polycyclic aromatic hydrocarbons (PAHs) has been developed. A functional derivative of dibenzothiophene was synthesized and covalently linked to carrier proteins that were used to produce monoclonal antibodies (mAbs). During the conjugation step, the conjugation efficiency was improved by the presence of 25% N,N-dimethylformamide (DMF). Antibodies were selected based on a competitive inhibition assay to isolate those with the highest sensitivity for free PAHs. When using the mAb in an ELISA format, free PAHs were detected at a concentration as low as 0.1 microg/L (0.1 ppb) in aqueous samples.

Antibody structural variation in rainbow trout fluids.

Rainbow trout (Oncorhynchus mykiss) were immunized with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) and the redox structure of induced anti-TNP antibodies from the serum, mucus, egg and ovarian fluid was examined. In conducting these studies it was determined that all TNP-specific antibody from each source possessed the mAb-specific H chain (1-14) epitopes, which facilitated the direct structural analysis of the induced antibodies. A protocol was developed which ensured complete adsorption of all specific anti-TNP antibody from each fluid. Together these protocols permitted the unbiased compositional analysis of all redox forms of the anti-TNP antibodies from each source. All antibodies, regardless of source, possessed the same molecular mass, characteristic of the trout tetramer (800 kDa). It was found that specific antibody titers were significantly higher in male than female trout, while the degree of disulfide polymerization was relatively invariant in male antibodies, while being highly variable in female antibodies. Within the females, no distinctively different redox ratios were between antibodies isolated from sera, ovarian fluid or eggs: however, mucus antibodies possessed a unique redox structure consisting of halfmeric constituents that were not observed in antibodies from other fluids.

Use of staphylococcal protein A in the analysis of teleost immunoglobulin structural diversity.

Staphylcoccal protein A (SpA) adsorption and sephacryl-S300 filtration were employed to isolate Ig from the sera of six aquaculturally important teleost species; Morone saxatilis (striped bass), Lates calcarifer (barramundi), Oreochromis mossambicus (Mozambique tilapia) and Oreochromis niloticus (Nile tilapia), Salmo salar (Atlantic salmon), and Oncorhynchus mykiss (rainbow trout). While both gel filtration (S300) and SpA adsorption could purify the 800 kDa tetrameric Ig, SpA demonstrated species-specific variability in the amount retrieved. Virtually 100% of this high molecular weight Ig could be isolated from Mosambique tilapia serum, while 84, 17, 10.7 and 0.5% could be isolated from barramundi, striped bass, Nile tilapia, and Atlantic salmon, respectively. Significant amounts of Ig could not be isolated (<0.1%) from rainbow trout (O. mykiss) serum. All SpA-isolated proteins were approximately 800 kDa in molecular weight and were solely composed of equimolar concentrations of H ( approximately 75 kDa) and L ( approximately 25 kDa) chains. Denaturing, non-reducing electrophoresis demonstrated differential disulfide polymerization (redox forms) consistent with those observed with other teleost species; however, SpA exhibited less affinity for Ig possessing completely polymerized tetramers than the more reduced forms, with the exception of Mossambique tilapia. The existence of three different molecular weight H chains (75, 85, 95 kDa) in Nile tilapia was also observed. Each redox form of Nile tilapia Ig incorporated only one size of H chain.

Validation of an antibody-based biosensor for rapid quantification of 2,4,6-trinitrotoluene (TNT) contamination in ground water and river water.

Nitroaromatics are common pollutants of soil and groundwater at military installations because of their manufacture, storage, and use at these sites. Long-term monitoring of these pollutants comprise a significant percentage of restoration costs. Further, remediation activities often have to be delayed, while the samples are processed via traditional chemical assessment protocols. Here we describe a rapid (<5 min), cost-effective, accurate method using a KinExA Inline Biosensor for monitoring of 2,4,6-trinitrotoluene (TNT) in field water samples. The biosensor, which is based on KinExA technology, accurately estimated the concentration of TNT in double-blind comparisons with similar accuracy to traditional high-performance liquid chromatography(HPLC). In the assessment of field samples, the biosensor accurately predicted the concentration of TNT over the range of 1-30,000 microg/L when compared to either HPLC or quantitative gas chromatography-mass spectrometry (GC-MS). Various pre-assessment techniques were explored to examine whether field samples could be assessed untreated, without the removal of particulates or the use of solvents. In most cases, the KinExA Inline Biosensor gave a uniform assessment of TNT concentration independent of pretreatment method. This indicates that this sensor possesses significant promise for rapid, on-site assessment of TNT pollution in environmental water samples.

Plasmablast and plasma cell production and distribution in trout immune tissues.

These studies describe the in vitro and ex vivo generation of plasmablasts and plasma cells in trout (Oncorhynchus mykiss) peripheral blood and splenic and anterior kidney tissues. Cells were derived either from naive trout and cultured with the polyclonal activator, Escherichia coli LPS, or from trout that had been immunized with trinitrophenyl-keyhole limpet hemocyanin. Hydroxyurea was used to resolve populations of replicating (plasmablast) and nonreplicating (plasma cell) Ab-secreting cells (ASC). Complete inhibition of Ig secretion was only observed within the PBL. Both anterior kidney and splenic lymphocytes possessed a subset of ASCs that were hydroxyurea resistant. Thus, in vitro production of plasma cells appears to be restricted to the latter two tissues, whereas peripheral blood is exclusively restricted to the production of plasmablasts. After immunization with trinitrophenyl-keyhole limpet hemocyanin, specific ASC could be isolated from all immune organs; however, the anterior kidney contained 98% of all ASC. Late in the response (>10 wk), anterior kidney ASC secreted specific Ab for at least 15 days in culture, indicating that they were long-lived plasma cells. Cells from spleen and peripheral blood lost all capacity to secrete specific Ab in the absence of Ag. Late in the Ab response, high serum titer levels are solely the result of Ig secretion from anterior kidney plasma cells.