IgM-bearing B cell affinity subpopulations possess differential antigen sensitivity in rainbow trout.

The need for accurate assessments of in vitro generated antibody prompted examination of the effect of antigen on secreted antibody concentrations and affinities. It was found that the antigen concentrations commonly employed for in vitro stimulation were able to significantly compromise IgM titer and affinity estimates in rainbow trout. Specifically, IgM titers were underestimated with the high affinity antibodies being specifically blocked. To remedy this, pulsed antigen cultures were employed, and it was found to reveal more accurate IgM antibody titers and affinity estimates. Additionally, pulsed dose responses provided evidence that high antigen concentrations specifically suppressed high affinity B cell induction. Optimal expression of high affinity antibodies required exposure to lower concentrations of antigen. Each affinity subpopulation appeared to possess a graded sensitivity to each dose of antigen, revealing the complex dynamic for differential IgM-bearing B cell induction that is possible during a response. These results reveal not only the need for antigen removal prior to in vitro antibody secretion, but also the possible role of high zone immunological tolerance on IgM affinity maturation in rainbow trout.

Production of a monoclonal antibody against of muskellunge (Esox masquinongy) IgM heavy chain and its use in development of an indirect ELISA for titrating circulating antibodies against VHSV-IVB.

This study reports the development of a monoclonal antibody (designated 3B10) against the muskellunge (Esox masquinongy) IgM. The 3B10 monoclonal antibody (mAb) belongs to the IgG3 kappa isotype. Western blotting demonstrated that 3B10 mAb reacted primarily to muskellunge IgM heavy chain. 3B10 also reacted strongly with the IgM heavy chain of other esocids, including the northern pike (Esox lucius), tiger muskellunge (E. masquinongy x E. lucius), and, to a much lesser extent, the chain pickerel (E. niger). The 3B10 mAb did not bind to IgM from 10 other fish species resident in the Great Lakes basin. Using the 3B10 mAb, it was possible to determine the muskellunge Ig ability to bind to antigens. Using trinitrophenyl hapten conjugated to keyhole limpet hemocyanin (TNP-KLH) as the eliciting antigen, muskellunge Ig subclasses exhibited a range of affinities with log aK values 5.56-6.25 that is considered intermediate compared to other fish species. 3B10 mAb was used to develop and evaluate an indirect ELISA for the detection and quantitation of circulating antibodies against the viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb). Using the newly optimized assay, anti-VHSV-IVb antibodies were detected in sera of VHSV-IVb vaccinated muskellunge as well as from those of wild muskellunge sampled from an endemic waterbody. In addition to its use in immunoassays, the developed 3B10 mAb will enable future investigation aiming at deciphering immune mechanism of this important fish species to pathogens.

Evaluation of a time efficient immunization strategy for anti-PAH antibody development.

The development of monoclonal antibodies (mAb) with affinity to small molecules can be a time-consuming process. To evaluate shortening the time for mAb production, we examined mouse antisera at different time points post-immunization to measure titer and to evaluate the affinity to the immunogen PBA (pyrene butyric acid). Fusions were also conducted temporally to evaluate antibody production success at various time periods. We produced anti-PBA antibodies 7 weeks post-immunization and selected for anti-PAH reactivity during the hybridoma screening process. Moreover, there were no obvious sensitivity differences relative to antibodies screened from a more traditional 18-week schedule. Our results demonstrate a more time efficient immunization strategy for anti-PAH antibody development that may be applied to other small molecules.

Differential compartmentalization of memory B cells versus plasma cells in salmonid fish.

The disposition of teleost memory and plasma cells (PCs) has essentially been un-explored. As the organization of the teleost immune system differs significantly from that of mammals (i.e. no bone marrow or lymph nodes, hematopoietic anterior kidney), this disposition could be essential in understanding how comparable functions are achieved. To address this question, the primary and secondary antibody-secreting cell, B memory cell, and antibody responses to T-independent and T-dependent antigens were analyzed in trout. Although the TI and TD antibody responses did not differ substantively from one another, the secondary responses to both were significantly prolonged compared with the primary responses. Logarithmic increases in titer and affinity were achieved for both antigens during the primary, with only modest increases during the secondary response. Antibody-secreting cells, both PCs and plasmablasts, quantitatively paralleled antibody production, with antibody-secreting cells skewing to the hematopoietic anterior kidney for both antigens. However, the enhanced antigen-inducible response of immune fish (indicative of the memory pool) skewed to the peripheral blood and spleen. This pattern of memory versus PC disposition parallels that observed in mammals even though the organization of the respective immune systems differs considerably.

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.

The differential dynamics of antibody subpopulation expression during affinity maturation in a teleost.

A compositional analysis of the antibody response in rainbow trout was conducted using an affinity-based immunopartitioning assay. Trout were immunized with TNP-keyhole limpet hemocyanin (TNP-KLH) and individual serum titers and their affinity distributions analyzed over a period of 27 weeks. The kinetics of antibody affinity subpopulation development revealed certain key features: 1) the lowest affinity subpopulation (log aK, 3.5-3.99) appears early, does not achieve high titer, and was more transient than the higher affinity subpopulations; 2) intermediate affinity subpopulations (log aK, 5.0-5.99) appear later (week 5), achieve relatively high titers and persist longer; and 3) the highest affinity subpopulations (log aK, 6.0-7.49) emerge much later (post week 15), and have comparable titers to the intermediate affinity group. We find that the affinity maturation of the serum antibody response can be resolved into each affinity subpopulation's contribution both in quantity and timing.

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.

A highly sensitive monoclonal antibody based biosensor for quantifying 3-5 ring polycyclic aromatic hydrocarbons (PAHs) in aqueous environmental samples.

Immunoassays based on monoclonal antibodies (mAbs) are highly sensitive for the detection of polycyclic aromatic hydrocarbons (PAHs) and can be employed to determine concentrations in near real-time. A sensitive generic mAb against PAHs, named as 2G8, was developed by a three-step screening procedure. It exhibited nearly uniformly high sensitivity against 3-ring to 5-ring unsubstituted PAHs and their common environmental methylated PAHs, with IC50 values between 1.68-31 µg/L (ppb). 2G8 has been successfully applied on the KinExA Inline Biosensor system for quantifying 3-5 ring PAHs in aqueous environmental samples. PAHs were detected at a concentration as low as 0.2 µg/L. Furthermore, the analyses only required 10 min for each sample. To evaluate the accuracy of the 2G8-based biosensor, the total PAH concentrations in a series of environmental samples analyzed by biosensor and GC-MS were compared. In most cases, the results yielded a good correlation between methods. This indicates that generic antibody 2G8 based biosensor possesses significant promise for a low cost, rapid method for PAH determination in aqueous samples.

Monoclonal antibody analysis of Perkinsus marinus extracellular products.

The protozoan oyster parasite Perkinsus marinus releases a complex set of extracellular products (ECP) during in vitro culture. These products have been previously implicated in parasite virulence, and their expression can be altered by medium supplementation with oyster tissue homogenate. Little is known regarding ECP function, regulation, or mechanism of storage and release. Perkinsus marinus ECP were purified from a protein-free medium and used to produce a panel of five monoclonal antibodies. Several of the antibodies recognised series of proteins implying that the ECP may originate from comparatively few parental molecules. The ECP are secreted by several pathways, including the release of one product from an external cell layer, and two other products from two morphologically distinct intracellular compartments. Antibodies against separate epitopes on one protein provided information about possible protein structure. A sandwich ELISA format allowed sensitive quantification of that protein and showed significantly reduced protein expression in oyster tissue homogenate supplemented cultures. Immunopurification allowed tandem mass spectroscopic amino acid sequencing of that protein. Another antibody was used to characterise the P. marinus cell wall. This antibody specifically bound to trophozoite and tomont walls, and was used to investigate the morphological and antigenic changes in these walls during Ray's fluid thioglycollate medium-induced formation of hypnospores. It was also used to confirm that oyster tissue homogenate supplementation could induce formation of hypnospores. This antibody labeled P. marinus cells in fixed oyster tissue in a species-specific manner.

Comparative analysis of mycobacterial infections in wild striped bass Morone saxatilis from Chesapeake Bay.

During an ongoing epizootic of mycobacteriosis, wild striped bass Morone saxatilis from Chesapeake Bay were analyzed using 3 methods for detection of either mycobacterial infection or associated granulomatous pathology. The specific detection techniques, which utilized aseptically collected splenic tissue, were histology, quantitative culture and nested PCR. Based on analysis of 118 samples, detection of infection differed significantly between the 3 methods (chi-square, p = 0.0007). Quantitative culture and nested PCR detected similar, higher rates of infection (69 and 75%, respectively) than the histological method (52%). Although primary PCR assays for a 924 to 940 bp segment of the mycobacterial 16S rRNA gene were positive for genomic DNA from mycobacterial cultures, a secondary, nested PCR reaction for an internal 300 bp gene segment was required in order to detect mycobacteria within splenic tissue. A similar rate of mycobacterial infection was present in fish collected from all sites tested. Although all detection methods found that striped bass age 4.0 to 4.9 yr had the highest positive incidence, nested PCR detected a higher frequency of mycobacterial infection in fish > or = 6.0 yr of age than the other 2 methods. Quantitative bacteriology was a more sensitive detection technique when the fish tissue contained < or = 10(3) mycobacteria g(-1).

Potential novel epitopes in the extracellular products of oyster homogenate-supplemented Perkinsus marinus cells are not detected by subtractive immunization.

The extracellular products (ECPs) of the oyster parasite Perkinsus marinus have been posed to contain virulence factors, including serine proteases. Supplementation of P. marinus cultures with oyster homogenates enhances infectivity and changes the ECP composition. Therefore, subtractive immunization was used to attempt creation of antibodies to proteins unique to ECPs produced following parasite exposure to oyster homogenates. While control mice remained competent to respond to an unrelated antigen, no serum titers against novel ECP epitopes were detected in experimental mice. Attempts to create discriminatory hybridomas resulted in no clones with anti-ECP specificity. These findings suggest that, because no unique epitopes can be found within ECPs generated following exposure of P. marinus to host homogenates, the changes to ECPs are greatly constrained.

Reduction of rainbow trout spleen size by splenectomy does not alter resistance against bacterial cold water disease.

In lower vertebrates, the contribution of the spleen to anti-bacterial immunity is poorly understood. We have previously reported a phenotypic and genetic correlation between resistance to Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) and spleen somatic index (spleen weight normalized to body weight, SI). Fish families with larger pre-challenge SI values were found to have greater BCWD survival (resistance) following intraperitoneal injection of a lethal dose of F. psychrophilum. Since the mammalian spleen is known to be crucial for capture and destruction of encapsulated bacteria, we tested the hypothesis that reduction of spleen size, by surgical splenectomy, should reduce the survival advantage of the larger-spleen, disease-resistant fish. Experiments were performed using two separate lines of fish that had previously been selected either based on BCWD survival (resistant and susceptible), or selected based on spleen size (high and low SI). Following 65 to 81 days post-surgical recovery, fish were challenged with F. psychrophilum and mortality monitored for a minimum of 21 days. No significant difference in the relative survival was detected between splenectomized or sham-operated groups, while SI of splenectomized fish was reduced to an average of 8-12% of control animals. A positive correlation was observed between the SI, measured at the time of splenectomy, and time-to-death post-challenge. In summary, these experiments argue that larger spleen size alone is not sufficient for greater BCWD resistance, but rather it is an indirect indicator of immunological status.

The humoral response to in vitro generated parasite antigens is enhanced by the removal of a defined media component prior to immunization.

Concentrated culture supernatants containing the extracellular products (ECP) of the protozoan oyster parasite Perkinsus marinus were used to immunize mice. This preparation, produced by ultrafiltration, was found to be both poorly immunogenic and toxic to experimental animals. The possibility that these effects were due to toxic parasite products and/or medium constituents was examined. Co-administration of this material with highly immunogenic oyster hemolymph caused a substantive suppression of the specific antibody response to hemolymph, as well as a decrease in the number of epitopes recognized. Potential protein/protease toxin-mediated causes of the immunosuppression were addressed by heat denaturation and proteolytic inhibition of the concentrate; neither substantially enhanced immunogenicity. Analysis of media constituents revealed that the known immunomodulatory surfactant, Pluronic F-68 (PF68), used in the defined lipid concentrate supplement, was capable of eliciting significant immunosuppression. Although isolated protein antigens from P. marinus ECP remain highly immunosuppressive, separation of the protein antigens from the PF68 has enabled production of polyclonal antisera with a broader recognition of antigens.

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.

Affinity maturation in trout: clonal dominance of high affinity antibodies late in the immune response.

The ability of an animal to develop a highly specific antibody response through affinity maturation has been considered an integral part of the adaptive immune response. However, much of the literature dealing with teleostean antibody responses suggests that little or no affinity maturation may occur within these taxa. As trout antibodies are similar to multimeric mammalian IgM, it has been reasoned that affinity maturational shifts in intrinsic affinity might be similarly small. Such small increases in affinity can, however, lead to potentially great avidity changes for multimeric antibodies. We therefore employed a partition-based immunoassay that permits the dissection of a single antiserum into discrete, affinity-based antibody subpopulations. Such partitioning assays provide for enhanced sensitivity and resolution of these affinity subpopulations over that which can be obtained by fluorescence quenching or equilibrium dialysis. Through the use of the partition-based immunoassay, we were able to detect a consistent increase in affinity within trout anti-TNP antisera. Furthermore, it was determined that trout are capable of generating new, higher affinity antibodies relatively late in the antibody response, which then come into dominance. Such evidence suggests that either somatic mutation does occur or that a unique form of affinity-based regulation of antibody expression is employed.

Status and opportunities for genomics research with rainbow trout.

The rainbow trout (Oncorhynchus mykiss) is one of the most widely studied of model fish species. Extensive basic biological information has been collected for this species, which because of their large size relative to other model fish species are particularly suitable for studies requiring ample quantities of specific cells and tissue types. Rainbow trout have been widely utilized for research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. They are distinctive in having evolved from a relatively recent tetraploid event, resulting in a high incidence of duplicated genes. Natural populations are available and have been well characterized for chromosomal, protein, molecular and quantitative genetic variation. Their ease of culture, and experimental and aquacultural significance has led to the development of clonal lines and the widespread application of transgenic technology to this species. Numerous microsatellites have been isolated and two relatively detailed genetic maps have been developed. Extensive sequencing of expressed sequence tags has begun and four BAC libraries have been developed. The development and analysis of additional genomic sequence data will provide distinctive opportunities to address problems in areas such as evolution of the immune system and duplicate genes.

Near real-time, on-site, quantitative analysis of PAHs in the aqueous environment using an antibody-based biosensor.

Rapid, on-site, quantitative assessments of dissolved polycyclic aromatic hydrocarbons (PAHs) were demonstrated for two field applications. The platform, a KinExA Inline Sensor (Sapidyne Instruments), employed the monoclonal anti-PAH antibody, 7B2.3, which has specificity for 3- to 5-ring PAHs. A spatial study was conducted near a dredging site where contaminated sediments were being removed, and a temporal study was performed during a rainfall event. Most importantly, the generation of near real-time data guided management decisions in the field and determined proper sampling protocols for conventional analyses. The method was able to determine PAH concentrations as low as 0.3 µg/L, within 10 min of sample acquisition, and to assess 80+ samples (not including standards and blanks) in less than 3 d. These results were compared with a laboratory-based gas chromatography-mass spectrometry method in which a wide array of PAHs, including alkylated homologs, were examined. This system shows great promise as a field instrument for the rapid monitoring of PAH pollution.

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.

Two new Ig VH gene families in Oncorhynchus mykiss.

Genes encoding the immunoglobulin heavy-chain variable region (Ig VH) in rainbow trout (Oncorhynchus mykiss) have been grouped into 11 families. While obtaining a baseline assessment of the various gene families utilized by trout in the production of secreted antibody, we discovered two new families. These proposed Ig VH families, Families XII and XIII, were rarely observed; only two VH sequence types were detected for each new family, suggesting that they may not be commonly used in response to antigens, or that the captive environment may not lead to typical exposures seen in the wild. Additionally, unlike preceding studies, we found at least one representative gene sequence for each of the 11 reported Ig VH gene families, possibly indicating that the repertoire of trout Ig VH gene families may be more universal among different stocks than previously realized.