Loss of TET2 increases B-1 cell number and IgM production while limiting CDR3 diversity.

Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell differentiation in B-2 cells, yet the role of TET2 in regulating B-1 cells is largely unknown. Here, B-1 cell subset numbers, IgM production, and gene expression were analyzed in mice with global knockout of TET2 compared to wildtype (WT) controls. Results revealed that TET2-KO mice had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow (B-1a) and spleen (B-1b). Consistent with this finding, circulating IgM, but not IgG, was elevated in TET2-KO mice compared to WT. Analysis of bulk RNASeq of sort purified peritoneal B-1a and B-1b cells revealed reduced expression of heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, the expression of IgM transcripts was the most abundant isotype in B-1 cells. Yet, only in B-1a cells there was a significant increase in the proportion of IgM transcripts in TET2-KO mice compared to WT. Analysis of the CDR3 of the BCR revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. V-D-J usage and circos plot analysis of V-J combinations showed enhanced usage of VH11 and VH12 pairings. Taken together, our study is the first to demonstrate that global loss of TET2 increases B-1 cell number and IgM production and reduces CDR3 diversity, which could impact many biological processes and disease states that are regulated by IgM.

Specific features of a scaffolding antibody light chain.

The antigen-binding sites in conventional antibodies are formed by hypervariable complementarity-determining regions (CDRs) from both heavy chains (HCs) and light chains (LCs). A deviation from this paradigm is found in a subset of bovine antibodies that bind antigens via an ultra-long CDR. The HCs bearing ultra-long CDRs pair with a restricted set of highly conserved LCs that convey stability to the antibody. Despite the importance of these LCs, their specific features remained unknown. Here, we show that the conserved bovine LC found in antibodies with ultra-long CDRs exhibits a distinct combination of favorable physicochemical properties such as good secretion from mammalian cells, strong dimerization, high stability, and resistance to aggregation. These physicochemical traits of the LCs arise from a combination of the specific sequences in the germline CDRs and a lambda LC framework. In addition to understanding the molecular architecture of antibodies with ultra-long CDRs, our findings reveal fundamental insights into LC characteristics that can guide the design of antibodies with improved properties.

Biophysical characterization of human-cell-expressed, full-length κI O18/O8, AL-09, λ6a, and Wil immunoglobulin light chains.

Immunoglobulin light chain (AL) amyloidosis involves the deposition of insoluble monoclonal AL protein fibrils in the extracellular space of different organs leading to dysfunction and death. Development of methods to efficiently express and purify AL proteins with acceptable standards of homogeneity and structural integrity has become critical to understand the in vitro and in vivo aspects of AL protein aggregation, and thus the disease progression. In this study, we report the biophysical characterization of His-tagged and untagged versions of AL full-length (FL) κI and λ6 subgroup proteins and their mutants expressed from the Expi293F human cell line. We used an array of biophysical and biochemical methods to analyze the structure and stability of the monomers, oligomerization states, and thermodynamic characteristics of the purified FL proteins and how they compare with the bacterially expressed FL proteins. Our results demonstrate that the tagged and untagged versions of FL proteins have comparable stability to proteins expressed in bacterial cells but exhibit multiple unfolding transitions and reversibility. Non-reducing SDS-PAGE and analytical ultracentrifugation analysis showed presence of monomers and dimers, with an insignificant amount of higher-order oligomers, in the purified fraction of all proteins. Overall, the FL proteins were expressed with sufficient yields for biophysical studies and can replace bacterial expression systems.

Facile One-Step Generation of Camelid VHH and Avian scFv Libraries for Phage Display by Golden Gate Cloning.

Since its development in the 1980s, the Nobel Prize-awarded phage display technology has been one of the most commonly used in vitro selection technologies for the discovery of therapeutic and diagnostic antibodies. Besides the importance of selection strategy, one key component of the successful isolation of highly specific recombinant antibodies is the construction of high-quality phage display libraries. However, previous cloning protocols relied on a tedious multistep process with subsequent cloning steps for the introduction of first heavy and then light chain variable genetic antibody fragments (VH and VL). This resulted in reduced cloning efficiency, higher frequency of missing VH or VL sequences, as well as truncated antibody fragments. With the emergence of Golden Gate Cloning (GGC) for the generation of antibody libraries, the possibility of more facile library cloning has arisen. Here, we describe a streamlined one-step GGC strategy for the generation of camelid heavy chain only variable phage display libraries as well as the simultaneous introduction of heavy chain and light chain variable regions from the chicken into a scFv phage display vector.

Amyloidogenic light chains impair plasma cell survival.

Systemic light chain amyloidosis (AL) is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains (LC) as insoluble fibrils in organs. The lack of suitable models has hindered the investigation of the disease mechanisms. Our aim was to establish AL LC-producing plasma cell lines and use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LC from patients suffering from AL amyloidosis. The AL LC-producing cell lines showed a significant decrease in proliferation, cell cycle arrest, and an increase in apoptosis and autophagy as compared with the multiple myeloma LC-producing cells. According to the results of RNA sequencing the AL LC-producing lines showed higher mitochondrial oxidative stress, and decreased activity of the Myc and cholesterol pathways. The neoplastic behavior of plasma cells is altered by the constitutive expression of amyloidogenic LC causing intracellular toxicity. This observation may explain the disparity in the malignant behavior of the amyloid clone compared to the myeloma clone. These findings should enable future in vitro studies and help delineate the unique cellular pathways of AL, thus expediting the development of specific treatments for patients with this disorder.

Complete variable domain sequences of monoclonal antibody light chains identified from untargeted RNA sequencing data.

Introduction: Monoclonal antibody light chain proteins secreted by clonal plasma cells cause tissue damage due to amyloid deposition and other mechanisms. The unique protein sequence associated with each case contributes to the diversity of clinical features observed in patients. Extensive work has characterized many light chains associated with multiple myeloma, light chain amyloidosis and other disorders, which we have collected in the publicly accessible database, AL-Base. However, light chain sequence diversity makes it difficult to determine the contribution of specific amino acid changes to pathology. Sequences of light chains associated with multiple myeloma provide a useful comparison to study mechanisms of light chain aggregation, but relatively few monoclonal sequences have been determined. Therefore, we sought to identify complete light chain sequences from existing high throughput sequencing data. Methods: We developed a computational approach using the MiXCR suite of tools to extract complete rearranged IGVL-IGJL sequences from untargeted RNA sequencing data. This method was applied to whole-transcriptome RNA sequencing data from 766 newly diagnosed patients in the Multiple Myeloma Research Foundation CoMMpass study. Results: Monoclonal IGVL-IGJL sequences were defined as those where >50% of assigned IGK or IGL reads from each sample mapped to a unique sequence. Clonal light chain sequences were identified in 705/766 samples from the CoMMpass study. Of these, 685 sequences covered the complete IGVL-IGJL region. The identity of the assigned sequences is consistent with their associated clinical data and with partial sequences previously determined from the same cohort of samples. Sequences have been deposited in AL-Base. Discussion: Our method allows routine identification of clonal antibody sequences from RNA sequencing data collected for gene expression studies. The sequences identified represent, to our knowledge, the largest collection of multiple myeloma-associated light chains reported to date. This work substantially increases the number of monoclonal light chains known to be associated with non-amyloid plasma cell disorders and will facilitate studies of light chain pathology.

Germline-encoded amino acid-binding motifs drive immunodominant public antibody responses.

Despite the vast diversity of the antibody repertoire, infected individuals often mount antibody responses to precisely the same epitopes within antigens. The immunological mechanisms underpinning this phenomenon remain unknown. By mapping 376 immunodominant "public epitopes" at high resolution and characterizing several of their cognate antibodies, we concluded that germline-encoded sequences in antibodies drive recurrent recognition. Systematic analysis of antibody-antigen structures uncovered 18 human and 21 partially overlapping mouse germline-encoded amino acid-binding (GRAB) motifs within heavy and light V gene segments that in case studies proved critical for public epitope recognition. GRAB motifs represent a fundamental component of the immune system's architecture that promotes recognition of pathogens and leads to species-specific public antibody responses that can exert selective pressure on pathogens.

Comparison of IGLV2-14 light chain sequences of patients with AL amyloidosis or multiple myeloma.

Light chain amyloidosis (AL) is one of the most common forms of systemic amyloidosis and is caused by the deposition of insoluble fibrils derived from misfolded and aggregated immunoglobulin light chains (LC). To uncover the causes leading to this aggregation, we compared AL LC sequences with those of patients with the related disease multiple myeloma (MM), which do not aggregate in insoluble fibrils in vivo. IGLV2-14 is one of the most common AL-associated IGLV subfamilies. Here, we analysed IGLV2-14 LC sequences of 13 AL and eight MM patients in detail. We found that AL-associated LCs presented a lower median mutation count (7.0 vs. 11.5 in MM; P = 0.045), as well as an overall composition of less charged amino acids than MM LCs. However, we did not find a mutation that was present in ≥ 50% of the AL and not in the MM sequences. Furthermore, we did not find a significant difference in the isoelectric point (pI) in general, suggesting similar stability of the LCs in AL and MM. However, the subgroup of patients without a detectable heavy chain stood out. Surprisingly, they are characterized by an increase in mutation count (median 7.0 vs. 5.5) and pI (median 7.82 vs. 6.44, P = 0.043). In conclusion, our data suggest that the amount of mutations and the introduction of charges play a crucial role in AL fibril formation, as well as the absence or presence of a potential heavy chain binding partner.

Common framework mutations impact antibody interfacial dynamics and flexibility.

Introduction: With the flood of engineered antibodies, there is a heightened need to elucidate the structural features of antibodies that contribute to specificity, stability, and breadth. While antibody flexibility and interface angle have begun to be explored, design rules have yet to emerge, as their impact on the metrics above remains unclear. Furthermore, the purpose of framework mutations in mature antibodies is highly convoluted. Methods: To this end, a case study utilizing molecular dynamics simulations was undertaken to determine the impact framework mutations have on the VH-VL interface. We further sought to elucidate the governing mechanisms by which changes in the VH-VL interface angle impact structural elements of mature antibodies by looking at root mean squared deviations, root mean squared fluctuations, and solvent accessible surface area. Results and discussion: Overall, our results suggest framework mutations can significantly shift the distribution of VH-VL interface angles, which leads to local changes in antibody flexibility through local changes in the solvent accessible surface area. The data presented herein highlights the need to reject the dogma of static antibody crystal structures and exemplifies the dynamic nature of these proteins in solution. Findings from this work further demonstrate the importance of framework mutations on antibody structure and lay the foundation for establishing design principles to create antibodies with increased specificity, stability, and breadth.

Impact of cytogenetic abnormalities on treatment outcomes in patients with amyloid light-chain amyloidosis: subanalyses from the ANDROMEDA study.

BACKGROUND: Cytogenetic abnormalities are common in patients with amyloid light-chain (AL) amyloidosis; some are associated with poorer outcomes. This post hoc analysis of ANDROMEDA evaluated the impact of certain cytogenetic abnormalities on outcomes in this patient population. METHODS: Patients with newly diagnosed AL amyloidosis were randomised 1:1 to daratumumab, bortezomib, cyclophosphamide, and dexamethasone (D-VCd) or VCd. Outcomes were evaluated in the intent-to-treat (ITT) population and in patients with t(11;14), amp1q21, del13q14, and del17p13. RESULTS: Overall, 321 patients had cytogenetic testing (D-VCd, n = 155; VCd, n = 166); most common abnormalities were t(11;14) and amp1q21. At a median follow-up of 20.3 months, haematologic complete response rates were higher with D-VCd vs VCd across all cytogenetic subgroups and organ response rates were numerically higher with D-VCd vs VCd across most subgroups. Point estimates for hazard ratio of major organ deterioration-PFS and -EFS favoured D-VCd over VCd for all cytogenetic subgroups. Deep haematologic responses (involved minus uninvolved free light chains [FLC] <10 mg/L or involved FLC ≤20 mg/L) were seen in more patients with D-VCd than VCd in all ITT and t(11;14) cohorts. CONCLUSIONS: These results support the use of D-VCd as standard of care in patients with newly diagnosed AL amyloidosis regardless of cytogenetic abnormalities.

A constant domain mutation in a patient-derived antibody light chain reveals principles of AL amyloidosis.

Light chain (AL) amyloidosis is a debilitating disease in which mutant antibody light chains (LC), secreted by aberrant plasma cell clones, misfold and form insoluble fibrils, which can be deposited in various organs. In the majority of cases, the fibrillar deposits consist of LC variable domains (VL) containing destabilizing mutations compared to their germline counterparts. This is also true for the patient LC FOR005. However, this pathogenic LC sequence contains an additional mutation in the constant domain (CL). The mechanistic impact of CL mutations is not yet understood in the context of AL amyloidosis. Our analysis reveals that the FOR005 CL mutation influences the amyloid pathway in specific ways: (1) folding and stability of the patient CL domain are strongly impaired; (2) the mutation disrupts the LC dimer interface and weakens dimerization; (3) the CL mutation promotes proteolytic cleavage of the LC monomers resulting in an isolated, amyloidogenic VL domain while dimeric LCs are not cleaved. The enhanced proteolysis rates and the inability of full-length LCs to form amyloid fibrils even in the presence of a destabilized CL domain support a model for AL amyloidosis in which the CL domain plays a protective role and in which proteolytic cleavage precedes amyloid formation.

Antibodies gone bad - the molecular mechanism of light chain amyloidosis.

Light chain amyloidosis (AL) is a systemic disease in which abnormally proliferating plasma cells secrete large amounts of mutated antibody light chains (LCs) that eventually form fibrils. The fibrils are deposited in various organs, most often in the heart and kidney, and impair their function. The prognosis for patients diagnosed with AL is generally poor. The disease is set apart from other amyloidoses by the huge number of patient-specific mutations in the disease-causing and fibril-forming protein. The molecular mechanisms that drive the aggregation of mutated LCs into fibrils have been enigmatic, which hindered the development of efficient diagnostics and therapies. In this review, we summarize our current knowledge on AL amyloidosis and discuss open issues.

The patterns and diagnostic significance of the lack of surface immunoglobulin light chain on mature B cells in clinical samples for lymphoma workup.

BACKGROUND: Surface immunoglobulin (sIg) light chains are not always detected on mature B cells. This may present as a challenge for clonality determination in clinical flow cytometry. METHODS: To explore the mechanism and diagnostic significance of sIg negative mature B cells, we retrospectively studied 14 cases of sIg negative reactive B-cell lymphocytosis and 89 cases of sIg negative mature B-cell lymphomas. The expression patterns of sIg and cytoplasmic immunoglobulin (cIg) light chains were studied by flow cytometry using both monoclonal and polyclonal antibodies. RESULTS: These 14 cases of sIg negative reactive B-cell lymphocytosis were proven to be polytypic based on cytoplasmic light chain studies. In 89 cases of sIg negative mature B-cell lymphomas, we described four distinct patterns of abnormal light chain expression including partial or complete loss of sIg or cIg, suggesting different underlying mechanisms. CONCLUSIONS: This study represents the first reported series of body or cystic fluids where reactive B cells do not have detectable sIg, arguing strongly against making a diagnosis of B-cell lymphoma based on lack of sIg in mature B cells. Since the lack of sIg does not always predict clonal/neoplastic mature B-cell proliferation, further cIg evaluation should be performed when sIg expression is not detected in mature B cells. The lack of both sIg and cIg in mature B cells may serve as a reliable surrogate clonality/neoplastic marker.

Bioinformatic Analysis of Natively Paired VH:VL Antibody Repertoires for Antibody Discovery.

Next-generation DNA sequencing (NGS) of human antibody repertoires has been extensively implemented to discover novel antibody drugs, to analyze B-cell developmental features, and to investigate antibody responses to infectious diseases and vaccination. Because the antibody repertoire encoded by human B cells is highly diverse, NGS analyses of antibody genes have provided a new window into understanding antibody responses for basic immunology, biopharmaceutical drug discovery, and immunotherapy. However, many antibody discovery protocols analyze the heavy and light chains separately due to the short-read nature of most NGS technologies, whereas paired heavy and light chain data are required for complete antibody characterization. Here, we describe a computational workflow to process millions of paired antibody heavy and light chain DNA sequence reads using the Illumina MiSeq 2x300 NGS platform. In this workflow, we describe raw NGS read processing and initial quality filtering, the annotation and assembly of antibody clonotypes relating to paired heavy and light chain antibody lineages, and the generation of complete heavy+light consensus sequences for the downstream cloning and expression of human antibody proteins.

Vaccination induces HIV broadly neutralizing antibody precursors in humans.

Broadly neutralizing antibodies (bnAbs) can protect against HIV infection but have not been induced by human vaccination. A key barrier to bnAb induction is vaccine priming of rare bnAb-precursor B cells. In a randomized, double-blind, placebo-controlled phase 1 clinical trial, the HIV vaccine-priming candidate eOD-GT8 60mer adjuvanted with AS01B had a favorable safety profile and induced VRC01-class bnAb precursors in 97% of vaccine recipients with median frequencies reaching 0.1% among immunoglobulin G B cells in blood. bnAb precursors shared properties with bnAbs and gained somatic hypermutation and affinity with the boost. The results establish clinical proof of concept for germline-targeting vaccine priming, support development of boosting regimens to induce bnAbs, and encourage application of the germline-targeting strategy to other targets in HIV and other pathogens.

Functional antibodies exhibit light chain coherence.

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens1. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

Evolution of surrogate light chain in tetrapods and the relationship between lengths of CDR H3 and VpreB tails.

In the mammalian immune system, the surrogate light chain (SLC) shapes the antibody repertoire during B cell development by serving as a checkpoint for production of functional heavy chains (HC). Structural studies indicate that tail regions of VpreB contact and cover the third complementarity-determining region of the HC (CDR H3). However, some species, particularly bovines, have CDR H3 regions that may not be compatible with this HC-SLC interaction model. With immense structural and genetic diversity in antibody repertoires across species, we evaluated the genetic origins and sequence features of surrogate light chain components. We examined tetrapod genomes for evidence of conserved gene synteny to determine the evolutionary origin of VpreB1, VpreB2, and IGLL1, as well as VpreB3 and pre-T cell receptor alpha (PTCRA) genes. We found the genes for the SLC components (VpreB1, VpreB2, and IGLL1) only in eutherian mammals. However, genes for PTCRA occurred in all amniote groups and genes for VpreB3 occurred in all tetrapod groups, and these genes were highly conserved. Additionally, we found evidence of a new VpreB gene in non-mammalian tetrapods that is similar to the VpreB2 gene of eutherian mammals, suggesting VpreB2 may have appeared earlier in tetrapod evolution and may be a precursor to traditional VpreB2 genes in higher vertebrates. Among eutherian mammals, sequence conservation between VpreB1 and VpreB2 was low for all groups except rabbits and rodents, where VpreB2 was nearly identical to VpreB1 and did not share conserved synteny with VpreB2 of other species. VpreB2 of rabbits and rodents likely represents a duplicated variant of VpreB1 and is distinct from the VpreB2 of other mammals. Thus, rabbits and rodents have two variants of VpreB1 (VpreB1-1 and VpreB1-2) but no VpreB2. Sequence analysis of VpreB tail regions indicated differences in sequence content, charge, and length; where repertoire data was available, we observed a significant relationship between VpreB2 tail length and maximum DH length. We posit that SLC components co-evolved with immunoglobulin HC to accommodate the repertoire - particularly CDR H3 length and structure, and perhaps highly unusual HC (like ultralong HC of cattle) may bypass this developmental checkpoint altogether.

A clonality assay in canine B cell tumors targeting the immunoglobulin light chain lambda locus.

Clonality assays for antigen receptor rearrangement have been used as adjunct examinations of lymphoproliferative diseases. These assays have been useful for differentiation between inflammation and clonal expansion of lymphocytes. Whereas the immunoglobulin heavy chain (IGH) and immunoglobulin light chain kappa (IGK) loci have been targeted in canine clonality assays previously, the immunoglobulin light chain lambda gene (IGL) locus has not yet been investigated. This study aimed to evaluate the usefulness of clonality assays in dogs using IGL. Canine diffuse large B cell lymphomas (DLBCL), cutaneous plasmacytomas, and pathologically diagnosed lymph nodes without lymphoma, were used in this study. Genomic DNA was extracted from formalin-fixed paraffin embedded sections. Sequences of IGLV and IGLJ were obtained from the ImMunoGeneTics database. Several primers against IGLVs and IGLJs were designed in the regions showing homology, by alignment of the gene segments. Products of polymerase chain reaction were analyzed on a capillary electrophoresis. In total, 20 of 23 cases of DLBCL showed clonality (87.0 %), whereas 8 of 30 cutaneous plasmacytomas were clonal (26.7 %). One of 23 lymph nodes without lymphoma showed clonality, thus the specificity was 95.7 %. These data indicate that the IGL locus could be a target for canine clonality assays and that the sensitivity of IGL-based clonality assays in cutaneous plasmacytomas was lower than that in DLBCL.

Different classes of genomic inserts contribute to human antibody diversity.

Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum. So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 104 to 105 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in in vitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.

Identification of a rabbit Ig light chain recombinant protein bound to serum immunoglobulins from different marine fish species.

The structures of fish serum immunoglobulin differ among different fish species. In this study, we accidently isolated a rabbit immunoglobulin (Ig) light chain bound to serum immunoglobulin from different marine fish species using phage display. Fish Ig was separated using a protein A column. The phage library was generated from variable regions of rabbit spleen B cells immunized with bluefin tuna Thunnus orientalis Ig. Fish Ig-specific phages were enriched using two rounds of bio-panning with yellowtail Seriola quinqueradiata serum Ig, followed by two rounds of bio-panning with red seabream Pagrus major serum Ig. The enriched phages demonstrated an increase in binding specificity to the tuna, yellowtail, and red seabream Igs compared to the phages listed in the unpanned library. A recombinant protein of a single clonal phage, which encodes the rabbit Ig light chain, was produced, and the binding specificities to fish Igs were analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting. The recombinant protein exhibited binding properties to fish Igs in the ELISA. However, the recombinant protein that bound to serum protein(s), but not IgM, was detected via western blotting. The recombinant protein may provide a novel information on the common structural feature in the fish immunoglobulins.