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Glycosylation of the IgG-Fc is essential for optimal binding and activation of Fcγ receptors and the C1q component of complement. However, it has been reported that the effector functions are down-regulated when the Fc glycans terminate in sialic acid residues and that sialylated IgG mediates anti-inflammatory effects of intravenous immunoglobulin (IVIG). Although recombinant IgG is hypo-sialylated, Fc sialylation is shown to be markedly increased when a mouse/human chimeric IgG3 Phe243Ala (F243A) variant is expressed in Chinese hamster ovary (CHO)-K1 cells. Here we investigate whether sialylation is increased in IgG1 F243A when expressed in CHO-K1, mouse myeloma J558L and human embryonic kidney (HEK) 293. Although the sialylation level was 2-5% for IgG1 wild type (WT), it was increased to 31%, 10% and 33% for the variant from CHO-K1, J558L and HEK293 cells, respectively. Interestingly, an increased addition of bisecting GlcNAc and α(1-3)-galactose residues to the Fc glycan was observed for HEK293-derived and J558L-derived IgG1 F243A, respectively. Fucosylation of HEK293-derived IgG1 F243A was maintained despite increased bisecting GlcNAc content. Although sialic acid and bisecting GlcNAc residues are reported to have an opposing effect on antibody-dependent cellular cytotoxicity (ADCC), IgG1 F243A showed 7 times lower ADCC activities than IgG1 WT, irrespective of bisecting GlcNAc residue. Thus, highly sialylated, human cell-derived IgG1 F243A with lowered ADCC activity may be of interest for the development of therapeutic antibodies with anti-inflammatory properties as an alternative to IVIG.

BACKGROUND Donor lymphocyte infusion (DLI) is used for treatment of hematologic malignancy relapse or mixed chimerism after allogeneic hematopoietic stem cell transplantation. Although graft‐versus‐host disease is well recognized as one of the adverse effects of DLI, there are limited reports on noninfectious pulmonary complications (NIPCs) after DLI. CASE REPORT A 55‐year‐old woman with acute myeloid leukemia received DLI for conversion from recipient predominant to complete donor chimerism on Day +193 after allogeneic HSCT. Eight weeks later, she complained of dyspnea with fever; chest computed tomography revealed diffuse, bilateral, ground glass opacity and reticular appearance. She was diagnosed as having NIPC based on serum and bronchoalveolar lavage fluid (BALF) findings. She was successfully treated with prednisolone (PSL) and completely recovered. DISCUSSION We analyzed the cell profile from the BALF and 27 cytokines and chemokines in the serum using the Bio‐Plex platform. The cells consisted of recipient predominant macrophages and T cells. The serum cytokine and chemokine profile showed significant elevation of interleukin (IL)−1β, IL‐6, IL‐8, tumor necrosis factor‐α, macrophage inflammatory protein (MIP)−1α, and MIP‐1β, which declined with the improvement of symptoms after initiation of PSL treatment. CONCLUSION Inflammatory effectors by recipient cells, rather than allogeneic responses by donor cells, played an important role in the pathogenesis of NIPCs after DLI in the present case.

The influence of sodium butyrate on the production and glycosylation of recombinant mouse/human chimeric antibody by transfected CHO-K1 cells was investigated. We selected cells expressing 'wild-type' antibody with a human IgG3 heavy chain and a mutant of this molecule in which Phe 243 is replaced by Ala. These proteins have previously been shown to exhibit very different glycoform profiles with the mutant IgG being comprised of glycoforms having a high galactose and sialic acid content. Cell culture with 0-5 mM butyrate was shown to effect a 2-4-fold increase in antibody production whilst the induction of apoptosis was observed in a dose-dependent manner. The optimal butyrate concentration was observed to be 2 mM. The glycoform profile of each antibody produced in the presence of butyrate was analyzed by HPAEC-PAD and shown to be unchanged, relative to that produced in the absence of butyrate. Biological activity was evaluated by the ability of the antibodies to trigger superoxide generation, through Fc gamma RI, and shown to be independent of production in the presence or absence of butyrate. A similar increase in production was observed for a high antibody-producing cell line when expanded in a hollow fibre bioreactor under low-serum conditions (1%). These results demonstrated that butyrate is of value for increasing the productivity of CHO-K1 for recombinant IgG and does not compromise either glycosylation or biological activity.

Antibodies are multifunctional molecules that following the formation of antibody antigen complexes, may activate mechanisms to effect the clearance and destruction of the antigen (pathogen). The IgG molecule is comprised of three globular protein moieties (2Fab+Fc) linked through a flexible hinge region. While the Fabs bind antigens, the Fc triggers effector mechanisms through interactions with specific ligands, e.g. cellular receptors (FcgammaR), and the C1 component of complement. Glycosylation of IgG-Fc has been shown to be essential for efficient activation of FcgammaR and C1. We report the generation of a series of truncated glycoforms of IgG-Fc, and the analysis of the contribution of the residual oligosaccharide to IgG-Fc function and thermal stability. Differential scanning microcalorimetry has been used to compare the stabilities of the homogeneous glycoforms of IgG1-Fc. The results show that all truncated oligosaccharides confer a degree of functional activity, and thermodynamic stability to the IgG1-Fc, in comparison with deglycosylated IgG1-Fc. The same truncated glycoforms of an intact IgG1 anti-MHC Class II antibody are shown to exhibit differential functional activity for FcgammaRI and C1 ligands, relative to deglycosylated IgG1. The minimal glycoform investigated had a trisaccharide attached to each heavy chain and can be expected to influence protein structure primarily in the proximity of the N-terminal region of the C(H)2 domain, implicated as a binding site for multiple effector ligands. These data provide a thermodynamic rationale for the modulation of antibody effector functions by different glycoforms.

The heavy and light chains of IgG monoclonal antibodies (mAbs) can be shown to be heterogeneous, with respect to isoelectric points, when analyzed by two-dimensional electrophoresis (2-DE). The molecular basis for this charge heterogeneity has not been clearly defined but it has been suggested that it could be due, in part, to differences in glycosylation. To investigate this possibility we have compared the 2-DE pattern of glycosylated and aglycosylated forms of the mouse IgG1 mAb (1B7-11), produced in vitro in the presence and absence of tunicamycin. Charge heterogeneity was shown not to be a consequence of glycosylation status. Intracellular and secreted IgG mAbs were also analyzed to investigate the time course of change in charge properties of the heavy and light chains. The charge heterogeneity was found to be generated intracellularly, and alterations in charge properties could be induced during incubation under physiological conditions. Semilogarithmic plots of the density of the principal heavy and light chain spots against incubation time showed linear relationships, suggesting that the charge shifts result from a first-order reaction. The semilogarithmic plot for the light chain correlated well with the time after IgG synthesis. These results suggest that the charge heterogeneity of an IgG mAb is due to intra- and extracellular modifications of the polypeptide chains which reflect "aging" of antibody molecules.

We proposed and demonstrated a method for reconstructing waveguide portions that were removed when dicing 45/spl deg/ mirrors onto other waveguides on the same substrate. The polymeric waveguide core divided by dicing at not optical input or output positions was repaired using a self-written waveguide formed with green laser irradiation. The optical coupling loss at the cut point was obtained to be 0.4 dB. We obtained optical output with low excess loss from the repaired waveguide.

The optical solder effect of the self-written waveguide (SWW) using Rhodamine 6G dye-dispersed ultraviolet (UV) curable resin by a green laser is demonstrated. This enables formation of SWW from the end of polyimide waveguide having strong UV absorption. High efficient coupling between an optical fiber and a vertical-cavity surface-emitting laser is successfully achieved

The immune response to different doses of a hapten of fluorescein isothiocyanate (FITC) in BALB/c mice was analyzed by two-dimensional affinity electrophoresis (2-D AEP). The mice were immunized with different doses (0.5 microgram, 5 micrograms, 50 micrograms, 500 micrograms and 2.5 mg) of FITC-conjugated bovine serum albumin (BSA). The antibodies to FITC bovine serum albumin (BSA) were separated into a large number of IgG spots due to differences in their isoelectric points (pI) and binding affinity to FITC ligand immobilized in the gel. The IgG spots, showing identical affinity to the ligand but different pI, have been considered as an IgG family. The affinity and quantity of IgG families changed with the increase in FITC-BSA dosage. With a low dose (5 micrograms) most of the families showed high affinity (Kd1 microMKd50 microM) and low affinity (Kd50 microM) antibodies were produced. The increase of FITC-BSA up to 500 micrograms markedly increased the quantity of IgG spots showing a variety of affinity to FITC. However, 2.5 mg FITC-BSA did not increase the quantity and heterogeneity of IgG spots significantly. The changes in the heterogeneity and quantity of anti-FITC antibodies and the subclass switch were observed over the course of immunization. The heterogeneity and the quantity of IgG1, IgG2b and IgG3 antibodies increased markedly during the first and the second immunization, whereas an increase in the heterogeneity and the quantity of IgG2a antibody was observed in the third immunization. This suggests that the subclass switch to IgG1, IgG2b and IgG3 and the somatic mutation of IgG1, IgG2b and IgG3 occur during the first and the second immunization, but the subclass switch to IgG2a and the somatic mutation of IgG2a seem to occur later than that of the other IgG subclasses.

Background: Lung cancer is one of the refractory malignancies and the leading cause of cancer-related death worldwide. Although prognosis of lung cancer was improved by the anticancer drugs and molecular targeted therapies, the results are not sufficient. Cellular senescence is irreversible cell cycle arrest. The drug-dependent induction of cellular senescence in neoplastic cells is considered an important tumor suppressive mechanism. 5-aza-2’-deoxycytidine (5-aza-dC) causes cellular senescence in solid tumors. Hypermethylation of CpG islands is well known as a major inactivation mechanism of tumor suppressor genes such as E-cadherin. Loss of E-cadherin confers a poor prognosis in lung cancer patients and is associated with in vitro resistance to epidermal growth factor receptor inhibitors. This study was designed to assess the relationship of E-cadherin expression cellular senescence induced by 5-aza-dC in non-small cell lung cancer (NSCLC) cell lines. Materials and Methods: We investigated two NSCLC cell lines; H1299 and A549 reported they have hypermethylation in promoter lesion of E-cadherin. There are two clinically approved DNA methyltransferase inhibitors (DNMTi), 5-aza-dC and 5-azacytidine (5-aza-CR). We also used Sodium Butyrate (NaB). We treated them with each drug for 120 hours. Flow cytometry analysis, immunofluorescence staining for E-cadherin and senescence associated beta-galactosidase assays and immunoblotting for senescence related proteins assessed NSCLC cell outgrowth and morphology. Results: Both cell lines with 5-aza-dC or NaB treatment changed morphology and increased size but decreased cell numbers. 5-aza-CR causes apoptosis in both cell lines. In A549, E-cadherin expression increased after 5-aza-dC treatment. There is no E-cadherin expression in H1299 with or without treatment. Both cell lines with 5-aza-dC were positive for Senescence-associated beta-galactosidase staining, H3K9me3 and gamma-H2AX. Conclusion: Taking together these findings strongly suggest the ability of 5-aza-dC to induce cellular senescence in NSCLC cells. The cellular senescence induced by a 5-aza-dC approach has to be considered a therapeutic option for NSCLC and an important phenomenon to further investigate in the next future. Citation Format: Masashi Furukawa, Yuka Mimura, Hiroyuki Tao, Yusuke Mimura, Kazunori Okabe. 5-aza-2’-Deoxycytidine induces cellular senescence in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2842.

Mouse antidextran monoclonal antibodies showed microheterogeneity which was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Not only the heavy (H) chains but also the light (L) chains were heter ogeneous in terms of isoelectric point (pI). The higher the pI, the more prominent the H chain spots. To demonstrate the cause of the microheterogeneity an IgG1 monoclonal antibody (mAb 35.8.2H) was examined especially for involvement of the sugar moiety in the microheterogeneity. The glycosylated region was determined in the Fc portion from serine 239 to methionine 309 by a glycan detection method using mild periodate oxidation, which confirms that the sugar chain is attached to the conserved glycosylation site of asparagine 297. However, charge heterogeneity of the H chain was not entirely attributed to the Fc because the papain digest of the antibody was separated into two Fc spots, a few Fd spots and two L chain spots by 2-D PAGE. This indicates that factors other than the sugar moiety are responsible for charge heterogeneity of IgG monoclonal antibody. On the other hand, the H chain isoforms of lower pI were shown to be more susceptible to V8 protease by peptide mapping. This result strongly suggests the occurrence of deamidation at glutamine or asparagine residues.

Immune response to a hapten of fluorescein isothiocyanate (FITC) in a single BALB/c strain mouse was analyzed by two-dimensional affinity electrophoresis (2D-AEP). Anti-FITC antibodies were induced by immunization with FITC-conjugated bovine serum albumin. The antibodies were separated into a large number of spots of IgG due to differences in their isoelectric points(pI) and binding affinities to the FITC ligand. These spots consisted of IgG families which were composed of several spots having an identical affinity to the ligand but a different pI. The spots were not clearly detected in the antiserum taken on day 7 after the primary immunization, but on day 21 the spots of IgG were clearly detected, with a high diversity and specificity for the ligand. The size and number of IgG spots were markedly increased by the secondary immunization; however, the third immunization did not increase the size and number of IgG spots. The IgG spots of each family were specifically stained with an antimouse IgG subclass antibody. Furthermore, a monoclonal antibody (FL-D6) was separated by 2D-AEP into a single family which consisted of seven IgG1 spots having an identical affinity to FITC but different pIs. Therefore, each of the IgG families of anti-FITC antibodies in the antiserum can be generated by a single clone of anti-FITC antibody-producing cells. The substitution of dextran T 2000 or lipopolysaccharide for bovine serum albumin as a carrier for FITC induced much smaller amounts of anti-FITC antibodies with a low diversity but high specificity to FITC.

SummaryGlycan recognition leading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a mass-spectrometric approach. Without any derivatization steps, the purified pig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.

CD4 + and CD8 + T cell responses to endogenous retroviral envelope glycoprotein gp90 generate protective immunity to murine colon carcinoma CT26. A panel of I-A d -restricted T cell hybridomas recognize gp90 synthesized by CT26 cells but not by other gp90-expressing tumors. Here we report that antigenicity resides in an incompletely folded form of gp90 that is unique to CT26. In contrast to more compact forms of gp90 that are present in other tumors, this open conformer is captured by recycling I-A d on antigen-presenting cells and is processed intracellularly. Thus, gp90 acquires immunodominance via MHC-guided processing, and the generation of an MHC class II-restricted response can be controlled by the intracellular folding environment of antigen-expressing cells.

A simple and efficient synthesis of N-linked glycoamino acids and glycopeptides from deprotected sugars using the Staudinger reaction.

We have developed "tapered self-written waveguide (SWW)" for coupling of optical components in the board level optical interconnection. Tapered waveguides have a possibility of achieving larger alignment tolerance for optical coupling. The fabrication condition of tapered SWW was studied by adjusting both the optical power and irradiation time of curing resin, and tapered SWW was successfully realized. The optical tolerance vertical to the optical axis twice as compared with straight SWW was obtained.

In the optical interconnection of board level, 45-degree micro mirrors are used to achieve 90-degree optical path change. These mirrors are often fabricated by using a rotating blade and this method has a serious problem of cutting other waveguides in the vicinity. A self-written waveguide ha been successfully applied to repari and connect these waveguides. So, it will be possible to allow optical signals to be input and output at a specific location on the board.

Class II molecules of the major histocompatibility complex (MHC class II molecules) bind peptides derived from protein antigens delivered into endocytic compartments and present these peptides to CD4+ T cells (Cresswell, 1994; Germain, 1994). In the endocytic pathway antigens are unfolded and cleaved into fragments during transport through the increasingly acidic endosomal network, from early endosomes to lysosomes. Newly synthesized MHC class II molecules associated with the invariant chains (Ii) are transported to a late endocytic compartment where the Ii is removed by proteolytic cleavage. MHC class II molecules bind polypeptide antigens by removal of the Ii-derived peptide CLIP (class II invariant chain-derived peptide) through a peptide-exchange process that is catalyzed by MHC-encoded DM molecules (Fig. 1) (Denzin and Cresswell, 1995; Sherman et al., 1995; Sloan et al., 1995; Wolf and Ploegh, 1995). The initial form of antigen that binds to class II molecules may be short peptides 10-20 amino acids in length, generated by acid endopeptidases (Watts, 2001). This pathway could be termed ‘‘cut/trim first, bind later’’ model and became the paradigm for the binding of peptides to MHC class II molecules. However, there is an alternative pathway independent of DM molecules that involves early capture of unfolded, extended sequence byMHC class II molecules (Pinet et al., 1994; Pinet et al., 1995; Sercarz et al., 1993). Subsequent trimming of

Human antibody 2G12 neutralizes a broad range of human immunodeficiency virus type 1 (HIV-1) isolates by binding an unusually dense cluster of carbohydrate moieties on the “silent” face of the gp120 envelope glycoprotein. Crystal structures of Fab 2G12 and its complexes with the disaccharide Manα1-2Man and with the oligosaccharide Man 9 GlcNAc 2 revealed that two Fabs assemble into an interlocked V H domain-swapped dimer. Further biochemical, biophysical, and mutagenesis data strongly support a Fab-dimerized antibody as the prevalent form that recognizes gp120. The extraordinary configuration of this antibody provides an extended surface, with newly described binding sites, for multivalent interaction with a conserved cluster of oligomannose type sugars on the surface of gp120. The unique interdigitation of Fab domains within an antibody uncovers a previously unappreciated mechanism for high-affinity recognition of carbohydrate or other repeating epitopes on cell or microbial surfaces.

Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Thus, when antigen-specific IgG antibodies form antigen/antibody immune complexes the effectively aggregated IgG can activate a wide range of effector systems. Multiple effector mechanisms result from cellular activation mediated through a family of IgG-Fc receptors differentially expressed on leucocytes. It is established that glycosylation of IgG-Fc is essential for recognition and activation of these ligands. IgG antibodies predominate in human serum and most therapeutic antibodies are of the IgG class. The IgG-Fc is a homodimer of N-linked glycopeptide chains comprised of two immunoglobulin domains (Cgamma2, Cgamma3) that dimerise via inter-heavy chain disulphide bridges at the N-terminal region and non-covalent interactions between the C-terminal Cgamma3 domains. The overall shape of the IgG-Fc is similar to that of a "horseshoe" with a majority of the internal space filled by the oligosaccharide chains, only attached through asparagine residues 297.To investigate the influence of individual sugar (monosaccharide) residues of the oligosaccharide on the structure and function of IgG-Fc we have compared the structure of "wild-type" glycosylated IgG1-Fc with that of four glycoforms bearing consecutively truncated oligosaccharides. Removal of terminal N-acetylglucosamine as well as mannose sugar residues resulted in the largest conformational changes in both the oligosaccharide and in the polypeptide loop containing the N-glycosylation site. The observed conformational changes in the Cgamma2 domain affect the interface between IgG-Fc fragments and FcgammaRs. Furthermore, we observed that the removal of sugar residues permits the mutual approach of Cgamma2 domains resulting in the generation of a "closed" conformation; in contrast to the "open" conformation which was observed for the fully galactosylated IgG-Fc, which may be optimal for FcgammaR binding. These data provide a structural rationale for the previously observed modulation of effector activities reported for this series of proteins.

Engagement of Fc gamma receptors (Fc gamma Rs) with the Fc region of IgG elicits immune responses by leukocytes. The recent crystal structure of Fc gamma RIII in complex with IgG-Fc has provided details of molecular interactions between these components (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). One of the most intriguing issues is that glycosylation of IgG-Fc is essential for the recognition by Fc gamma Rs although the carbohydrate moieties are on the periphery of the Fc gamma RIII-Fc interface. To better understand the role of Fc glycosylation in Fc gamma R binding we prepared homogeneous glycoforms of IgG-Fc (Cri) and investigated the interactions with a soluble form of Fc gamma RIIb (sFc gamma RIIb). A 1:1 complex stoichiometry was observed in solution at 30 degrees C (K(d), 0.94 microm; Delta G, -8.4 kcal mol(-1); Delta H, -6.5 kcal mol(-1); T Delta S, 1.9 kcal mol(-1); Delta C(p), -160 cal mol(-1) K(-1)). Removal of terminal galactose residues did not alter the thermodynamic parameters significantly. Outer-arm GlcNAc residues contributed significantly to thermal stability of the C(H)2 domains but only slightly to sFc gamma RIIb binding. Truncation of 1,3- and 1,6-arm mannose residues generates a linear trisaccharide core structure and resulted in a significantly decreased affinity, a less exothermic Delta H, and a more negative Delta C(p) for sFc gamma RIIb binding, which may result from a conformational change coupled to complex formation. Deglycosylation of the C(H)2 domains abrogated sFc gamma RIIb binding and resulted in the lowest thermal stability accompanied with noncooperative unfolding. These results suggest that truncation of the oligosaccharides of IgG-Fc causes disorder and a closed disposition of the two C(H)2 domains, impairing sFc gamma RIIb binding.

The molecular specificity and mechanisms for the interaction of immunoglobulins with Fc binding ligands has been a subject of intensive investigation for many years. Significant information has been gained for human IgG-Fc from x-ray crystallographic analysis of IgG-Fc and IgGFc/ligand complexes. The crystal complexes of IgG-Fc with FcRn, SpA, SpG and rheumatoid factor autoantibody ligands revealed binding sites at the CH2/CH3 interface and an IgG-Fc:ligand stoichiometry of 1:2; reflecting the two fold symmetry of the IgG molecule [1]. For FcγR and C 1 q monomeric IgG must necessarily be functionally monovalent since IgG, FcγR bearing cells and Cl co-exist in the blood, in the absence of activation and the consequent inflammatory reactions.

To characterize local hyperfibrinolysis in the pathogenesis of chronic subdural hematoma (CSDH), fibrin degradation products (FDPs) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify each FDP fragment (fragments HMW, YY, DY and DD) by immunoblotting. The electrophoretic patterns of FDP in 40 hematomas from 33 patients could be categorized into 3 patterns; (i) type 1, the sharp DY and DD bands; (ii) type 2, the broad DY and DD bands; and (iii) type 3, the sharp DY and DD with two bands with higher molecular weights than fragment DY and DD. The broadening of DY and DD bands was reproduced by incubating the sample containing sharp DD and DY bands with the hemolyzed peripheral blood cells. These results indicated that the hyperfibrinolysis in CSDH could be characterized by the constant and incomplete proteolysis of fibrinogen and fibrin by plasmin and further degradation of FDPs by non-plasmin proteases released from hemolyzed blood cells.

Affinity maturation of anti-hapten antibodies in an inbred mouse (BALB/c) was analyzed by two-dimensional affinity electrophoresis (2D-AEP) in which the antibodies specific to a hapten of dinitrophenyl (DNP) group were separated into a large number of IgG families according to the differences in their isoelectric points (pI) and in their affinities for the ligand of DNP immobilized in the gel matrices. Each of the IgG families consisted of several spots showing an identical affinity for the ligand but different pI, and the spots were specifically stained by immunostaining with an anti-murine IgG subclass antibody. These results lead us to the conclusion that each of the IgG families is derived from a single clone of antibody-producing cells specific to DNP. The mass and affinity of the IgG families varied in the course of immunization with DNP-conjugated chicken serum albumin (DNP-CSA). The first injection of DNP-CSA induced a small amount of IgG families showing a wide variety in their affinity for DNP. The second injection induced a large amount of IgG families, especially of families having a medium affinity for DNP. Additional injections, however, did not change the mass or affinity of the IgG families significantly. This suggests that an antigen-induced somatic mutation of the immunoglobulin gene does not occur frequently to mature the affinity of antibodies by the additional injections after the second injection. 2D-AEP enables us to analyze the affinity maturation of antibodies in vivo in a single mouse and to document the subclass switch of the antibody in the course of immunization.

Carbohydrates are common environmental antigens. As dextran B512 is composed of a repeating structure of simple antigenic determinants, it is widely used to study the immunochemical properties of immunoglobulins. Two-dimensional affinity electrophoresis patterns of a mouse monoclonal antidextran antibody (35.8.2H; IgG1, BALB/c) were produced to obtain insights into the microheterogeneity of the monoclonal antibody. The monoclonal antibody was separated into about six spots which had an identical affinity to dextran B512, but differed in their isoelectric points (pI). In addition, the pH dependence of the binding affinity of this antidextran to dextran B512 was examined. By comparing affinities obtained by affinity electrophoresis between weakly basic (pH 9.5) and weakly acidic (pH 3.8) discontinuous buffer systems, the latter showed an affinity about 500 times lower than the former. The change in the affinity was investigated with a continuous pH gradient by an affinity titration curve and was seen to change markedly at about pH 6. This suggests that the histidine at residue 34 in the light-chain CDR1 is largely responsible for the dextran binding.