AL Lambda Amyloidosis Activates Acute Liver Failure in the Absence of Plasma Cell Dyscrasia.

A patient with systemic amyloidosis developed portal hypertension, acute liver failure and multiorgan dysfunction. Extensive testing was unrevealing for paraproteinemia, plasma cell dyscrasia, infectious, or inflammatory conditions. He was transferred to our institution for orthotopic liver transplant evaluation but was ultimately declined given clinical instability and dysautonomia. Post-mortem evaluation revealed extensive amyloid deposition in multiple organs determined to be AL-lambda amyloidosis.

Immunoglobulin-binding protein-based affinity chromatography in bispecific antibody purification: Functions beyond product capture.

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.

Mass spectrometry analyses of κ and λ fractions result in increased number of complementarity-determining region identifications.

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.

A novel approach for the purification and proteomic analysis of pathogenic immunoglobulin free light chains from serum.

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.

Clinical comparison of new monoclonal antibody-based nephelometric assays for free light chain kappa and lambda to polyclonal antibody-based assays and immunofixation electrophoresis.

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.

Chromogenic in situ hybridization for the detection of lambda and kappa immunoglobulin light chains as a potential auxiliary diagnostic technique in canine plasmacytomas.

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.

Analytical validation of new ELISAs for the quantitation of polyclonal free light chains and comparison to existing assays for healthy and patient samples.

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.

Serum free-light chain removal by high cutoff hemodialysis: optimizing removal and supportive care.

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.

Comparison of immunohistochemistry and immunofluorescence techniques using anti-lambda light chain antibodies for identification of immune complex deposits in canine renal biopsies.

Comprehensive renal biopsy evaluation of canine glomerular disease uses immunofluorescence (IF) labeling of fresh frozen tissue to detect immune complexes that are confirmed with transmission electron microscopy. This methodology requires the veterinarian to harvest additional tissue samples, whereas sections for immunohistochemistry (IHC) could be performed on paraffin sections. If adequate IHC labeling of formalin-fixed, paraffin-embedded tissue was possible, the additional tissue samples would be unnecessary. We compared the specificity and sensitivity of IHC to IF for diagnosis of immune complex-mediated glomerulonephritis (ICGN). Commercial anti-canine IHC and IF antibodies targeting the lambda light chain component of immunoglobulins were evaluated, using previously diagnosed cases of ICGN and cases without immune complexes (non-ICGN). Because the pattern of IF labeling is crucial for accurate interpretation, sections were evaluated by a trained nephropathologist and a novice to assess the impact of experience in the diagnosis of ICGN. Unfortunately, our attempts to develop an IHC protocol that could improve the workflow for clinicians and laboratory personnel were unsuccessful; the IHC protocol did not demonstrate staining patterns that could be detected reliably by either evaluator. Moreover, the IHC antibody demonstrated abundant nonspecific staining in non-ICGN cases, and 60% of true ICGN cases were misdiagnosed as non-ICGN. We did not achieve a reliable IHC protocol for the anti-lambda light chain antibody and, therefore, IF for lambda light chain remains the method of choice for ICGN detection.

Stabilizing an amyloidogenic λ6 light chain variable domain.

Light chain amyloidosis is a lethal disease where vital organs are damaged by the fibrillar aggregation of monoclonal light chains. λ6a is an immunoglobulin light chain encoded by the germ-line gene segment implicated in this disease. AR is a patient-derived germ-line variant with a markedly low thermodynamic stability and prone to form fibrils in vitro in less than an hour. Here, we sought to stabilize this domain by mutating some residues back to the germ-line sequence, and the most stabilizing mutations were the single-mutant AR-F21I and the double-mutant AR-F21/IV104L, both located in the hydrophobic core. While mutation Arg25Gly in 6aJL2 destabilized the domain, mutating Gly25 back to arginine in AR did not contribute to stabilization as expected. Crystallographic structures of AR and 6a-R25G were generated to explain this discrepancy. Finally, 6a-R25G crystals revealed an octameric assembly which was emulated into 6aJL2 and AR crystals by replicating their structural parameters and suggesting a common assembly pattern. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession numbers 5IR3 and 5C9K.

A novel approach for the chromatographic purification and peptide mass fingerprinting of urinary free light chains.

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.

Angioimmunoblastic lymphadenopathy with paraproteinemia. Analysis of the clinical and biological characteristics and their prognostic significance.

We report a case of a double M component appearing in the course of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD). Lymphocyte function, especially the decrease in T-suppressor cells, could play an essential role in the mechanisms of the disease. In the presence of an M component, the prognosis for AILD seems to be more pessimistic. Arguments for considering AILD as a premalignant disease are reviewed.

Molecular characterization of the human immunoglobulin V lambda I germline gene repertoire.

To advance our understanding of the human immunoglobulin V lambda germline gene contribution to normal as well as autoimmune responses, we have isolated and sequenced six germline genes of the V lambda I subgroup. These genes can be divided into three sub-subgroups on the basis of greater than or equal to 93% nucleotide sequence homology and greater than or equal to 88% deduced amino acid sequence similarity. Examination of all cDNA and protein sequences available for expressed V lambda I genes supports the assignment of these three sub-subgroups. Sequence comparisons also suggest that germline gene members of two of these sub-subgroups, I-a and I-b, are preferentially utilized in the expressed V lambda I repertoire. This finding may be at least partially attributable to regulatory sequence abnormalities apparent in two of the other V lambda I germline genes (Humlv101 and Humlv104) which may interfere with their expression.

The second riboflavin-binding myeloma IgG lambdaDOT. I. Biochemical and functional characterization.

A human monoclonal immunoglobulin, IgGDOT, with flavin-binding capacity has been obtained from an elderly woman with multiple myeloma who developed yellow skin and yellow hair. The case presented a remarkable similarity with that previously reported by Farhangi and Osserman [N. Engl. J. Med. 294, 177-183 (1976)]. Purified IgGDOT was bright yellow and the ligand was identified as riboflavin and its oxidation products by thin layer chromatography, proton nuclear magnetic resonance and mass spectroscopy. Competitive binding studies with different haptens demonstrated highest affinity for riboflavin, followed by flavin mononucleotide and flavin adenine dinucleotide; no significant binding was detected for several other non-flavin compounds tested. The hapten was associated with the protein in vivo, as well as with the purified antibody. Removal of the already bound riboflavin from the combining site was associated with irreversible denaturation of the monoclonal protein. By the fluorescence quenching technique it was determined that there were 0.68 available combining sites for riboflavin molecule in IgGDOT with a binding constant of 8.5 x 10(8)/M, while FabDOT presented 0.27 available combining sites with a binding constant of 5.1 x 10(8)/M. The fact that 1.2 and 0.81 mol riboflavin/mol protein were already bound to IgGDOT and FabDOT, respectively, is consistent with the usual hapten/antibody stoichiometry. The heavy chain subclass of IgGDOT was identified as gamma 2, as in the previously reported case of riboflavin-binding protein IgGGAR. However, the lambda chain subclass was different and no idiotype cross-reactivity was found.

Molecular cloning and characterization of human thyroid peroxidase autoantibodies of lambda light chain type.

IgG class thyroid peroxidase (TPO) autoantibodies with kappa light (L) chains predominate in serum and the genes for a large repertoire of such autoantibodies have been characterized. The present study was performed to clone and characterize TPO autoantibodies with lambda L chains which comprise approximately 20% of serum TPO autoantibodies. From a combinatorial IgG H/lambda L chain cDNA library in the phage display vector pComb3, 24 TPO-binding clones with lambda L chains were isolated, comprising three different heavy (H) and light (L) chain combinations. These combinations utilized two genes from the Vlambda II and IIIb families (closest germline genes DPL11 and hsigg11150) and three genes from the VH1, VH3 and VH4 families (VH26, 4.34 and hv1L1). The deduced amino acid sequences of these H chains were quite different from those of kappa F(ab) isolated using the same H chain library. We expressed the proteins for these three lambda F(ab), as well as for a lambda F(ab) (Humlv318 L chain/DP10-like H chain) previously isolated from another patient. The affinities for TPO of the lambda F(ab) (Kd 8 x 10(-10) M to 10(-7) M) were lower than those of the kappa F(ab) (Kd approximately 10(-10) M). For two lambda F(ab), both H and L chain genes were close to germline configuration, but there was no straightforward relationship between the extent of somatic mutation from germline configuration and affinity for TPO. All four lambda F(ab) bound less well to denatured TPO as to native TPO. The three F(ab) for which sufficient protein could be expressed for competition studies all recognized domain B within the immunodominant region on TPO previously identified using F(ab) with kappa L chains. Aside from these TPO-specific F(ab), only a few other human IgG class, organ-specific autoantibodies with lambda L chains have been characterized at the molecular level. Our study significantly augments the small database on this category of autoantibodies in general.

Rapid molecular weight estimation and separation of selected immunoglobulin chains by high speed gel filtration.

Rapid and reliable molecular weight estimations of reduced and alkylated immunoglobulin heavy or light chains were performed by high speed gel filtration in 6 M guanidinium chloride using a short (30 cm X 7.5 mm) TSK 3000 SW type column. Molecular weight estimations based on Kav values of eluted polypeptides and glycopolypeptides were generally unaffected by protein bound carbohydrate. Rapid separation of immunoglobulin H and L chains was also achieved during high speed gel filtration.

The use of cellulose carbonate-based immunoadsorbents in the isolation of minor allotypic components of rabbit immunoglobulin populations.

Cellulose trans-2,3-carbonate has been used as a new insoluble matrix for the simple coupling of a1- and b4-positive rabbit immunoglobulin to make immunoadsorbents capable of purifying from serum, with great efficiency, alloantibodies to these allotypic determinants. The antibodies have themselves been conjugated to prepare specific antibody immunoadsorbents of high binding activity for their allotypic target molecules. With these anti-allotypic solid-phase reagents it has been possible to affinity purify a1- and b4-positive immunoglobulin molecules and to deplete serum immunoglobulin of these molecules to leave in the eluates only the allotypically uncontaminated minor immunoglobulin components which are a-negative or b-negative (lambda chain-bearing) molecules. lambda chain molecules were also purified in very small quantities by affinity chromatography on a sheep anti-rabbit lambda chain column. This method of purifying minor populations of rabbit immunoglobulin from normal serum by special immunoadsorbent applications offers new opportunities to study the products of rarely expressed immunoglobulin genes in normal rabbits.

Non-IgM monoclonal gammopathy in patients with Sjögren's syndrome.

Two Japanese patients with Sjögren's syndrome with non-immunoglobulin M(IgM) class monoclonal gammopathy are described. The monoclonal IgA lambda detected in the serum and saliva was confirmed to possess rheumatoid factor activity in the first patient with a hypergammaglobulinemic purpura and hyperviscosity syndrome. Idiotype specificity was present on the surface membrane of peripheral blood lymphocytes as well as in the cytoplasm of infiltrating cells in the salivary glands. Common idiotypic specificity was found in four of 60 other patients who had rheumatoid factors. In the serum and saliva of the other patient, a monoclonal immunoglobulin G, kappa type (IgG kappa), was detected. Kappa type IgG was found in most of the infiltrating cells in the salivary glands and also in the saline extract from a resected submandibular gland. Our findings indicate that non-IgM class monoclonal gammopathy is also one of the complications of Sjögren's syndrome.

Isolation and characterization of a monoclonal human thyroid peroxidase autoantibody of lambda light chain type.

Thyroid peroxidase (TPO) autoantibodies, a hallmark of human autoimmune thyroid disease, may have kappa or lambda light chains. Monoclonal human TPO autoantibodies with kappa light chains have previously been developed by cloning and expressing "combinatorial" libraries of immunoglobulin genes in bacteria. In the present study, an IgG1/lambda combinatorial library was generated from thyroid cDNA of a Graves' patient whose serum contained lambda TPO antibodies. Screening the bacteriophage library with 125I-TPO yielded one clone, TR1.41. The oligonucleotide sequence of TR1.41 was determined and the nature of its interaction with TPO was investigated. The affinity of TR1.41 for TPO is high (Kd approximately 10(-9) M), comparable to that of monoclonal kappa TPO autoantibodies derived from the same patient. The genes encoding the heavy and light chains of TR1.41 differ in a number of respects from the closest available germline genes. Such differences are consistent with somatic mutation in a high-affinity antibody. An important characteristic of TR1.41 is its interaction with the immunodominant domain on TPO recognized by approximately 80% of serum TPO autoantibodies. The frequency of TPO-specific F(ab) generated from the thyroid gland of patient TR was much lower for F(ab) with lambda light chains (1:150,000) than for F(ab) with kappa light chains (1:13,000). Despite this low frequency, the high affinity of TR1.41 and its recognition of the immunodominant region on TPO indicate that lambda autoantibodies of this type may represent an important constituent of the TPO autoantibody response in man. In conclusion, this is the first report on the molecular cloning and characterization of a thyroid autoantibody of lambda L chain type by the combinatorial library approach.