Growth and body composition of preterm infants: influence of nutrient fortification of mother''s milk in hospital and breastfeeding post-hospital discharge

Enhanced oxidative stress is a feature of inflammatory and infectious conditions. Proteins may be important targets of oxidation and this may alter their function. We evaluated whether metal-catalyzed oxidation of IgG could alter its ability to bind to Fc receptors on macrophages. Human IgG incubated with an FeCl3/EDTA/ascorbate metal-catalyzed oxidation system resulted in a significant increase in carbonyl content, a measure of protein oxidation, compared to IgG treated with EDTA alone (control). Western blot analysis using an antibody to oxidized protein revealed an increase in antibody binding to both the heavy (Fc portion-containing) and light chains of IgG treated with the oxidizing system. Western blot analysis of papain-digested IgG confirmed oxidative modification of the Fc portion. Binding studies carried out with J774.16 macrophages demonstrated significantly diminished ability of the oxidized IgG to bind to macrophage Fc receptors compared to control IgG. These data demonstrate that IgG is susceptible to metal-catalyzed oxidation and that this impairs its ability to bind to macrophage Fc receptors. Oxidation of IgG might play a role in modulating immune function in infection and disorders associated with immune complex formation by diminishing IgG binding to phagocytic cells.     

Human IgG3 can adopt the disulfide bond pattern characteristic for IgG1 without resembling it in complement mediated cell lysis

In this paper we describe the construction of mouse-human IgG3 mutant antibodies resembling IgG1 in their disulfide bond pattern between the heavy and light chain (H-L) and between the two heavy chains (H-H). The effector functions of these mutant antibodies were compared to normal IgG3 and IgG1. Changing only the disulfide bond pattern between the heavy and light chains did not alter the ability to induce complement mediated cell lysis (CML), regardless of the amount of corresponding antigen that had been introduced to the surface of the target cells. However, alteration of the disulfide bond pattern between the two heavy chains had a large effect on CML due to shortening of the hinge from 62 to 15 amino acids. No difference between the mutants and normal antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC) was observed. This suggests that IgG3 can adopt the H-L disulfide bond pattern of IgG1 without obtaining the CML activity characteristic for IgG1.     

An efficient route to human bispecific IgG

Production of bispecific IgG (BsIgG) by coexpressing two different antibodies is inefficient due to unwanted pairings of the component heavy and light chains. To overcome this problem, heavy chains were remodeled for heterodimerization using engineered disulfide bonds in combination with previously identified "knobs-into-holes" mutations. One of the variants, S354C:T366W/Y349'C:T366'S:L368'A:Y407++ +'V, gave near quantitative (approximately 95%) heterodimerization. Light chain mispairing was circumvented by using an identical light chain for each arm of the BsIgG. Antibodies with identical light chains that bind to different antigens were identified from an scFv phage library with a very restricted light chain repertoire for the majority (50/55) of antigen pairs tested. A BsIgG capable of simultaneously binding to the human receptors HER3 and cMpI was prepared by coexpressing the common light chain and corresponding remodeled heavy chains followed by protein A chromatography. The engineered heavy chains retain their ability to support antibody-dependent cell-mediated cytotoxicity as demonstrated with an anti-HER2 antibody.     

Biosynthesis of the carbohydrate units of immunoglobulins. 1. Purification and properties of galactosyltransferases from swine mesentary lymph nodes

Galactosyltransferase (UDPgalactose:glycoprotein galactosyltransferase EC 2.4.1.22) was isolated from swine mesentary lymhromatography on Sepharose 4B colums containing covalently bound p-aminophenyl-beta-D-N-acetylglucosamine. The homogenous enzyme showed a single band on disc gel electrophoresis and had a specific activity of 35 nmol min-1 (mg of protein)-1 at 37 degrees C. A molecular weight of 57 000 was obtained by exclusion chromatography, sucrose density centrifugation, and sodium dodecyl sulfate-gel electrophoresis. The same molecular weight was obtained after reduction and alkylation which indicates that the enzyme is composed of only a single polypeptide chain. The enzyme catalyzed the formation of beta1 leads to 4 bonds between galactose and free terminal N-acetylglucosaminyl residues of soluble preparations of porcine IgG immunoglobulin heavy chain, fetuin, ovalbumin, and ovomucoid. An endogenous glycoprotein, present in particulate subcellular preparations, was also a very good substrate for the enzyme, and it was identified as incomplete IgG immunoglobulin heavy chain. The Km of the purified enzyme was 2.9 x 10(-5) M for fetuin, 5.4 x 10(-5) M for ovalbumin, 2.0 x 10(-5) M for IgG immlnoglobulin heavy chain, and 2.2 x 10(-5) M for UDP-galactose. About 20% of the total galactosyltransferase activity in lymph node homogenates was present in the cytosol fraction, and 80% was in the microsomal and Golgi fractions. The kinetic properties of the bound and soluble galactosyltransferases were similar,and both required Mn2+ for maximal activity. However, the bound enzyme required the addition of detergent, lysolecithin, GDP-mannose, and UDP-N-acetylglucosamine for maximum activity. These compounds did not influence the activity of the soluble transferase. The membrane preparations catalyzed the transfer of galactose from UDP-galactose and N-acetylglycosamine from UDP-N-acetylglucosamine to incomplete oligosaccharide chains of endogenous IgG immunoglobulin bound to these particles. The labeled products of these reactions were isolated, and the structures of their oligosaccharide chains were determined and compared with those isolated from the heavy chain of porcine IgG immunoglobulin. The glycopeptide prepared from the endogenous acceptor and the major glycopeptide prepared by proteolytic digestion of the heavy chain of porcine IgG immunoglobulin has identical structures. The following structure for the carbohydrate chains of porcine IgG immunoglobulin was determined by sequential enzymatic hydrolysis and methylation studies.     

Magnetic resonance angiography in trauma

The heavy and light chain genes (mu and lambda) of the human anti-adenocarcinoma monoclonal antibody HB4C5 (MAb-C5) were cloned from the human-human hybridoma cell line HB4C5 and co-expressed in Chinese hamster ovary (CHO) and COS-7 cells. ELISA assay and the RI imaging of the cancer tissue xenografted into nude mice showed that the recombinant MAb-C5 retained the original antigen binding activity. We then replaced the IgM constant region of the MAb-C5 heavy chain gene with the human IgG1 constant region gene and co-expressed it with the light chain gene. This recombination was confirmed by a complete DNA sequencing and Western-blot analysis. Despite the fact that the DNA sequence, the expressed protein size, and the assembly of heavy and light chains were indicated to be normal, the IgG1 type MAb-C5 could not bind to the original antigen. This result suggest that this antibody alters its antigen binding property upon class-switching.     

Molecular phylogeny of the families Campulidae and Nasitrematidae (Trematoda) based on mtDNA sequence comparison

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.     

Prevalence and characterization of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCA) directed against HMG1 and HMG2 in ulcerative colitis (UC)

To examine the structural relationship among autoantibodies produced by individuals with anti-GBM antibody-mediated disease, a polyclonal anti-idiotype directed against human anti-alpha3(IV)NC1 antibodies was produced and then used to study autoantibodies from other patients. For this purpose, anti-alpha3(IV)NC1 antibodies (anti-GBM), derived from a single patient (LL) with high titer and typical anti-GBM antibody specificity, were isolated using recombinant alpha3(IV)NC1-sepharose affinity chromatography. Following hyperimmunization of rabbits with anti-GBM IgG, irrelevant rabbit anti-human IgG antibodies were removed from the antiserum using a human IgG-sepharose column. The rabbit anti-alpha3(IV)NC1 antibodies (anti-Id GBM) effluent bound to human anti-GBM antibodies, but it did not bind to either normal human IgG or recombinant alpha3(IV)NC1 protein. The Id-anti-Id interaction was conformationally dependent on intact heavy and light chains of the anti-alpha3(IV)NC1 antibodies (ELISA and Western blotting). A competitive immunoassay was developed to evaluate structural and potential genetic relationships among anti-alpha3(IV)NC1 antibodies from different patients. All patients tested (9 of 9) had a substantial fraction (producing > 50% inhibition) of anti-GBM antibodies expressing Id-GBM. The results indicate that shared determinants are expressed by anti-GBM antibodies from different individuals, and they raise the possibility that common genetic elements are used to encode them. These regions are potential targets for design of reagents to regulate autoreactive B cells and/or interfere with pathogenic antibody-GBM interactions, in individuals with anti-GBM antibody mediated diseases.     

Fitting a hand-glove prosthesis to enhance the reconstructed mutilated hand

We report a unique case of tubular polyclonal immunoglobulin G (IgG) deposition disease (PIDD) superimposed on diabetic nephropathy in an 84-year-old man presenting with subacute renal failure and proteinuria. The deposits were located exclusively between the tubular epithelial cells and the tubular basement membranes (TBMs) and stained intensely with antisera to IgG heavy chain and both kappa and lambda light chains. Electron microscopy revealed large predominantly extracellular electron-dense deposits with a distinctive curvilinear substructure. The associated light microscopic findings of tubular simplification with features of acute tubular necrosis implicate this tubulopathy as the cause of the acute renal failure. This appears to represent a unique entity that does not fit into any previously described category of renal tubular immune complex or immunoglobulin deposition disease.     

The roles of gamma 1 heavy chain membrane expression and cytoplasmic tail in IgG1 responses

In antibody responses, B cells switch from the expression of immunoglobulin (Ig) mu and delta heavy (H) chains to that of other Ig classes (alpha, gamma, or epsilon), each with a distinct effector function. Membrane-bound forms of alpha, gamma, and epsilon, but not mu and delta, have highly conserved cytoplasmic tails. Mutant mice unable to express membrane gamma1 H chains or producing tailless gamma1 H chains failed to generate efficient IgG1 responses and IgG1 memory. H chain membrane expression after class switching is thus required for these functions, and class switching equips the B cell antigen receptor with a regulatory cytoplasmic tail that na?ve B cells lack.     

Characterization of platelet-reactive antibodies in children with varicella-associated acute immune thrombocytopenic purpura (ITP)

Urinary excretion of trypsin inhibitor increased after injection of a carcinogen, N-nitrosobis(2-oxopropyl)amine, into Syrian hamsters. Two inhibitors were purified to apparent homogeneity from urine collected during the course of the carcinogenesis experiment. Their complete amino acid sequences were determined by Edman degradation of the intact proteins and partially degraded fragments. One corresponded to a hamster liver cDNA clone that hybridized with human bikunin probe [Ide et al, (1994) Biochim, Biophys. Acta 1209, 286-292], except that the protein sequence lacked C-terminal serine and the other was trypstatin, the C-terminal half of the bikunin molecule. Three proteins containing covalently linked bikunin were also identified in pooled blood plasma. They were all dissociated into heavy and light chains by treatment with chondroitinase ABC or 50 mM NaOH, but not by heating at 100 degrees C in the presence of sodium dodecyl sulfate and dithiothreitol, N-terminal amino acid sequence analyses of the native chains and partially degraded fragments thereof revealed that these proteins are (i) human-type inter-alpha-trypsin inhibitor, consisting of heavy chains 1 and 2 and bikunin, (ii) bovine-type inter-alpha-trypsin inhibitor, consisting of heavy chains 2 and 3 and bikunin, and (iii) pre-alpha-trypsin inhibitor, consisting of heavy chain 3 and bikunin. Heterodimer of bikunin/heavy chain 1 or bikunin/heavy chain 2 was not detected. These results suggest that the composition, and hence function, of the inter-alpha-trypsin inhibitor family differs considerably from species to species.     

Radiodiagnosis of elbow and shoulder--questions by the clinician

Anti-double stranded(ds) DNA antibody is one of markers of systemic lupus erythematosus (SLE). Two human monoclonal anti-DNA antibody-producing cell lines were established from two SLE patients. One cell line secreted IgG isotype antibody (KSUG) and the other secreted IgM isotype antibody (KSUN). The light chains of the two immunoglobulins were lambda chains. The nucleotide sequences for the immunoglobulin variable region genes of the two antibodies were determined and compared to germline sequences. The heavy and lambda light chains of KSUG were VH3 family and V lambda IIIb, respectively. The heavy and lambda light chains of KSUN were VH4 family and V lambda IX, respectively. Antibody KSUG, IgG isotype, showed somatic mutations, whereas KSUN, IgM isotype, used the germline gene without mutation. These findings reconfirm the current paradigms that IgM anti-DNA antibodies are produced by utilizing germline genes whereas IgG anti-DNA antibodies are produced by somatic mutations.     

Purification and characterization of novel whey glycoprotein WGP-88 which binds to a monoclonal antibody to PAS-4 glycoprotein in the bovine milk fat globule membrane

A monoclonal antibody to the PAS-4 glycoprotein (78 kDa) of the bovine milk fat globule membrane (MFGM) specifically recognized PAS-4, and was named KAS4. A component recognized by KAS4 was found in whey protein, this being a glycoprotein of 88 kDa by SDS-PAGE and named WGP-88. WGP-88 was purified and characterized in comparison with PAS-4. WGP-88 had apparent pI values of 6.45 and 6.39, while those of PAS-4 were 7.39 and 7.35. Neuraminidase digestion shifted the pI values of WGP-88 to 6.57 and of PAS-4 to 7.52. WGP-88 was rich in polar amino residues (44.9 mol%), while PAS-4 was abundant in nonpolar amino acid residues (48.7 mol%). WGP-88 contained 17.1% of carbohydrate and PAS-4 had 7.2%. The results of reductive hydrolysis, N-glycanase digestion, and a lectin blot analysis suggested that N- and O-linked sugar chains were contained in both glycoproteins. WGP-88 and PAS-4 had a different N-terminal amino acid sequence. WGP-88 and PAS-4 respectively inhibited competitively the binding of KAS4 to PAS-4 and WGP-88. Our studies revealed WGP-88 recognized by KAS4 mAb to be a novel whey protein and to have different biochemical properties from those of PAS-4.     

High-level expression of a 56-kilodalton protein gene (bor56) of Rickettsia tsutsugamushi Boryong and its application to enzyme-linked immunosorbent assays

The 56-kDa protein of Rickettsia tsutsugamushi, which is located on the rickettsial surface, has been shown to be an immunodominant antigen. The gene that encodes the 56-kDa protein of R. tsutsugamushi Boryong (bor56) was cloned. Sequencing revealed an open reading frame of 1,602 bp encoding 534 amino acids with a molecular weight of 56,803. The 56-kDa protein of R. tsutsugamushi Boryong (Bor56) was expressed as a fusion protein with the maltose-binding protein of Escherichia coli by deleting 252 bp from the 5' end of the open reading frame and subcloning it into the StuI site of pIH821. The recombinant fusion protein was purified by amylose column chromatography for application in an enzyme-linked immunosorbent assay to evaluate the ability of the method to detect the antibody to R. tsutsugamushi in human patient sera. By using sera from 100 patients with scrub typhus and 70 patients with other febrile diseases, a high diagnostic sensitivity (95%) and a high diagnostic specificity (100%) were demonstrated, suggesting the suitability of the recombinant antigen for use as an immunodiagnostic tool.     

The antigen-binding domain of a human IgG-anti-F(ab')2 autoantibody

Recent studies revealed an immunoregulatory role of natural IgG-anti-F(ab')2 antibodies in both healthy individuals and patients with certain diseases. The implication of anti-F(ab')2 antibodies in the pathogenesis of diseases prompted us to study the gene segment structure of their antigen-binding domains and their binding characteristics. cDNA was prepared from the lymphocytes of a patient with a high IgG-anti-F(ab')2 serum titer. Variable heavy and light gene segments were amplified by PCR and inserted into a phagemid surface expression vector. Single-chain antibodies displayed on the phage surface were screened for binding to F(ab')2 fragments. The subsequent analysis of 95 single clones demonstrated that they all bound specifically to F(ab')2. Sequence analyses of 12 clones showed that 11 were identical and 1 contained a silent point mutation in the heavy chain and three amino acid exchanges in the light chain. The heavy chains belonged to the V(H)3 and the light chains to the V(kappa)2 gene family. The 11 identical light-chain genes were completely homologous to a germ-line sequence (DPK-15). Binding assays showed that the single-chain antibodies bind to F(ab')2, but not to Fab, Fc, or intact IgG. This binding pattern was confirmed by surface plasmon resonance studies, which revealed a relatively high affinity (Ka = 2.8 x 10(7) M(-1)). The strong binding capacity was further demonstrated by competitive inhibition of the serum anti-IgG antibody's interaction with antigen. The present study defines for the first time to our knowledge the gene segment structure of the antigen-binding domain of two human IgG-anti-F(ab')2 autoantibody clones and describes the binding kinetics of the purified monomeric fragments.     

Increased synthetic peptide specificity of tissue-CSF bound anti-MBP in multiple sclerosis

Free and bound hydrosoluble protein extracts were prepared from four anatomical areas of a multiple sclerosis (MS) cerebrum and from corresponding anatomical areas of a normal (non-MS) control. Increased levels of IgG and anti-myelin basic protein antibodies (anti-MBP) were detected in all MS samples and they were undetectable in the controls. IgG and anti-MBP from free (unbound) hydrosoluble protein extracts are defined as free IgG and free anti-MBP while IgG and anti-MBP from tissue bound protein extracts are defined as bound IgG and bound anti-MBP. IgG was purified from free protein extracts by protein G Sepharose affinity chromatography and anti-MBP was further isolated from purified IgG by antigen specific (MBP) Sepharose affinity chromatography. Free and bound anti-MBP were reacted with 20 synthetic peptides of human MBP prepared by the Fmoc method. Free anti-MBP, whether in the context of whole protein extracts, or as purified IgG or as purified antibody was completely neutralized by peptides #12, #15, #56 and #56* containing overall residues 75-106, partially neutralized by peptides #27, #16 and #21 containing overall residues 61-83 and did not react with the remaining 13 peptides. Tissue bound anti-MBP was completely neutralized only by peptides #12, #15, #56 and #56* (overall residues 75-106) and showed no reactivity towards the remaining 16 peptides including peptides #27, #16 and #21. Synthetic peptide specificity of free anti-MBP purified from MS cerebrum was identical to previously reported specificity of free anti-MBP from MS cerebrospinal fluid (CSF), while tissue bound anti-MBP, as well as bound anti-MBP from CSF had a more restricted synthetic peptide specificity than free anti-MBP. This suggests that the most likely epitope of anti-MBP is located between residues 84 and 95 of human MBP just proximal to the tri-proline sequence (99-101).     

Heterogeneity of antibodies reactive with the dominant antigen of Actinobacillus actinomycetemcomitans

The serotype b-specific carbohydrate antigen (SbAg) of Actinobacillus actinomycetemcomitans Y4 is reported to be the O antigen of lipopolysaccharide, and the highest titers of serum antibody reactive with A. actinomycetemcomitans in early-onset periodontitis (EOP) patients bind SbAg. These high titers of serum antibody reactive with SbAg are associated with a lesser extent and severity of periodontal disease. The aim of this study was to determine if a limited number of genes code for anti-SbAg antibodies as has been shown for immunoglobulin G (IgG) reactive with the type b polysaccharide from Haemophilus influenzae. Serum IgG reactive with the SbAg was prepared from 20 high-titer EOP patients by affinity chromatography. The IgG subclass concentrations were determined, and heterogeneity was analyzed by isoelectric focusing (IEF). IgG2 was the dominant subclass (83% of total IgG) in the anti-SbAg IgG fraction and represented an average of 1.33% of total serum IgG2. The IgG2 reactive with SbAg was isolated from the affinity-purified IgG fraction by affinity chromatography with protein A and subclass-specific monoclonal antibodies. On IEF gels, only 4 to 20 bands were observed in the anti-SbAg IgG fractions, indicating limited heterogeneity. N-terminal amino acid sequence analysis of eight representative anti-SbAg IgG2 preparations indicated that variable heavy and light chains consisted largely of V(H)III and V(kappa)II, respectively. However, a significant fraction of anti-SbAg may use V(H) and V(lambda) genes with blocked N termini. In short, these findings indicate that IgG reactive with SbAg is very much like the antibody reactive with H. influenzae type b polysaccharide. Similarities include IgG2 dominance, limited bands on IEF gels, supporting an oligoclonal response, and use of genes from V(H)III and V(kappa)II regions.     

Immune complexes from sera of patients with juvenile rheumatoid arthritis reveal novel 40 and 60 kd bands

OBJECTIVE: Immune complexes (IC) have been detected in the majority of sera of patients with juvenile rheumatoid arthritis (JRA). Our aim was to determine the possible constituents of these IC. METHODS: One hundred and four sera from patients with JRA were separated on a Sepharose 4B column to which were bound F(ab')2 fragments of goat anti-human IgM antibody to separate IgM-containing IC. The column was sequentially eluted with 1 M ammonia and 0.1 M glycine-HCl buffer, pH 3. The eluted material was treated with sodium dodecyl sulfate (SDS) and simultaneously reduced with 2-mercaptoethanol. Individual components were then separated by SDS-gradient polyaclylamide gel electrophoresis and were transferred to nitrocellulose by Western blotting. The nitrocellulose strips were overlaid with the patient's own serum and developed with specific antiserum to human IgM and IgG. RESULTS: Four bands were noted in the majority of JRA patients, including a band in the 70-80 kD area to the IgM heavy chain and in the 50 kD area to the IgG heavy chain. Additional bands specific for JRA patients' sera were noted in the 40 kD and 60 kD areas. Overall, 61 of 104 sera demonstrated all four bands; 78 showed the 40 kD band and 73 the 60 kD band. CONCLUSION: These IC were previously shown to contain IgM rheumatoid factor (RF), IgG-RF, and IgG in addition to the 40 and/or 60 kD constituents present in the isolates. Preliminary studies on these constituents suggest the possibility of the 60 kD band being the human 60 kD mitochondrial heat shock protein. Further investigations of these immune complex components may aid in the understanding of the pathophysiology of JRA.     

Proton nuclear magnetic resonance sequential assignments and secondary structure of an immunoglobulin light chain-binding domain of protein L

The 1H NMR assignments have been made for the immunoglobulin (Ig) light chain-binding B1 domain of protein L from Peptostreptococcus magnus. The secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone coupling constants, and slowly exchanging amide protons. The B1 domain was found to be folded into a globular unit of 61 amino acid residues, preceded by a 15 amino acid long disordered N-terminus. The folded portion of the molecule contains a four-stranded beta-sheet spanned by a central alpha-helix. The fold is similar to the IgG-binding domains of streptococcal protein G, despite the fact that the binding sites on immunoglobulins for the two proteins are different; protein G binds IgG through the constant (Fc) part of the heavy chain, whereas protein L has affinity for the variable domain of Ig light chains.     

Evaluation of butanone, carbon dioxide, and 1-octen-3-OL as attractants for mosquitoes associated with north central Florida bay and cypress swamps

In the sera of (NZB x NZW)F1 (B/W) mice that develop a lupus-like syndrome, increased levels of IgG antibodies (Ab) reacting with TNP have been detected before the appearance of IgG anti-DNA Ab and clinical symptoms. A single injection of trinitrophenyl-bovine serum albumin (TNP/BSA) in physiological saline into a young B/W mouse (3 months old), followed by fusion of its splenocytes 3 days later, gave rise to hybridomas simultaneously secreting IgM and IgG Ab with anti-TNP reactivity. Both mu and gamma chains were detected in culture supernatants by ELISA, and double isotype-producing cells were labeled by immunofluorescence. Molecular analysis of two of these double isotype-producing hybridomas showed the presence of mRNA coding for both mu and gamma chains of Ig, and this gamma mRNA could be translated in vitro into a gamma heavy (H) chain. Comparison of the H chain variable-region sequences of IgM and IgG revealed 100% homology between mu and gamma V(H) genes in one clone, while mu and gamma V(H) genes showed only 80% homology in the other clone. Both clones produced a single kappa light (L) chain. These two hybridomas, isolated from a B/W mouse, thus represent two different mechanisms of double isotype expression: the first one corresponds to an IgM to IgG switch, while the second one reflects a lack of allelic exclusion.     

Analysis of heavy and light chain pairings indicates that receptor editing shapes the human antibody repertoire

In the bone marrow, diversity in the primary antibody repertoire is created by the combinatorial rearrangement of different gene segments and by the association of different heavy and light chains. During the secondary response in the germinal centres, antibodies are diversified by somatic mutation and possibly by further rearrangements, or "receptor editing". Here, we have analysed the pairings of heavy and light chain variable domains (VH and VL) in 365 human IgG+ B cells from peripheral blood, and established that these pairings are largely random. The repertoire is dominated by a limited number of pairings of segments and folds. Among these pairings we identified two identical mutated heavy chains in combination with two different mutated light chains (one kappa and one lambda). This shows that receptor editing occurs in the human periphery and that the same antibody lineage can be subjected to both receptor editing and somatic hypermutation. This suggests that receptor editing may be used together with somatic mutation for the affinity maturation of antibodies. We also propose that receptor editing has shaped variable gene segment use and the evolution of V gene families.