Elsevier

Fish & Shellfish Immunology

Volume 64, May 2017, Pages 401-406
Fish & Shellfish Immunology

Full length article
Production, characterization and application of monoclonal antibody against immunoglobulin D heavy chain of flounder (Paralichthys olivaceus)

https://doi.org/10.1016/j.fsi.2017.03.043Get rights and content

Highlights

  • Monoclonal antibody 5G7 against the heavy chain of flounder IgD was produced.

  • MAb 5G7 could recognize the native IgD on the membrane of lymphocytes in flounder.

  • Only IgD+/IgM + lymphocytes subset was found in the PBLs of healthy flounder.

Abstract

Immunoglobulin D (IgD) is considered to be an enigmatic Ig molecule because of the lack understanding of its immunological functions. In the present study, a partial δ region of the flounder IgD was recombinantly expressed, purified and used as an immunogen to produce monoclonal antibodies (MAbs) against the H chain of flounder IgD. After fusion, a total of 97 hybridomas were generated and observed under an inverted microscope One of the hybridomas, designated 5G7, gave strong positive results in an indirect enzyme-linked immunosorbent assay (ELISA) and was cloned and subcloned by limiting dilution. Western blot analysis showed that MAb 5G7 could specifically recognize a 118 kDa protein from peripheral blood lymphocytes (PBLs), which was identified to be the H chain of flounder IgD by mass spectrometric analysis. Indirect immunofluorescence assay tests (IIFAT) showed that specific fluorescence signals were observed on the membranes of the PBLs, which suggests that MAb 5G7 could recognize the membrane-bound IgD molecule. Moreover, only the subset of IgD+/IgM + B cells were observed in the PBLs of healthy flounder when tested by flow cytometry analysis. Consistent with the results of flow cytometry, a double immunofluorescence assay test (DIFAT) showed that the positive lymphocytes were stained with both green and red fluorescence signals, which represent the IgM+/IgD + lymphocytes subset. These results demonstrate that the produced MAb 5G7 could specifically recognize the flounder IgD, which provides a useful tool to study the functions of flounder IgD.

Introduction

Immunoglobulins (Igs) are important molecules in the innate and adaptive immune systems of jawed vertebrates. Igs are useful in protecting against a wide range of pathogens [1], [2]. In mammals, five Ig isotypes were identified based on the constant domains of their H chains. In teleost fish, there are three major Ig isotypes: IgM, IgD and IgT, defined by the heavy chains μ, δ and τ, respectively. Among the three Ig types, IgM is the earliest identified Ig isotype, which presents as the main antibody in serum and is secreted into the mucus via the polymeric Ig receptor (pIgR) [3], [4]. IgZ/T was found to play an important role in mucosal immunity similar to IgA in mammals [5], [6]. In contrast, IgD has remained an enigmatic isotype since its discovery over 50 years ago. IgD is a primitive class of immunoglobulin present in most jawed vertebrates and conserved through evolution [7]. Initially, IgD was only detected as a membrane-bound form in teleosts, but a secreted form was subsequently discovered in catfish [8] and rainbow trout [9]. In recent decades, research on IgD in teleost fish has attracted much attention, and many IgD genes have been cloned and characterized, such as cod (Gadus morhua) [10], [11], Japanese flounder (Paralicthys olivaceus) [12], fugu (Fugu rubripes) [13], Atlantic salmon (Salmo salar) [14] and zebrafish (Danio rerio) [15]. Teleosts demonstrate remarkable plasticity in their δ genomic arrangement, with many species possessing repeated blocks of exons encoding Cδ2–4 as well as duplicated δ loci [16], [17], [18].

To date, information regarding the immunological properties and biological functions of IgD is very limited. In mammals, IgD was reported to function as an antigen receptor optimized for efficient recruitment of B cells in antigen-driven responses [19]. Moreover, IgD can largely increase in B cells, in which IgM function is suppressed, suggesting that IgD is largely able to substitute for IgM functions [20]. In addition, previous studies have demonstrated that the association of secretory IgD with basophils and mast cells results in the production of antimicrobial factors and the enhancement of respiratory immune resistance [8], [21]. Taken together, these facts demonstrate a crucial role for IgD in coordinating the immune surveillance of the host at the intersection of the innate and adaptive immune systems. Teleost IgD is thought to play similar roles to mammalian IgD in the immune system [22]. However, in teleost fish, the available information about IgD is mainly its gene structure and expression profile after stimulation [23], [24]. Flounder is an economically important fish popularly cultured in Asian countries, and accumulating studies on its immune system have been conducted in recent years. The cDNA encoding flounder IgD has been cloned and analysed previously [12], though no further research was performed at the protein level.

In the present study, the conserved δ1−δ4 region was recombinantly expressed and purified, which was then used as an immunogen to produce MAbs against the H chain of flounder IgD. Furthermore, the produced MAbs were used to investigate the distribution of IgD and IgM molecules by the double immunofluorescence assay test (DIFAT), and the percentage of IgD+ and IgM + lymphocyte subsets were determined by flow cytometric immunofluorescence analysis (FCIA).

Section snippets

Expression and purification of recombinant IgD

According to the gene sequence encoding the IgD heavy chain of flounder (GenBank No. AB052658), specific primers were designed to amplify the δ1−δ4 region of flounder IgD (F: 5′-CGGGATCCAAAAGTCGGGTTGTCTCTCC-3’; R: 5′-CCAAGCTTTGAGCAGAGGCTGATATTC-3′). The PCR product was purified and digested with specific restriction enzymes and subsequently ligated into the pET-28a vector to construct recombinant plasmids. The recombinant plasmid pET-28a-IgD was transformed into E. coli BL21 (DE3) cells. The

Expression and purification of rIgD

The δ1−δ4 region of flounder IgD was expressed in E. coli BL21 (DE3) cells with the pET-28a system. SDS-PAGE revealed that the δ1−4 region of IgD was successfully expressed after IPTG induction with a distinct band at approximately 45 kDa (Fig. 1, lane 2), which is in accordance with the predicted molecular mass of the δ1−δ4 region of flounder IgD. After purification with a Ni2+ affinity chromatography, high purity rIgD was obtained (Fig. 1, lane 3).

Production and selection of MAbs

A total of 97 hybridomas were observed under

Discussion

IgD is considered to be an enigmatic molecule because of its low level of expression, the difficulty of detecting its antibody activity and its diverse gene structures among species. In the past several decades, the production of monoclonal antibodies against marine and freshwater fish Igs has greatly advanced studies of the immune system of teleost fish. MAbs against fish Igs provide a powerful tool for investigating the immunological functions of different Igs and the immune response of

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31302216; 31672685; 31672684; 3142295), the Taishan Scholar Programme of Shandong Province, Science and Technology Development Project of Shandong Province (2014GNC111015) and the Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (No.2015ASKJ02).

References (31)

  • A.H. Al-harbi et al.

    Production and characterization of monoclonal antibodies against tilapia Oreochromis niloticus immunoglobulin

    Aquaculture

    (2000)
  • E.S. Edholm et al.

    Insights into the function of IgD

    Dev. Comp. Immunol.

    (2011)
  • Y.J. Liu et al.

    Normal human IgD+IgM- germinal center B cells can express up to 80 mutations in the variable region of their IgD transcripts

    Immunity

    (1996)
  • M.F. Flajnik

    Comparative analyses of immunoglobulin genes: surprises and portents

    Nat. Rev. Immunol.

    (2002)
  • M.R. Wilson et al.

    The immunoglobulin M heavy chain constant region gene of the channel catfish, ictalurus punctatus: an unusual mRNA splice pattern produces the membrane form of the molecule

    Nucleic Acids Res.

    (1990)
  • Cited by (5)

    View full text