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In general, purification of bispecific antibody (bsAb) is more challenging than that of monospecific antibody due to the increased complexity in byproduct profile. Like in the case of monospecific antibody purification, immunoglobulin-binding protein-based affinity chromatography is an indispensable tool for bsAb purification. For example, Protein A affinity chromatography has been widely used to capture Fc-containing bsAbs whereas other affinity media such as Protein L and KappaSelect, which bind kappa light chain, are used to capture bsAbs that do not contain a Protein A-binding site. In fact, affinity chromatography also possesses the capability of removing certain product-related impurities in bsAb purification when it is conducted with suitable medium and under appropriate conditions. Fully exploring the potential of affinity chromatography in bsAb purification to achieve both product capture and byproduct removal is highly desirable, as this can greatly alleviate the purification burden on subsequent polishing steps and hence improves the overall robustness of the downstream process. This article briefly reviews the byproduct clearance potential of several commonly used affinity media under relevant bsAb purification scenarios., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: A new medium cut-off (MCO) membranes has been designed to achieve better removal capacities for middle and large middle molecules in hemodialysis (HD) treatment., Aim: The aim of this study was to evaluate the removal efficacy of Theranova® in standard HD in comparison with standard high-flux HD., Methods: Four HD patients (M/F 1/4) were included in 12-week observational pilot study in HD with Theranova® 400 and Theranova® 500 dialyzers. Each patient was assessed 4 times, T0 with high-flux dialyzers, T1 at 1 month, T2 at second month, and T3 at third month, by measuring pre- and post-HD samples of urea, Cr, β2-microglobilin (β2M), myoglobin, albumin, free light chains kappa (FLC-k), and free light chains lambda (FLC-λ)., Results: The data showed a higher average removal rate for all the uremic toxins with Theranova® dialyzers for β2M, myoglobin, FLC-k, and FLC-λ (62.7, 56.9, 63.5, and 54.6%, respectively) during the 3 months. Albumin retention was observed and did not change between T0 and T3 (p = 0.379)., Conclusion: Compared to high-flux membranes, MCO membranes show greater permeability for middle molecules in midterm report., (© 2020 S. Karger AG, Basel.)

The heterogeneous morphologic features of canine plasmacytomas (PCTs) can make their differentiation from other round cell tumors challenging. Immunohistochemistry (IHC) for lambda (λ) and kappa (к) immunoglobulin (Ig) light chains is often equivocal because of high background staining. The chromogenic in situ hybridization (CISH) technique for light chains has shown higher sensitivity compared to IHC in human plasma cell tumors. Therefore, we aimed to validate automated CISH for light chains in canine tissues and to evaluate its diagnostic potential in canine PCTs, in conjunction with routinely used IHC markers. CISH for light chains demonstrated a clear signal in plasma cell populations of canine control tissues (lymph nodes, lymphoplasmacytic inflammation) showing a polyclonal pattern with a prevalence of λ-producing cells. CISH detected monotypic light chain expression in 33 of 53 (62%) PCTs, 31 expressing λ and 2 expressing к. CISH was more sensitive than IHC for λ light chain (58% vs. 47%, respectively) and more easily interpretable given the absence of confounding background staining. The absence of CISH staining for both λ and к in a considerable subset of tumors may be the result of lower light chain production by neoplastic cells. Multiple myeloma oncogene 1 (MUM1) was expressed by all but 2 PCTs (96%), which showed λ expression by CISH and IHC. The identification of poorly differentiated canine PCTs requires the assessment of a panel of IHC markers, with the potential support of CISH for Ig light chains.

KappaSelect is an affinity medium that specifically binds to the constant region of the kappa light chain (LC). Obviously, KappaSelect can be used to separate antibody species containing the kappa LC constant region from those lacking that region. However, it is not clear whether this resin can readily separate species containing one kappa LC constant region from those containing two kappa LC constant regions although the former are assumed to bind weaker than the latter. In this work, we demonstrated that antibody species with two kappa LC constant regions binds to the KappaSelect resin much tighter than species with only one kappa LC constant region. Consequently, these two species can be readily separated using this resin. This information not only enriches our knowledge with KappaSelect but also has important practical value. In addition to the sample case used in the current study, there are other cases in which the target protein contains one kappa LC constant region whereas a byproduct contains two kappa LC constant regions. Our finding provides a convenient means for removing such two kappa LC binding site containing byproduct in these cases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Polyclonal FLCs can be used as a biomarker of inflammation and immune activation in a range of diseases. This study evaluated the performance of new FLC ELISAs (Seralite FLC ELISA) for the quantitation of polyclonal κ and λ FLC, including comparisons to existing assays., Methods: Technical performance was assessed for the ELISA and reference ranges were generated using healthy donor serum (N = 91). Patients with a range of conditions associated with polyclonal FLC dysregulation (N = 164) were measured across platforms., Results: The ELISAs generated references ranges of: 8.72-23.0 mg/L κ FLC, and 8.52-25.24 mg/L for λ FLC. ELISAs demonstrated linearity across the calibration range and intra-assay (≤ 8.7%) and inter-assay (≤ 12.3%) imprecision was low. The limit of detection was 0.63 mg/L for κ and 0.57 mg/L for λ FLC. Minimal cross-reactivity was observed for interference agents, alternate FLC and whole immunoglobulin (median change ≤3.6 mg/L). Assays showed good batch-to-batch consistency. For patient samples, methods generated different κ and λ FLC concentrations and differences were seen between methods for the number of patients classified as below, with and above references ranges for κ and λ FLC. There was no significant difference in the FLC sum between the different techniques., Conclusions: The ELISAs displayed good analytical and technical performance. The quantification of individual κ and λ FLC appears inherently different between platforms. These differences are attenuated if using the FLC sum, which was similar between methods and provided agreement in relation to patients having normal or elevated FLCs., Competing Interests: Declaration of Competing Interest MS is an employee of Abingdon Health Ltd. MD has an advisory role with Abingdon Health Ltd. and reports personal fees from Abingdon Health Ltd. JC, MG, and TP own shares in Abingdon Health Ltd. CH is Chairman of Scientific & Medical Advisory Board of Abingdon Health Ltd., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 10 9 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.

We describe a chromatographic approach for the purification of urinary free light chains (FLCs) viz., lambda free light chains (λ-FLCs) and kappa free light chains (κ-FLCs). Isolated urinary FLCs were analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular masses of λ-FLC and κ-FLC are 22,933.397 and 23,544.336Da respectively. Moreover, dimer forms of each FLC were also detected in mass spectrum which corresponds to 45,737.747 and 47,348.028Da respectively for λ-FLCs and κ-FLCs. Peptide mass fingerprint analysis of the purified λ-FLCs and κ-FLCs has yielded peptides that partially match with known light chain sequences viz., gi|218783338 and gi|48475432 respectively. The tryptic digestion profile of isolated FLCs infers the exclusive nature of them and they may be additive molecules in the dictionary of urinary proteins. This is the first report of characterization and validation of FLCs from large volume samples by peptide sequencing. This simple and cost-effective approach to purification of FLCs, together with the easy availability of urine samples make the large-scale production of FLCs possible, allowing exploration of various bioclinical as well as biodiagnostic applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Multiple myeloma is a malignant plasma cell dyscrasia that is becoming more prevalent in an increasingly ageing population. It is a complex disease with clinical phases ranging from the premalignant monoclonal gammopathy of undetermined significance to asymptomatic (smouldering) myeloma and then symptomatic myeloma; the latter occasionally terminating in the clonal proliferation of plasma cells outside the bone marrow. We present a patient whose clonally evolved disease from monoclonal gammopathy of undetermined significance to multiple myeloma demonstrated the presence of an unusual combination of monoclonal immunoproteins. Capillary electrophoresis demonstrated the presence of three paraproteins in the gamma region (γ-region), two of which were additional to the IgGk paraprotein which migrated in the slow γ-region at initial diagnosis. Subsequent isotypic identification of the new paraproteins was not possible by immunotyping and initial immunofixation studies failed to definitively characterize the monoclonal proteins. After reduction with beta-mercaptoethanol, two paraproteins were detected by both capillary and gel electrophoresis. However, only immunofixation was able to resolve three distinct monoclonal bands, confirming the presence of free monoclonal kappa light chains in the mid-gamma region and free monoclonal heavy chains in the fast gamma region. Triple gammopathies in themselves are uncommon; this case presents a very unusual combination of paraproteins which required various electrophoretical and immunochemical techniques to identify and characterize them. The change of electrophoretic signature from the monoclonal gammopathy of undetermined significance phase to the diagnosis of multiple myeloma suggested that a number of genetically distinct subclones were present in the pretreatment clonal evolution of the disease.

Recombinant antibody single-chain variable fragments (scFv) are difficult to purify homogeneously from a protein complex mixture. The most effective, specific and fastest method of purification is an affinity chromatography on Protein L (PpL) matrix. This protein is a multi-domain bacterial surface protein that is able to interact with conformational patterns on kappa light chains. It mainly recognizes amino acid residues located at the VL FR1 and some residues in the variable and constant (CL) domain. Not all kappa chains are recognized, however, and the lack of CL can reduce the interaction. From a scFv composed of IGKV10-94 according to IMGT®, it is possible, with several mutations, to transfer the motif from the IGKV12-46 naturally recognized by the PpL, and, with the single mutation T8P, to confer PpL recognition with a higher affinity. A second mutation S24R greatly improves the affinity, in particular by modifying the dissociation rate (kd). The equilibrium dissociation constant (KD) was measured at 7.2 10(-11) M by surface plasmon resonance. It was possible to confer PpL recognition to all kappa chains. This protein interaction can be modulated according to the characteristics of scFv (e.g., stability) and their use with conjugated PpL. This work could be extrapolated to recombinant monoclonal antibodies, and offers an alternative for protein A purification and detection.

In this study, the light chain (κ) and heavy chain (γ) sequences of the monoclonal antibody against vascular endothelial growth factor (VEGF) were sub-cloned into the eukaryotic pcDNA3.1 (+) (Hygro) and the pcDNA3.1 (+) (Neo) expression vectors using the traditional and homologous recombination methods. To express the antibody, the recombinant plasmids were transfected into the Chinese hamster ovary (CHO) and the K562 cell lines. The recombinant antibody was then purified using the protein A affinity chromatography. Furthermore, in order to demonstrate the inhibition of VEGF-induced mitogenesis of the recombinant antibody, the bovine aorta endothelial like cells were employed. The results showed specialization and conjunction of the recombinant antibody to the VEGF. It was also indicated that the antibody expression in the K562 cell lines was higher than the CHO cell lines. Furthermore, the in vitro VEGF inhabitation of the recombinant antibodies which were produced from the K562 cell line, and the CHO cell line, were similar. This proved that the K562 cell line is a good substitute for the CHO cell line in the production of the recombinant antibodies.

The combination of marked hypersideremia, hypertransferrinemia, and monoclonal gammopathy of underdetermined significance (MGUS) should alert clinicians to the possible presence of an anti-transferrin immunoglobulin, an uncommon acquired disorder also defined as transferrin-immune complex disease (TICD). The authors have previously described a case of TICD with 100% transferrin saturation and liver iron overload. However, the findings in the few cases so far reported are heterogeneous, and the presence of high transferrin saturation and liver iron overload is not universal. In this article, the authors have described the identification of two additional patients with anti-transferrin monoclonal gammopathy, hypersideremia, and hypertransferrinemia, but with incomplete transferrin saturation and no hepatic iron overload. The autoantibodies were purified by using transferrin as affinity bait and characterized. One subject showed a high-titer monoclonal anti-transferrin IgM with a κ-type light chain. This finding is the first observation of IgM autoantibodies against transferrin. The other patient developed the disease after pregnancy. In this study, monoclonal antibody was an IgG mounting a κ-type light chain with altered molecular weight. These results highlight that transferrin might induce the development of a monoclonal immune response of different classes and specificity. The identification, in a single hematologic center, of three different subjects with anti-transferrin monoclonal gammopathy suggests that the disease probably represents a still underdiagnosed condition. From a clinical standpoint, these patients must be followed up both as MGUS and as hemochromatosis., (Copyright © 2013 Wiley Periodicals, Inc.)

An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of three patients with systemic lupus erythematosus was used. Phage particles displaying DNA binding light chains were isolated by affinity chromatography on DNA-cellulose, and the fraction eluted by an acidic buffer (pH 2.6) was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Thirty three of 687 individual colonies obtained were randomly chosen for study of MLCh DNase activity. Nineteen of 33 clones contained MLChs with DNase activity. Four preparations of MLChs were expressed in Escherichia coli in soluble form, purified by metal chelating chromatography followed by gel filtration, and studied in detail. Detection of DNase activity after SDS-PAGE in a gel containing DNA demonstrated that the four MLChs are not contaminated by canonical DNases. The MLChs demonstrated one or two pH optima. They were inactive after the dialysis against ethylenediaminetetraacetic acid but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mg(2+) , Mn(2+) , Ni(2+) , Ca(2+) , Zn(2+) , and Co(2+) was individual for each MLCh preparation. K(+) and Na(+) inhibited the DNase activity of various MLChs at different concentrations. Hydrolysis of DNA by all four MLCh was saturable and consistent with Michaelis-Menten kinetics. These clones are the first examples of recombinant MLChs possessing high affinity for DNA (Km  = 3-9 nM) and demonstrating high kcat values (3.4-6.9 min(-1) ). These observations suggest that the systemic lupus erythematosus light chain repertoire can serve as a source of new types of DNases., (Copyright © 2013 John Wiley & Sons, Ltd.)

Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production.

Sera from lung cancer patients contain antibodies against tumor-associated antigens. Specific amino acid sequences of the complementarity-determining regions (CDRs) in the antigen-binding fragment (Fab) of these antibodies have potential as lung cancer biomarkers. Detection and identification of CDRs by mass spectrometry can significantly be improved by reduction of the complexity of the immunoglobulin molecule. Our aim was to molecular dissect IgG into κ and λ fragments to reduce the complexity and thereby identify substantially more CDRs than by just total Fab isolation. We purified Fab, Fab-κ, Fab-λ, κ and λ light chains from serum from 10 stage I lung adenocarcinoma patients and 10 matched controls from the current and former smokers. After purification, the immunoglobulin fragments were enzymatically digested and measured by high-resolution mass spectrometry. Finally, we compared the number of CDRs identified in these immunoglobulin fragments with that in the Fab fragments. Twice as many CDRs were identified when Fab-κ, Fab-λ, κ and λ (3330) were combined than in the Fab fraction (1663) alone. The number of CDRs and κ:λ ratio was statistically similar in both cases and controls. Molecular dissection of IgG identifies significantly more CDRs, which increases the likelihood of finding lung cancer-related CDR sequences., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: New monoclonal antibody-based assays for serum-free light chains (FLC) have become available., Methods: In a clinical study with 541 patients, the new N Latex FLC assays were compared with the Freelite FLC assays and immunofixation electrophoresis (IF)., Results: Comparison of the different FLC kappa (κ) assays showed a slope of 0.99 with a deviation of 5.0%, rs=0.92, for FLC lambda (λ) a slope of 1.22, deviation 13.8%, rs=0.90 and for the κ/λ ratio a slope of 0.72, deviation -4.6%, rs=0.72. The concordance for the FLC κ assays was 91%, for FLC λ 85% and κ/λ ratio 95%. The clinical sensitivity and specificity of the κ/λ ratios in the study were comparable: 60% and 99% for the N Latex FLC assay and 61% and 97% for the Freelite assay. In IF-FLC positive samples, the N Latex FLC κ/λ ratio scored 20/23 (87%) samples outside the reference range and Freelite 21/23 (91%). For IF-FLC negative samples, N Latex FLC assay κ/λ ratio scored 338/350 (97%) within the reference range and Freelite scored 332/350 (95%)., Conclusions: The concordance scores and the clinical sensitivity and specificity of the new N Latex FLC assays and Freelite assays appeared comparable, but there are some differences in measurement of concentrations between the methods.

Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA' specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA' in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened--demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA' conjugate., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain-intein tag for purification via a chitin-agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and β-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the ΔI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins., (Copyright © 2010 Elsevier Inc. All rights reserved.)

An excess of circulating monoclonal free immunoglobulin light chains (FLC) is common in plasma cell disorders. A subset of FLC, as amyloidogenic ones, possess intrinsic pathogenicity. Because of their complex purification, little is known on the biochemical features of serum FLC, possibly related to their pathogenic spectrum. We developed an immunopurification approach to isolate serum FLC from patients with monoclonal gammopathies, followed by proteomic characterization. Serum monoclonal FLC were detected and quantified by immunofixation and immunonephelometry. Immunoprecipitation was performed by serum incubation with agarose beads covalently linked to polyclonal anti-κ or λ FLC antibodies. Isolated FLC were analyzed by SDS-PAGE, 2D-PAGE, immunoblotting, mass spectrometry (MS). Serum FLC were immunoprecipitated from 15 patients with ALλ amyloidosis (serum λ FLC range: 98-2350mg/L), 5 with ALκ amyloidosis and 1 with κ light chain (LC) myeloma (κ FLC range: 266-2660mg/L), and 3 controls. Monoclonal FLC were the prevalent eluted species in patients. On 2D-PAGE, both λ and κ FLC originated discrete spots with multiple pI isoforms. The nature of eluted FLC and coincidence with the LC sequence from the bone marrow clone was confirmed by MS, which also detected post-translational modifications, including truncation, tryptophan oxidation, cysteinylation, peptide dimerization. Serum FLC were purified in soluble form and adequate amounts for proteomics, which allowed studying primary sequence and detecting post-translational modifications. This method is a novel instrument for studying the molecular bases of FLC pathogenicity, allowing for the first time the punctual biochemical description of the circulating forms., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In multiple myeloma the predominant cause of irreversible renal failure is cast nephropathy, secondary to excess kappa or lambda serum free light chains (FLCs). These molecules are efficiently cleared by hemodialysis (HD) using the Gambro HCO 1100 dialyzer. To optimize the removal of FLCs by this dialyzer we have studied the effect of dialyzers in series, dialyzer change, and hemodiafiltration in 14 patients with multiple myeloma and renal failure. The clearance rates of both kappa FLCs and lambda FLCs were significantly increased on two dialyzers from 19 (7.3-34)-15.3 (9-28) mL/min to 47 (17-79)-35.5 (20-57) mL/min, respectively. Clearance rates of both FLCs decreased over the course of the dialysis sessions (both P < 0.001). Changing the dialyzer during a HD session increased lambda FLC clearance rates (22.5 [6-41] to 37.6 [9-52] mL/min; P < 0.001) and decreased kappa FLC clearance rates (39.6 [9-72] to 19 [8-59] mL/min; P < 0.003). Ultrafiltration during HD increased the clearance rates of kappa FLCs (R 0.52, P < 0.01) but not lambda FLCs (R -0.25; P < 0.076). Hemodiafiltration increased the clearance rates of both kappa (19 [SD 6.8] to 32 [SD 9.8] mL/min) and lambda FLCs (15 [SD 7.8] to 20 [SD 7.7] mL/min). Albumin replacement requirements for 8 h of HD increased from 12 g for a single dialyzer to 45 g for two dialyzers in series (P < 0.001). Different protocols are required to optimize the removal of kappa and lambda FLCs in patients with myeloma and renal failure.

Two unusual but similar cases of very high serum iron, extremely high transferrin concentrations (5.4 to 6.5 g/L), and increased transferrin saturation are reported. No anemia or microcytosis was observed. The ferritin concentration remained within the normal range, and no iron overload was observed. In one case, the in vivo half-life of 59Fe-labeled transferrin was shown to be prolonged (206 minutes versus 75 minutes for controls). In both patients, a monoclonal IgG was observed in the serum. The association between serum transferrin and monoclonal IgG was demonstrated by Western blot analysis and affinity chromatography. We suggest that the transferrin bound to the monoclonal IgG molecule has a prolonged half-life in the circulation, leading to high transferrin concentrations, and that the increased serum iron values prevent the onset of anemia. The antitransferrin activity of monoclonal antibody should be added to the list of situations accounting for elevated serum iron with elevated transferrin saturation.

We have recently shown that Neovastat, an antiangiogenic extract from shark cartilage, stimulates the in vitro activation of plasminogen by facilitating the tissue-type plasminogen activator (tPA)-dependent conversion of plasminogen to plasmin. In this report, we describe the purification and characterization of the stimulatory molecules. Neovastat was subjected to a three-step purification procedure including gel filtration, preparative isoelectric focusing, and preparative SDS-PAGE. Two 28-kDa proteins with pIs of approximately 4.5 and 6.5 were purified to apparent homogeneity and identified as immunoglobulin (Ig) kappa light chains by N-terminal microsequencing. Ig light chains do not directly stimulate the activity of tPA or plasmin, suggesting a mechanism of action involving an interaction with plasminogen. Kinetic analysis showed that both Ig light chains accelerate the in vitro tPA-dependent conversion of plasminogen in plasmin by increasing the affinity of tPA for plasminogen by 32- and 38-fold (Km decrease from 456 nM to 12-14 nM). Shark Ig light chains also stimulated the degradation of fibrin by the tPA/plasminogen system in an in vitro assay. A direct interaction between Ig light chains and plasminogen (KA=4.0-5.5 x 10(7) M(-1); KD=18-25 nM) and with tPA (KA=2.8 x 10(7) M(-1); KD=36 nM) was demonstrated using real time binding measured by surface plasmon resonance. Ig light chain is the first molecule associated with the antiangiogenic activity of Neovastat to be purified and identified.

Aim: To prepare monoclonal antibody (mAb) against erythropoietin(EPO), characterize its biological properties and use it to purify the rhEPO from transgenic goat milk., Methods: A crude rhEPO product was used as the antigen to immunize BALB/c mice for preparing mAbs against rhEPO. The mAbs were characterized by Western blot and indirect ELISA. Purified mAb 2E6 was coupled with pre-activated Sepharose 4B to prepare the immunoaffinity chromatography column for purifying the rhEPO from transgenic goat milk., Results: Two hybridoma cell lines(1E7 and 2E6)were obtained. mAbs 1E7 and 2E6 were shown to be IgG1 and IgG2b respectively, and their light chains were both kappa. Western blot analysis confirmed that the two mAbs could bind to rhEPO. The immunoaffinity chromatography column could adsorb 70% of rhEPO in purifying the rhEPO from transgenic goat milk., Conclusion: Two hybridoma cell lines secreting anti-rhEPO mAbs were successfully established. The mAb-immunoaffinity chromatography column could be used to purify the rhEPO from transgenic goat milk.

The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti-TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP kappa L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while kappa L chains act via another C-independent but mast cell-dependent pathway.

Glomerulonephritis with organized microtubular monoclonal immunoglobulin deposits (GOMMID) and glomerulonephritis related to type I cryoglobulin are well-known but rare complications of B cell derived chronic lymphocytic leukaemia. In these disorders, monoclonal Ig have never been studied at the molecular level. We conducted a pathological and molecular analysis in a patient with chronic lymphocytic leukaemia, glomerulonephritis and a single circulating monoclonal Ig. Unusual IgG1kappa kidney deposits were observed. The heavy and light chain variable region sequences of that cryoprecipitating monoclonal Ig were characterized. Light microscopy revealed glomerulonephritis typical of cryoglobulinaemia, with neutrophil and macrophage infiltration, endocapillary hyperplasia and few protein thrombi. Electron microscopic study clearly evidenced numerous subepithelial mixed granular and organized deposits with a unique microtubular organization, reminiscent of the GOMMID. The Ig molecule sequence revealed alterations of charge and hydrophobicity potentially promoting a crystal-like aggregation and the aggregation of microtubules. This description suggests that common mechanisms are involved in various forms of precipitation and/or deposition of complete Ig molecules, with a variable extent of organization and with a possible overlap between pathological patterns of either glomerulonephritis with microtubular deposits or type I cryoglobulinic glomerulonephritis.

We present the case of a 69-years-old man who was admitted to hospital with multiple myeloma. IgG-kappa type monoclonal protein was detected in the serum. When we separated the serum obtained from blood sample of the patient and the lid of the collecting tube was opened, the patient's serum became gelled immediately. When the lid of the collecting tube remained closed, the patient's serum did not become gelled even at 4 degrees C. Moreover, the gelled serum of the patient did not resolve at 56 degrees C. Taken together, these results indicated that gel formation of the patient's serum may not be due to cryoglobulin. It was found that the pH of the patient's serum elevated to pH 8.0 quickly after exposed to air. It was also found that the patient's serum, but not the sera of other IgG-kappa multiple myeloma patients, became gelled as soon as PBS of pH 8.0 was added. These results highly suggest that the patient's serum becomes gelled at pH 8.0. However, the isoelectric focusing of isolated precipitation in the patient showed fractions around the pH 8.5-8.7 zone, which was different from the pH at which the precipitation began to form. We think that this may be the first report of a multiple myeloma patient whose serum becomes gelled after exposed to air.

While amyloid infiltration of articular structures is rare, the 'shoulder-pad' sign resulting from periarticular soft tissue amyloid deposition is essentially pathognomonic for immunogloblin (Ig) (AL) amyloidosis. We report the characterization of an amyloid protein (GRA) which produced articular amyloid deposits and the shoulder-pad sign. Amyloid fibrils were isolated from soft tissue shoulder mass of a patient with systemic AL amyloidosis. The fibrils were solubilized in guanidine HCl and proteins separated by Sepharose chromatography. Amino acid sequence of fractionated protein was determined after tryptic digestion. Sequence analysis of the major amyloid protein yielded a kappa III Ig light chain structure. The entire variable region (VL) plus the constant region (CL) to residue 207 was identified; but lesser amounts of CL than VL were present. A number of amino acid residues previously not observed in kappa III VL proteins, plus a two amino acid insert (95A, 95B), were identified. Kappa III VL amyloid proteins are rare and may show an increased predilection for soft tissue deposition. While several unique amino acid residues that were identified in protein GRA may contribute to soft tissue amyloid deposition, no definite pattern is obvious from comparison with other reported kappa III amyloid proteins.

Amyloid-deposited light chain (AL) amyloidosis is correlated with the overproduction of a monoclonal immunoglobulin light chain protein by a B-lymphocyte clone. Since the amyloid fibril deposits in AL amyloidosis most often consist of the N-terminal fragments of the light chain, the majority of studies have focused on the determination of the primary structure of the protein, and reducing agents have been used routinely in the initial purification process. In this study, two light chain proteins were isolated and purified, without reduction, from the urine of a patient diagnosed with kappa 1 (kappa1) AL amyloidosis. One protein had a relative molecular mass of 12,000 and the other 24,000. Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry, in combination with enzymatic digestions, were used to verify the amino acid sequences and identify and locate posttranslational modifications in these proteins. The 12-kDa protein was confirmed to be the N-terminal kappa1 light chain fragment (variable region) consisting of residues 1-108 or 1-109 and having one disulfide bond. The 24-kDa protein was determined to be the intact kappa1 light chain containing a cysteinyl posttranslational modification at Cys214 and disulfide bonds located at Cys23-Cys88, Cys134-Cys194, and Cys214-Cys. The methods used in this report enable high-sensitivity determination of amino acid sequence and variation in intact and truncated light chains as well as posttranslational modifications. This approach facilitates consideration of the effect of cysteinylation on the native protein structure and the potential involvement of this modification in AL amyloidosis., (Copyright 2001 Academic Press.)

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.

Abs to U1 RNA are frequently found in patients suffering from systemic lupus erythematosus overlap syndromes and Ab titers correlate with disease activity. We describe the isolation of the first human anti-U1 RNA autoantibodies from a combinatorial IgG library made from the bone marrow of a systemic lupus erythematosus patient. With the use of phage display technology, two anti-U1 RNA single-chain variable fragment (scFv) Abs were selected. Both high affinity anti-U1 RNA Ab fragments (Kd approximately 1 nM) recognize stem II of U1 RNA and were derived from the same heavy chain gene (VH3-11) and the same lambda (3r) light chain gene although somatic mutations, predominantly present in the complementarity-determining regions, are different. Experiments, in which the heavy chain genes of both anti-U1 RNA scFvs were reshuffled with the original light chain repertoire of the patient resulted, after selection on stem loop II, in a large number of RNA-binding Ab fragments. All these stem loop II-specific RNA binding clones used a similar, but not identical, 3r lambda light chain. When scFvs were selected from the reshuffled libraries by stem loop IV, representing the other autoantigenic site of U1 RNA, most selected Ab clones did react with stem loop IV, but no longer with stem loop II. The stem loop IV-reactive Ab clones contained different, not 3r-related, light chains. These results point to a major role for the light chain in determining the sequence specificity of these disease-related anti-U1 RNA Abs. The possibility that secondary light chain rearrangements are involved in this autoimmune response is discussed.

Human Fab (IgG1kappa) phage display libraries were constructed from bone marrow RNA from 2 donors who recovered from infection with Ebola (EBO) virus during the 1995 outbreak in Kikwit, Democratic Republic of the Congo. The libraries were initially panned against a radiation-inactivated EBO virus-infected Vero cell lysate, but only weak binders were identified. In contrast, panning against secreted EBO glycoprotein (SGP) resulted in Fabs showing very strong reactivity with SGP in ELISA. These Fabs also reacted with a virion membrane preparation. The Fabs were strongly positive in IFAs with cells infected with EBO (subtype Zaire) virus but negative with uninfected cells, with a characteristic punctate staining pattern in the cytoplasm. The Fabs showed weak or no reactivity with the virus cell lysate although donor serum did react. The Fabs are now being characterized in structural and functional terms. Major interest will focus on the ability of antibodies to neutralize EBO virus and, later, to protect animals against infection.

The beta-sandwich structure of immunoglobulin variable domains is characterized by a typical kink in the first strand, which allows the first part of the strand to hydrogen bond to the outer beta-sheet (away from the VH-VL interface) and the second part to the inner beta-sheet. This kink differs in length and sequence between the Vkappa, Vlambda and VH domains and yet is involved in several almost perfectly conserved interactions with framework residues. We have used the selectively infective phage (SIP) system to select the optimal kink region from several defined libraries, using an anti-hemagglutinin single-chain Fv (scFv) fragment as a model system. Both for the kink with the Vkappa domain length and that with the Vlambda length, a sequence distribution was selected that coincides remarkably well with the sequence distribution of natural antibodies. The selected scFv fragments were purified and characterized, and thermodynamic stability was found to be the prime factor responsible for selection. These data show that the SIP technology can be used for optimizing protein structural features by evolutionary approaches., (Copyright 1998 Academic Press.)

Immunoglobulin light chains are the precursor proteins of AL-amyloidosis. In the fibril formation process properties of the variable part of the immunoglobulin light chains are believed to be of major importance. In this work it is shown that fragments of the constant part of the immunoglobulin light chain are a constituent of the AL-amyloid proteins of kappa type. A specific antiserum has identified these fragments in gel filtration fractions where the absorbance approached the base line after the main retarded peak. The fragments are small and have been overlooked previously in the purification process. The significance of the constant part in AL-proteins is unclear, but adds new aspects to the discussion of pre- or post-fibrillogenic cleavage of the immunoglobulin light chains., (Copyright 1998 Academic Press.)

The mouse has approximately 140 germline V kappa genes, and functional V kappa exons are expressed at roughly equivalent levels in the preimmune repertoire. We have examined the expression of individual members of the V kappa 10 family. V kappa 10A and V kappa 10B genes have been utilized in numerous hybridomas and myelomas, while V kappa 10C has not. In this study, we have cloned the V kappa 10C gene and shown that it is structurally functional, has the expected promoter elements and recombination signal sequences, and that it is capable of recombination. V kappa 10C mRNA, however, is present at levels at least 1000-fold lower than V kappa 10A and V kappa 10B in adult spleens. While there are no sequence differences in the octamer or TATA box between V kappa 10C and V kappa 10A, there are three nucleotide changes in the promoter region. These promoters equally drive the expression of a reporter gene in B cells or plasma cells, but the V kappa 10A promoter is able to drive expression in pre-B cell lines significantly better than the V kappa 10C promoter (p < 0.05). V kappa 10C rearrangements can be detected in bone marrow and splenic DNA. Therefore, the lack of V kappa 10C expression may reflect the inability of V kappa 10C-rearranged cells to undergo positive or negative selection. Our results suggest that the available Ab repertoire is shaped not only by the number of structurally functional genes, but also by the ability of assembled genes to be expressed at critical points during B cell maturation.

We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 10(4)-10(5)-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries.

Protein L (IgG kappa-chain-binding bacterial protein) showed a precipitate line(pseudo-immuno-reaction) with IgG and F(ab')2 fragment, but did not show any line with the Fab fragment, the Fc fragment and free kappa-chains in the micro-Ouchterlony method. The IgG and Fab fraction obtained from pa-pain-digested IgG (from the sera of patients with chronic thyroiditis), followed by Protein A-Sepharose, were separated by Protein L-Sepharose affinity chromatography. The unbound fraction (UF) consisted of IgG(lambda) or Fab(lambda) and the bound fraction (BF) consisted of IgG(kappa) or Fab(kappa) were obtained. Anti-thyroglobulin and anti-thyroid peroxidase antibody activities were found equally in both the UF and the BF. When Fab(kappa) was reduced with dithiothreitol (DTT), the Fd fragment in the UF could be separated from the free kappa-chain and the unreduced Fab(kappa) in the BF with a Protein L-Sepharose column. A separation method of human IgG fragments such as free kappa-chain, combined forms of kappa-chain [Fab or F(ab')2], and the Fd region, using Protein L, is described.

The human red cell Rh(D) antigen elicits the production of high-affinity IgG antibodies, which can prevent blood transfusion and cause hemolytic disease of the newborn. It has been known for 20 years that Rh(D) antibodies are among the most positively charged human serum IgGs. Analysis by IEF of 9 human anti-Rh(D) monoclonal antibodies showed that their isoelectric points (pI) (8.3 to 8.6) were also significantly higher than the average pI of serum IgGs (7.0 to 8.5). Sequencing of the anti-Rh(D) H and L chains cDNAs showed a preferential use of V(H)1, V(H)3, J(H)6, and V(kappa)1 gene segments. The high pIs in IEF were correlated with a higher number of cationic amino acid residues in the H chain V regions without clustering in the complementary determining region. Computer analysis indicated that the germline V(H) used in anti-Rh(D) was selected among the most cationic segments available in the human V(H) repertoire or expressed in normal B cells. These results indicate that the selection of cationic V(H) segments may be an important early step in the formation of clinically relevant anti-Rh(D) and other red cell antibodies, possibly to facilitate epitope binding in the negatively charged red cell membrane environment.

This study examined the hypothesis that certain immunoglobulin light chains directly altered mesangial cell calcium homeostasis. Intracellular Ca2+ concentration (intracellular [Ca2+]) signaling was determined in suspensions of rat mesangial cells using the acetoxymethyl ester of fura 2 with a calcium removal/replacement protocol. Pretreatment of cultured rat mesangial cells with a glomerulopathic kappa-light chain (gle) produced reversible dose- and time-dependent attenuation of ATP- and thrombin-evoked [Ca2+] transients (189 +/- 24 vs. 126 +/- 10 nM, P < 0.05 with ATP; 198 +/- 5 vs. 117 +/- 3 nM, P < 0.05 with thrombin) and capacitative calcium influx (199 +/- 14 vs. 142 +/- 17 nM, P < 0.05 for ATP; 252 +/- 19 vs. 198 +/- 18 nM, P < 0.05 for thrombin). Mesangial cells treated with gle and supplemented with myo-inositol (450 microM) did not demonstrate the attenuation of the ATP-evoked [Ca2+] transient and capacitative calcium influx. Gle also decreased mean [Ca2+] transient (80 +/- 7 vs. 56 +/- 1 nM, P < 0.05) and capacitative calcium influx (306 +/- 10 vs. 241 +/- 4 nM, P < 0.05) in response to thapsigargin, a Ca2+-adenosinetriphosphatase inhibitor. This inhibition was not reversed by exogenous myo-inositol. Another kappa-light chain (10 microg/ml) did not affect mesangial cell calcium signaling. Deranged mesangial cell calcium homeostasis by certain light chains may play a central pathogenetic role in glomerulosclerosis associated with deposition of immunoglobulin light chains.

A systematic detection method for the single performance of cellulose acetate (CA) membrane isoelectric focusing to detect six different types of information on protein abnormalities was developed. High-voltage isoelectric focusing was carried out on six layers of CA membrane using a thermoelectric cooling apparatus. After electrophoresis, the proteins on the top, the third, the fourth, the fifth, and the bottom CA membrane were transferred to a polyvinylidene difluoride (PVDF) membrane by a simple contact printing procedure to detect IgM, kappa-chain, lambda-chain, IgA, and IgG, respectively. Each PVDF membrane revealed the microheterogeneity of these immunoglobulins using specified anti-serum and enzyme immunostaining. The second CA membrane was stained with Coomassie brilliant blue G250 to detect serum protein patterns. All stained membranes showed clear electrophoretic patterns of immunoglobulin microheterogeneity. By our method, immunoglobulin abnormalities in serum could be screened out using six different types of information obtained simultaneously.

Antibodies to thyroglobulin (Tg) are commonly found in patients with the autoimmune thyroid diseases Graves' disease and Hashimoto's thyroiditis as well as in individuals with apparently normal thyroid function. Although it is not clear how Tg Abs are involved in the pathology of the diseases, the study and analysis of these Abs may nevertheless be instructive in allowing the development of an Ab response to an autoimmune disease-associated self Ag to be followed. We have prepared IgG kappa and lambda phage display combinatorial libraries from the cervical lymph node of a single Hashimoto's thyroiditis patient with a high anti-Tg titer. These were selected with purified human Tg, and 10 IgG kappa and 9 IgG lambda clones were analyzed further. Sequence analysis of the clones showed a very highly restricted heavy chain usage and a less restricted light chain usage. There was a variable degree of divergence from germ-line sequence in the light chain sequences, with a clear relationship between relatively higher affinity of the Fab for human Tg and an increased degree of somatic hypermutation. The Tg-selected Fab did not bind to Tg from other species, to reduced denatured Tg, or to thyroid peroxidase. The Fab inhibited patient serum binding to human Tg by between 39 and 79%. In summary, we have isolated 19 high affinity, human Tg-specific Fab and shown that the relative affinity of the Fab is directly related to the pattern of somatic hypermutation.

In continuation of our efforts to elucidate the immunoglobulin kappa locus of the mouse we analyzed 46 yeast artificial chromosomes (YACs) containing V kappa, J kappa and C kappa genes. The YACs, which were derived from DNA of C57BL/6 and C3H mice, ranged from 0.3-1.9 Mb in size. On the basis of hybridization with probes specific for the V kappa gene families a group of 13 YACs was selected for detailed analysis. The V kappa genes of the YACs were then characterized by hybridization to the family-specific probes and by the sizes of the EcoRI fragments on which they were found. This way evidence was obtained for 140 different V kappa gene signals on the YACs. Of these 63 had been characterized before on clones from a cosmid library of total mouse DNA (I. Zocher et al., Eur. J. Immunol. 1995. 25: 3326-3331) and 22 others were found now on cosmid clones derived from the YACs. Six V kappa genes of the previous study which were not found on the YACs are probably located outside of the kappa locus. The YACs were arrayed in a unique order establishing a YACs panel which most likely contains the whole kappa locus. The cosmid contigs and solitary cosmid clones which contain the 63 plus 22 V kappa gene signals mentioned above comprise about 2.0 Mb. Assuming that the remaining 55 V kappa genes are spaced at the same average distance of 24 kb, one may extrapolate to a locus size of 3.3 Mb.

Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.

In a woman suffering from IgE myeloma, hay fever and polyvalent respiratory and skin allergy the IgE monoclonal protein VL was isolated and investigated with respect to structural and functional properties. The amino acid sequence of 22 isolated peptides--especially of the biologically significant C2-C3 part--corresponded with that originally described by Bennich et al. (Immunol Rev 1978;41:3-23; Prog Immunol 1974;13:49-58). However, in mass spectrometry the sugar residues on ASN 99 (219) and 252 (371) were deficient in sialic acids. The native IgE VL protein precipitated with high intensity all mannose-specific lectins as concanavalin A (Con A) and was able to release histamine after triggering by these lectins. The same lectins also elicited more histamine release and more positive skin reactions in atopic than in healthy persons. In sera from atopic patients the binding of IgE on Con A Sepharose 4B column was stronger than in normal persons. It is suggested that changes in the IgE glycosylation state may contribute to IgE-mediated pictures of clinical allergy by the nonimmunological pathway.

Cardiomyopathy due to monoclonal light chain deposits is a complication of plasma cell disorders. The deposits may be either fibrillar as in light chain amyloid or nonfibrillar as in light chain deposition disease. The reasons for these structural differences are still unknown. We characterized the myocardial deposits by immunohistochemical examination of sections and extraction and biochemical analysis of the tissue deposits in a patient (MCM) who died of myeloma and systemic light chain deposition disease. Amino acid sequence analysis of the extracted nonfibrillar MCM kappa-light chain reveals that it belongs to the L12a germline subset of the kappa(I) protein and contains five distinctive amino acid substitutions (three in the framework region III and two in the complementarity-determining region III) that have not been reported previously in the same positions in other kappa(I) light chains. The theoretically determined isoelectric point (pI 8.21) of the MCM light chain is high compared with the low isoelectric point of other Bence Jones proteins from subjects without light chain deposition disease. The diffuse binding to basement membranes and the high isoelectric point of the MCM kappa-light chain suggest electrostatic interaction as a possible mechanism of tissue deposition. The spatial locations of the five distinctive residues and a sixth rare substitution of the MCM protein modeled on the backbone structure of REI, a kappa(I)-soluble Bence Jones light chain of known three-dimensional structure, may be responsible for protein destabilization, partial unfolding, and aggregation leading to tissue deposition.

In our previous experimental work we suggested that the frequent nephritogenicity of type II cryoglobulins could depend on a particular affinity of the immunoglobulin (Ig) M kappa rheumatoid factor (RF) component for mesangial matrix. Since cellular fibronectin (cFN) in the human kidney is mainly represented in glomerular mesangium, we studied the binding capacity to cFN of IgM kappa RFs from type II cryoglobulins compared with other different monoclonal and polyclonal IgM and IgM RFs. We purified 13 IGM kappa from human IgM kappa/IgG cryoglobulins, eight monoclonal IgM from patients with Waldenström's macroglobulinemia, nine polyclonal IgM from normal donors, and eight polyclonal IgM RFs from patients with rheumatoid arthritis. Purified IgM were used at the same concentration in enzyme-linked immunosorbent assay (ELISA) on cFN-coated plates. All the cryoglobulin IgM showed high specific binding to cFN while IgM from Waldenström's macroglobulinemia, normal IgM, and polyclonal IgM RFs had low or absent binding. These data were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cFN followed by Western blot analysis with purified IgM. The IgM kappa binding to cFN persisted using IgM kappa monomers, and was inhibited by cFN but not by plasma FN in a specific inhibition test. Further enzyme-linked immunosorbent assay studies showed that cryoglobulin IgM kappa RFs are still able to bind IgG in a dose-dependent manner once linked to solid-phase cFN. The data suggest that the affinity of cryoglobulin IgM kappa RFs for immobilized cFN could be involved in the particular high nephritogenicity of type II cryoglobulins and might lead to in situ immune complex formation.

The immunoglobulin kappa chain gene of human lymphoma cell line HF-1.3.4 was partially sequenced from the 3' end of the leader exon 2.0 kb downstream. The sequenced stretch of DNA included 1.5 kb of the non-coding JK region 3' to the JK2 element of the mature gene. Among the known VK germline genes the closet relative was KV328, which was 91% homologous to HF-1.3.4. In the 1.5 kb JK region homology with JK allele of Whitehurst et al. (allele 2) was 89%, with the allele of Hieter et al. (allele 1) 87%. The vast majority of the differences located in the leader intron, the VJ exon or 0.6 kb 3' to the exon, a localization characteristic of somatic hypermutations of immunoglobulin genes. Another indication that most of the differences observed were due to somatic hypermutations is that the 153 bp stretch of the kappa constant gene (CK) sequenced from the mRNA was 100% homologous with the published CK sequence. The most differences between the JK region sequence and that of Whitehurst et al. probably represent somatic mutations: 43% were transversions, 55% transitions and 2% deletions. In the non-coding JK region transversions of C.G to G.C rather than to A.T were heavily over-represented. This is possibly a feature of B-cell hypermutations in humans and mice.