Ig Molecules Research Articles

Expression of protectin (CD59) in human melanoma and its functional role in cell‐ and complement‐mediated cytotoxicity

Immunohistochemical and/or indirect immunofluorescence analysis with monoclonal antibody (MAb) H19 demonstrated the expression of protectin (CD59) in 54 surgically removed metastatic melanoma lesions and on 8 out of 12 melanoma cell lines. CD59 expression had a low degree of intra- and intertumor heterogeneity. SDS-PAGE analysis showed that the molecular weight of CD59 expressed on melanoma cells is about 20 kDa. Treatment of melanoma cells with 5U/ml of phosphatidylinositol-specific phospholipase C completely abolished cell-surface expression of CD59. Interferon-gamma and/or tumor necrosis factor-alpha or phorbol 12-myristate 13-acetate neither modulated the expression of CD59 by melanoma cells nor influenced the amounts of CD59-specific mRNA. F(ab')2 fragments of anti-CD59 MAb YTH53. I did not inhibit the lysis of melanoma cells by allogeneic natural killer (NK) cells or lymphokine-activated killer (LAK) cells. In contrast, the whole Ig molecule of MAb HI9 or YTH53.I significantly (p < 0.05) enhanced NK-cell-mediated lysis of melanoma cells, suggesting the induction of antibody-dependent cell-mediated cytotoxicity. Lastly, masking of CD59 by MAb YTH53.I or its F(ab')2 fragments significantly (p < 0.05) enhanced, in a dose-dependent fashion, the lysis of anti-GD3-sensitized melanoma cells by homologous complement. These data demonstrate that CD59 expressed by human melanoma cells might regulate host-tumor interaction by protecting neoplastic cells from complement-mediated lysis.

Sequence analysis of the human alpha beta T-cell receptor CDR3 region.

Although the three-dimensional structure of the T-cell receptor (TCR) has not yet been determined, several groups have proposed that the outline structure of the TCR will closely resemble that of immunoglobulin (Ig). Hypervariable regions can be identified within the TCR variable (V) domains, and by analogy to similar regions in the Ig molecule which together form the antigen combining site these have been termed the complementarity determining regions (CDR) 1, 2, and 3. By far the greatest extent of variability occurs at CDR3 and this has led to the proposal that CDR3 is involved in interaction with the peptide bound within the cleft of a major histocompatibility complex (MHC) molecule. We have cloned and sequenced the CDR3 region of several hundred human TCRA and TCRB transcripts from different T-cell populations and studied the amino acid usage in this region. Results show that the average length of the CDR3 region is 10 amino acids with less variation in length than is seen for the Ig heavy chain. There is no difference in CDR3 length between fetal and adult T cells or between CD4 and CD8 populations. The pattern of amino acid usage in the CDR3 region is dissimilar between TCRA and TCRB transcripts. In particular there is a predominance of charged and polar residues in the region of the TCRA transcript thought to interact with peptide. These data provide information on the general pattern of CDR3 length and composition for both TCRA and TCRB.

Inhibition of complement-mediated red cell lysis by immunoglobulins is dependent on the IG isotype and its C1 binding properties.

We have investigated the effect on complement activation of human immunoglobulins (Ig) using several therapeutic Ig preparations including two for intravenous use (IVIG), and various purified myeloma proteins. Ig inhibited lysis in a dose-dependent manner in the classical pathway assay whereas no alternative pathway inhibition was observed. The Fc part of the molecule was responsible for all the inhibitory effect. Purified IgG3 myeloma proteins were potent inhibitors whereas IgG1 inhibited to a lesser extent and IgG2 and IgG4 did not inhibit at all. Inhibition was obtained both when Ig was added to the solution and when it was coated onto a solid matrix. Analysis of the soluble and solid phase Ig after incubation revealed binding of C1q and activated C4 and C3 to the isotypes which inhibited lysis. Using selectively depleted sera and reconstitution with their respective purified components, efficient inhibition of lysis was seen when Ig was added prior to serum (C1), some inhibition was seen at the C4 level, whereas no effect was seen when Ig was added at the C9 level. We conclude that the complement-modulatory effect of Ig in vitro is isotype specific and dependent mainly on competitive C1 binding by the Ig molecule in the absence of antigen.

Production, characterisation and applicability of monoclonal antibodies to European eel ( Anguilla anguilla L., 1758) immunoglobulin

Monoclonal antibodies (mAbs) to European eel ( Anguilla anguilla L., 1758) immunoglobulin (Ig) were produced, characterised and tested for applicability in a number of immuno(cyto)chemical assays. The selected mAbs, WEI 1 and WEI 2, were specifically reactive with Ig heavy and light chain, respectively. WEI 1 appeared to react with all or nearly all Ig molecules, B cells and plasma cells. WEI 2 was reactive with a subpopulation of those cells, indicating that European eel possesses at least two antigenically different light chain types. Both mAbs could be used for detection of antigen-specific antibodies in plasma by means of an enzyme-linked immunosorbent assay (ELISA).

Three new immunoglobulin kappa variable (Igk-V) gene segments in the mouse.

In the mouse, the immunoglobulin (Ig) kappa variable (Igk-V) gene segments make a large contribution to the antibody repertoire, because the light chain in over 90% of serum Ig molecules is of the kappa type. The number of Igk-V gene segments has been the subject of some controversy. Here we report three new Igk-V gene segments found among rearranged kappa genes from day 14 - 16 fetal liver of the C57BL/6J mouse. As the segments are potentially productive, the new Igk-V segments are presumable used. 8 refs., 1 fig.

Recombinant antibodies containing an engineered B-cell epitope capable of eliciting conformation-specific antibody responses

The immunogenicity of a soluble, non-self protein or peptide can be greatly enhanced by injecting this antigen coupled to an antibody specific for class II MHC molecules in the recipient. This adjuvant-independent immunization strategy is known as immunotargeting. We have investigated the ability of a mouse anti-class II MHC antibody to provide the three-dimensional framework for the reconstitution of a heterologous conformational B-cell epitope, specifically the A loop epitope from the influenza virus hemagglutinin (HA). From a panel of three anti-class II MHC immunoglobulin (Ig)-A loop constructs, we found that one of these, an insertion into the FR3 region of the Ig molecule, retained its specificity for mouse I-A k. Although mouse monoclonal antibodies specific for the A loop region in the HA molecule were unable to react with the Ig-A loop variants, we did find that the heavy chain CDR3 insertion construct was able to elicit an A loop-specific, HA-reactive antibody response when used as an immunogen in rabbits. These results demonstrate the potential for the Ig molecule to function successfully as a structural framework for the reconstitution and presentation of heterologous conformational B-cell epitopes.

B cell and immunoglobulin heterogeneity in carp ( Cyprinus carpio L); An immuno(cyto)chemical study

B cell and immunoglobulin (Ig) heterogeneity was demonstrated in carp, Cyprinus carpio L., using two monoclonal antibodies (MAbs; WC14, WCI12) produced against carp serum Ig. Immunochemical results showed that both WCI4 and WCI12 react with a protein determinant on the heavy chain of Ig (relative molecular mass ~70,000). Immunofluorescence microscopic and flow cytometric analyses of lymphoid cells suggest three distinct subpopulations of B cells and plasma cells: WCI4 +12 − cells, WCI4 −12 + cells, and WCI4 +12 + cells. WCI4 −12 + and WCI4 +12 + anti-DNP antibody-secreting cells were also demonstrated with the ELISPOT assay in pronephros and spleen cell suspensions from primary immunised carp. Affinity chromatography of carp serum and sequential immunoprecipitation of 125I-labelled peripheral blood leucocyte (PBL) membrane proteins only indicated the presence of two antigenically different Ig molecules i.e., WCI4 −12 + and WCI4 +2 + molecules. WCI4 +12- molecules could not be detected by affinity chromatography or immunoprecipitation. During ontogeny, a shift in percentages of WCI4 +12 − and WCI4 −12 + cells was found in the spleen and the pronephros. In these organs, WCI4 +12 − cells formed the majority of B cells at 2 weeks of age, but the percentages of this cell type decreased during ontogeny. On the other hand, the percentages of WCI4 −12 + cells increased during development, and these cells became the major population of B cells from 13 weeks Address correspondence to Dr. J.H.W.M. Rombout. onward. The proportion of WCI4 +12 + cells remained almost constant during ontogeny. The distribution of B cell subpopulations in blood was more or less stable at all ages. The functional significance of Ig heterogeneity in fish and in particular carp is discussed.

Interplay between the human TCR/CD3ϵ and the B-cell antigen receptor associated Ig-β (B29)

T and B lymphocytes are characterized by the surface expression of highly variable antigen receptors called the T-cell (TCR) and B-cell (BCR) receptor. In both B and T cells, binding of antigen to their respective surface receptors results in transmembrane signaling which leads either to programmed cell death or to proliferatin and differentiation. The human and murine TCR consist of the highly variable TCR α and β or γ and δ chains recognizing antigen and the non-covalently associated invariable CD3 complex (γ, δ, ϵ) and ζ. The antigen-recognizing surface membrane-bound immunoglobulin (Ig) molecules on the surface of human and murine B cells are non-covalently associated with a heterodimeric protein complex of Ig-α (MB-1) and Ig-β (B29). Like the CD3 complex associated with TCR the Ig-associated proteins are predicted to regulate the assembly and transport of the Ig complex to the cell surface and to couple membrane-bound Ig to intracellular signal transduction pathways. To gain more insight into structure/function relationships between CD3 proteins and Ig-α and Ig-β, we transiently co-transfected pairs of expression vectors encoding either TCR/CD3 chains on the one hand and Ig-α or Ig-β on the other into Cos cells. Thus we found a very strong interaction between CD3ϵ and Ig-β mediated by the extracellular domains. Experiments in which we could stain Jurkat T cells with soluble Ig-β but not Ig-α protein indicated the recognition of CD3ϵ by Ig-β even in the context of the whole TCR/CD3 complex. The staining of Jurkat cells by soluble Ig-β protein could specifically be inhibited by blocking the extracellular domains of either CD3ϵ or Ig-β with antibodies. These data hint at a possible function for CD3ϵ and Ig-β in constituting a novel pair of surface molecules involved in T-/B-cell interactions.

Production of stable bovine-murine interspecies hybrids.

AbstractAn understanding of the processes involved in immunity to infection requires a clear knowledge of the different immunoglobulin (Ig) classes and subclasses, and their interaction with cells of the myeloid and lymphocytic lineages. Use has been made of Ig-secreting myelomas and plasmacytomas that occur in mice, humans, and a few other species (1) to provide a source of Ig. Although it is possible that such myelomas may exist in cattle (2), none has been described to date. Fortunately, it has been possible to induce fusion between bovine B-cells and nonsecreting murine myeloma cell lines, resulting in the generation of stable, monoclonal cell lines that secrete functional bovine Ig molecules. Because each B-lymphocyte is committed to producing Ig of a single isotype and specificity, this approach leads to the production of pure and homogeneous Ig for detailed study (3) and provides a powerful tool for the precise functional analysis of specific Ig molecules.KeywordsFusion PartnerRepeat CloningCluster PlateUnwanted Immune ReactionMonoclonal Cell LineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The differential effects of dithiothreitol and 2-mercaptoethanol on the secretion of partially and completely assembled immunoglobulins suggest that thiol-mediated retention does not take place in or beyond the Golgi.

Dithiothreitol (DTT) blocks the endoplasmic reticulum (ER)-Golgi transport of newly synthesized immunoglobulin (Ig) molecules, whereas 2-mercaptoethanol (2ME) allows secretion of unpolymerized Igs otherwise retained intracellularly by disulphide interchange reactions. To understand this dichotomy, we have compared the effects of DTT and 2ME on the assembly, intracellular transport, and secretion of a panel of chimeric Igs that are either constitutively secreted or retained intracellularly. Our results demonstrate that DTT, but not 2ME, reduces some of the inter- and intrachain disulphide bonds and causes partial disassembly of H2L2 complexes and unfolding of individual chains in the ER. Upon DTT removal, heavy (H) and light (L) chains reform hapten-binding H2L2 molecules, which are later secreted. Reduction of the H2L2 interchain disulphide bonds can occur along the entire secretory pathway; however, in or beyond the Golgi this does not result in efficient H-L disassembly or unfolding. As a consequence, DTT does not block the exit from the Golgi. Moreover, unpolymerized Igs--normally retained in a pre-Golgi compartment--no longer require reducing agents to be secreted once they have reached the Golgi. Thus, little if any thiol-mediated retention seems to take place in or beyond the Golgi complex.

Structural and functional evolution of the extracellular regions of T cell receptors

The primary structure of T cell receptor (TcR) heterodimers has been elucidated to varying degrees in 11 vertebrate species and the availability of these sequences provides a broader perspective for reassessing TcR evolution. Many features of the αβ TcR have been well conserved between divergent taxa whereas the structure and diversity of the γδ TcR is more variable. An alternative evolutionary scheme is presented whereby the γδ TcR and Ig molecules represent the earliest separation between specific cell mediated and humoral immunity with the αβ TcR emerging later. It is proposed that a set of ligands expressed on epithelia mediated the earliest selection of γδ T cells and B cells and was used as an antigen presentation substrate by γδ T cells. A subset of the earliest selection ligands became expert at binding small peptide fragments and the emergent 'classical' MHC molecules then co-evolved with the αβ TcR as obligate ligand-receptor pairs. Progression between the major stages in this sequence followed from adaptations in the selecting and presenting ligands and can be characterized as MHC 'capture'.

Idiotype-cytokine fusion proteins as cancer vaccines. Relative efficacy of IL-2, IL-4, and granulocyte-macrophage colony-stimulating factor.

Idiotypic determinants, antigenic sites expressed on the variable region of Ig molecules of malignant B cells, represent tumor-specific Ags but are weak immunogens. We have previously shown that the immunogenicity can be dramatically increased by fusing tumor Id to granulocyte macrophage (GM)-CSF. Here, we demonstrate that fusion proteins with IL-2 or IL-4 can also be highly immunogenic. Co-immunization of these fusion proteins with another Id demonstrated the importance of physical linkage between the cytokine and relevant Ag for this enhancement. All three fusion proteins are capable of eliciting significant levels of specific Abs against the Id without the use of carrier proteins or adjuvants, although the GM-CSF fusion protein appeared to be unique in its ability to induce higher titers of anti-Id Abs in the primary response. Furthermore, the Id-IL-2 fusion protein induced high titers of IgG2a and IgG3 anti-Id Abs, whereas the Id-IL-4 and Id-GM-CSF fusion proteins did not. Despite the differences, tumor protection was comparable in all mice having significant titers of anti-Id Abs, regardless of the fusion protein used. We concluded that Id-cytokine fusion proteins are potent immunogens that can elicit significant antitumor immunity. The general approach of fusing a cytokine to a potential Ag may be applicable to the design of vaccines for immunotherapy of other types of tumors as well as for other pathogens and disease states.

A mutation of the mu transmembrane that disrupts endoplasmic reticulum retention. Effects on association with accessory proteins and signal transduction.

The mu heavy chain has an unusually high content of hydroxyl-containing amino acids in its membrane-spanning region. We have examined the involvement of two of these hydrophilic residues in endoplasmic reticulum (ER) retention, interactions with Ig-alpha/Ig-beta, and transmembrane signaling. Neighboring tyrosine and serine residues were mutated to either phenylalanine and alanine (mutant YS/FA) or valine and valine (mutant YS/VV). Membrane Ig (mIgM) molecules containing these mutant mu chains were expressed on the surface of transfected B lymphoma cells. Anti-Ig-induced signaling by the YS/FA mutant mIgM was equivalent to wild-type (wt) mIgM, whereas signaling by the YS/VV mutant mIgM was notably diminished. Association between mutant YS/VV mIgM and Ig-alpha/Ig-beta was detectable but reduced in comparison to YS/FA or wt mIgM. Signaling by YS/VV mutant mIgM appeared to involve Ig-alpha/Ig-beta, because these proteins were tyrosine phosphorylated on receptor cross-linking. When YS/VV and wt mu chains were cotransfected with light chains into nonlymphoid cells, mutant mIgM was expressed at the cell surface in the absence of Ig-alpha/Ig-beta, whereas wt mIgM was not. These data suggest that the mutated residues contribute to ER retention and directly or indirectly to association with Ig-alpha/Ig-beta. Moreover, ER retention can be disrupted without preventing functional association with Ig-alpha/Ig-beta. In addition, these data indicate that the hydroxyl groups of the mutated residues are not required for functional association between mu and Ig-alpha/Ig-beta because their removal did not reduce the ability of the YS/FA mutant mIgM to associate with accessory proteins or to participate in signal transduction.

B cell antigen receptors

Antigen receptors have a dual role on the surface of B lymphocytes, namely the binding and internalization of antigens and the activation of the antigen-recognizing B cell. The structural requirements for internalization are still a matter of controversy; but as far as signalling is concerned it is becoming increasingly apparent that the antigen receptor is functionally divided into the ligand-binding immunoglobulin (Ig) molecule and the signal -transducing Ig-α/Ig-β heterodimer. This knowledge has been exploited to design variant antigen receptors which combine both functions in a single molecule.

Immature surface Ig+ B cells can continue to rearrange kappa and lambda L chain gene loci.

Pro and pre B cells possess the long-term capacity to proliferate in vitro on stromal cells and interleukin 7 (IL-7) and can differentiate to surface immunoglobulin (sIg+) cells upon removal of IL-7 from the cultures. A key event in this differentiation is the extensive cell loss due to apoptosis. Because the proto-oncogene bcl-2 can promote cell survival, we established pre-B cell lines from E mu-bcl-2 transgenic mice. These pre-B cells have the same properties as those derived from non-bcl-2 transgenic mice except that they do not die by apoptosis. This allowed us to study the fate of newly formed B cells in vitro for a longer period of time. Here we show that early during the differentiation of pre-B cells, upregulation of RAG-1 and RAG-2 expression go hand in hand with rearrangements of the Ig gene loci. Moreover, the newly formed sIg+ B cells continue to express RAG-1 and RAG-2 and continue to rearrange L chain gene loci, even in the absence of proliferation, in an orderly fashion, so that kappa L+ sIg+ cells can become lambda L+ sIg+ or sIg- cells, whereas lambda L+ sIg+ cells can become sIg-, but not kappa L+ sIg+ cells. Thus, deposition of a complete Ig molecule on the surface of a B cell does not automatically stop the Ig-rearrangement machinery.

Polymorphism of the functional immunoglobulin variable region genes in the chicken by exchange of sequence with donor pseudogenes.

We have isolated a number of new allelic variants of the unique functional genes encoding chicken immunoglobulin heavy and light chain variable regions (VH1 and VL1, respectively). The distribution and nature of nucleotide variation among these and previously identified VH1 and VL1 alleles demonstrates that random point mutations are likely not the predominant cause of allelic variation at these loci. Comparison of the variant nucleotides with sequences from the pseudo-VH and pseudo-VL gene families, which lie 5' to VH1 and VL1, respectively, suggests that the great majority of allelic variants can be accounted for by segmental transfer of sequence from donor pseudogenes into the germ-line VH1 and VL1 genes. These results demonstrate that the chicken VH1 and VL1 genes are susceptible to sequence replacement at the germ-line level as well as somatically during antibody diversification. The limited repertoire of B cell specificities produced by gene rearrangement in the chicken has led to speculation that these specificities may play a critical role in the progression of chicken B cell development. The results presented here do not support this hypothesis since many of the allelic variant nucleotides described here encode non-conservative amino acid substitutions within the antigen-binding sites of the Ig molecule.

Simultaneous involvement of all six predicted antigen binding loops of the T cell receptor in recognition of the MHC/antigenic peptide complex.

The TCR is predicted to resemble the Fab fragment of an Ig molecule and by analogy to possess six Ag binding loops that contact MHC proteins bound with antigenic peptides. We have identified residues in the predicted Ag binding loops (beta 1, beta 2, and beta 3) on a TCR beta-chain that are important in the recognition of the MHC/antigenic peptide complex. Using site-directed mutagenesis, we altered the residues forming the predicted Ag binding site on the beta-chain expressed by the T lymphocyte clone D5, which specifically recognizes p-azobenzenearsonate-conjugated peptides presented by the class II MHC molecule I-Ad. Amino acid substitution of individual residues in each loop affected Ag recognition, demonstrating that all three putative Ag binding loops of the D5 TCR beta-chain are important in interaction with I-Ad/arsonate-conjugated Ag. Taken together with our previous work on the D5 TCR alpha-chain (Nalefski et al., J. Exp. Med. 175:1553), these results suggest that all six Ag binding loops of the D5 TCR alpha- and beta-chains interact simultaneously with the MHC/peptide complex. Consequently, the area of interaction between the TCR and the MHC/antigenic peptide complex is predicted to be extensive.

Production and Immunodiagnostic Applications of Antihuman Light Chain Monoclonal Antibodies

Hybridomas producing antihuman light chain monoclonal antibodies (MoAbs) were derived from fusion of SP2/O mouse myeloma cells with splenic lymphocytes from mice repeatedly immunized with purified kappa- and lambda-type Bence Jones proteins representative of the major V kappa (V kappa I, V kappa II, V kappa III, V kappa IV) and V lambda (V lambda I, V lambda II/V, V lambda III, V lambda IV, V lambda VI) subgroups or gene families. Monoclonal antibodies were obtained that had specificity for constant-region (CL) determinants common to all kappa or lambda light chains (C kappa and C lambda, respectively) as well as for variable-region (VL) epitopes unique to each of the V kappa or V lambda subgroups. The capability of these reagents to recognize CL and VL determinants on monoclonal immunoglobulin (Ig) molecules was demonstrated in fluid-phase antigen-capturing enzyme-linked immunosorbent assay (ELISA), solid-phase ELISA, and immunoblotting. In addition, these antilight chain MoAbs were used to establish immunocytochemically the kappa or lambda type and VL-subgroup nature of light chains expressed by the cytoplasmic Ig of monoclonal plasma cell and surface Ig of B-lymphocyte populations, respectively. These antibodies facilitated the immunohistochemical detection and characterization of light-chain-associated amyloid (AL amyloid) and other types of light-chain-related tissue deposits. Furthermore, the anti-CL-specific MoAbs were used to measure serum and urinary Ig kappa and Ig lambda concentrations. Quantification of Bence Jones protein excretion, even in the presence of other urinary proteins, was possible using the highly sensitive anti-C kappa and anti-C lambda MoAbs reactive only with free light chains. The ability to identify and characterize, through the use of these antihuman light chain MoAbs, light-chain-related epitopes at the protein, cellular, and tissue level has clinical importance in the diagnosis and treatment of patients with monoclonal plasma cell and related B-cell immunoproliferative diseases.

Normal and atopic IgE responses.

"Isotype switch", the capacity of B lymphocytes to change expression from one to another class of immunoglobulin (Ig) molecule while retaining the same antigen specificity, has been known to exist for a long time. However, it was not known whether this D N A rearrangement in the Ig constant region gene cluster is simply a random event or a response to a specific exogenous signal. The IgE switch now stands out as a paradigm, both for (1) Ig class expression in response to a defined signal [interleukin(IL)-4 binding to its specific receptor] [6, 10, 14, 42] and (2) for a biological response which in a rather unique manner depends on one particular cytokine; mice with two deleted IL-4 genes make no IgE [20]. However, IL-4 leads to IgE switch only in conjunction with other B cell activation signals. In murine B cells, the stimulation with lipopolysaccharide (LPS) and IL-4 is sufficient for the induction o f an IgE response [6]. Human B cells are more demanding in that they require cell contact with helper T cells in addition to IL-4 [13, 28, 38]. The recently cloned ligand on T cells [2, 3] for the CD40 cell surface molecule on B cells can act as a potent co-signal for IgE switch [31], as well as for B cell activation in general [30]. For our own studies on the regulation of the human B cell response, we have used a culture system in which the direct contact with cells of a mutagenized subclone of the mouse EL4 thymoma cell line, in conjunction with either human T cell supernatant (T-SN) or phorbol myristate acetate (PMA), induces a particularly potent, polyclonal human B cell response [41, 45]. Our EL4 ceils are now known also to express the CD40 ligand, which reacts with human CD40 [2, 3], and this may at least in part explain their antigenand lectin-independent, "universal" B cell stimulatory activity. The questions which were addressed with regard to the IgE response are (1) how much IgE can B cells maximally produce, how do various parameters of this response (switch frequency, amount of Ig produced per clone, cellular Ig secretion rate, cytokine dependence) compare with IgG and IgA responses, and (2) is increased IgE production in atopic individuals related to an

Tryptic digestion of the serum immunoglobulin of the flounder, Platichthys flesus

Purified serum immunoglobulin (Ig) from the flounder has been fragmented by treatment with trypsin at 56°C. The major product of the digestion is a protein which resists further degradation over a two-hour period. This fragment constitutes approximately 40% of the Ig molecule and on fully reducing SDS electrophoresis is composed of a single polypeptide chain of apparent molecular weight 33 kD. This is larger than the light chains and therefore indicates that the fragment is derived only from the heavy chains of the Ig molecule. If this fragment is composed of eight polypeptide chains, consistent with the tetrameric nature of the flounder Ig, then its molecular weight is 264 kD. Although its electrophoretic mobility on 4% non-reducing SDS gels suggests a molecular weight of 180 kD, densitometric data support the 264 kD value. The kinetics of the digestion indicate that the first event is the liberation of a 50 kD fragment which is a dimer of two 25 kD polypeptides and which may be a Fabµ fragment.