Polyclonal free light chain of Ig may interfere with interpretation of monoclonal free light chain κ/λ ratio.

There is controversy about whether a sensitive assay for the serum Ig free light chain (FLC) κ/λ ratio can replace urine immunofixation electrophoresis (UIFE). This report describes two untreated patients in whom monoclonal FLCs were identified in urine despite normal serum FLC κ/λ ratios. Unlike the classical serum electrophoretic patterns in multiple myeloma, both serum samples showed adequate amounts of polyclonal Ig. The most likely explanation is a masking effect by polyclonal FLC on the serum κ/λ ratio when sufficient concentrations of polyclonal FLC exist. These cases illustrate this likely effect and attest to the continued importance of UIFE for initial screening of patients for Bence-Jones protein.

Phosphoester hydrolysis by cerium(IV)-thiacalix[4]arene complexes and its application to immunoassay.

Thiacalix[4]arenetetrasulfonate was treated with Ce(IV) in water at pH 9.5 to give novel phosphoester-hydrolyzing complexes. The dinuclear Ce(IV) complex promoted the hydrolysis of p-nitrophenyl phosphate with a turnover frequency of 6.8 h(-1) at 50 degrees C, showing fourfold higher activity than the mononuclear complex. The dinuclear complex was readily immobilized onto an antibody by simply mixing them in water, hence its phosphatase-like activity was applied to the color-developing reaction in immunoassay. The model assay using an antibody labeled with the dinuclear complex allowed the detection of as little as 10 ng mL(-1) of a tumor marker, Bence-Jones protein, in a 96-well microtiter plate format. Analysis of urine for Bence-Jones protein was performed by the proposed method.

Immunodiagnostic capabilities of anti-free immunoglobulin light chain monoclonal antibodies.

Overproduction of plasma cell-derived monoclonal free kappa or lambda immunoglobulin light chains (FLCs) is a hallmark of multiple myeloma, AL amyloidosis, and light chain deposition disease. Because these components serve as unique cellular and serologic biomarkers, their detection and quantitation has diagnostic, therapeutic, and prognostic import. In this regard, we have developed monoclonal antibodies (mAbs) that specifically recognize the kappa or lambda FLC products of all known human variable and constant region light chain genes. We now report the results of our studies that have demonstrated the capability of these reagents to measure, in a modified fluid-phase capture enzyme-linked immunosorbent assay (ELISA), serum kappa and lambda FLCs at concentrations as low as 5 and 15 ng/mL, respectively. The mAb-based ELISA has greater sensitivity and reproducibility than does the commercially available immunoturbidimetric assay that uses polyclonal anti-FLC antibodies. In addition, the mAbs can immunostain monoclonal FLC-producing plasma cells and pathologic light chain-related amyloid and nonfibrillar tissue deposits. Our anti-FLC mAbs, with their high degree of reactivity and versatility, may provide an invaluable tool in the diagnosis and management of light chain-associated disease.

Deposition-associated diseases related with a monoclonal compound.

Up to 3% of adults over 50 years of age show a monoclonal peak values in blood or urine. Findings and prognosis will be distinct in view of the nature of this factor. In B-cell neoplasias (multiple myeloma, Waldeström macroglobulinaemia, chronic myeloid leukaemia and non-Hodgkin lymphoma) the clinical pattern is dominated by the systemic effects produced by the expansion of the malign clone; the monoclonal protein may result in hyperviscosity syndrome or renal damage. On the other hand, there are other less frequent processes called diseases associated to monoclonal components, where the main clinical manifestations and prognosis depend of the biological effects of the monoclonal protein. With reference to this last group, which is the objective of this revision, no bone lesions, anaemia or a greater tendency to infections usually occur when compared with the first group. Even so, there are some cases of interposition between both groups: for instance, type IgM immunoglobulin present in Waldeström macroglobulinaemia may have cold agglutinin activity, and in the case of multiple myeloma, the clone may secrete amyloidogenic light chains.

Deposition-associated diseases related with a monoclonal compound

Up to 3% of adults over 50 years of age show a monoclonal peak values in blood or urine. Findings and prognosis will be distinct in view of the nature of this factor. In B-cell neoplasias (multiple myeloma, Waldeström macroglobulinaemia, chronic myeloid leukaemia and non-Hodgkin lymphoma) the clinical pattern is dominated by the systemic effects produced by the expansion of the malign clone; the monoclonal protein may result in hyperviscosity syndrome or renal damage. On the other hand, there are other less frequent processes called diseases associated to monoclonal components, where the main clinical manifestations and prognosis depend of the biological effects of the monoclonal protein. With reference to this last group, which is the objective of this revision, no bone lesions, anaemia or a greater tendency to infections usually occur when compared with the first group. Even so, there are some cases of interposition between both groups: for instance, type IgM immunoglobulin present in Waldeström macroglobulinaemia may have cold agglutinin activity, and in the case of multiple myeloma, the clone may secrete amyloidogenic light chains (AU)

Phage display and peptide mapping of an immunoglobulin light chain fibril-related conformational epitope.

Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human kappa4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O'Nuallain, B., et al. (2006) Biochemistry 46, 1240-1247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vkappa and Vlambda N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody's cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1-4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both kappa and lambda light chain fibrils. We posit that the associated binding site involves a rare type VI beta-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis.

Pathogenicity of catalytic antibodies: catalytic activity of Bence Jones proteins from myeloma patients with renal impairment can elicit cytotoxic effects.

Some Bence Jones proteins (BJPs) can display catalytic activity. Although the catalytic activity of BJPs might be associated with the pathogenesis of disease, this relationship has not yet been established. We tested the effects of seven BJPs on LLC-PK1 cells to assess their pathogenicity. Two out of the seven BJPs showed cytotoxic activity, as assessed by microscopic analysis, the WST method and TUNEL staining. Moreover, the cytotoxic BJPs were excreted by patients who presented with renal impairment. The cytotoxic BJPs displayed 20- to 40-fold higher catalytic activities (kcat of 3.5-2.2 min(-1)) in hydrolyzing a chromogenic substrate compared to the other BJPs. By treating the cytotoxic BJPs with diisopropylfluorophosphate, they lost not only their catalytic activity, but also the cytotoxic effects. These results indicate a direct link between cytotoxicity and the catalytic activity of the BJPs. The catalytic activity of BJPs contributes to the pathogenesis, as well as to development, of symptoms of multiple myeloma. Inhibition of the catalytic activity of BJPs may form the basis of a novel treatment for multiple myeloma patients with renal dysfunction.

[Lectin-enzyme assay as a method of estimation of immunoglobulins' glycosylation].

The mechanism of interaction of lectins with IgG molecules by the method of the lectin-enzyme assay has been described that allows to register a degree of human serum IgG molecules' glycosylation (mannosylation in case of lectin of Pisum sativum) in norm and at pathology. To detect an authentic difference in a glycosylation degree between control and pathological IgG, the wells of an ELISA plate were coated with an antibody in concentration of 1 microg/ml. Introducing alpha-D-mannose between the stages of incubation of immunoglobulin and lectin showed, that alpha-D-mannose inhibits the affinity of lectins for IgG. The preliminary incubation of lectin with IgG molecules stabilizes the activity of horseradish peroxidase, which labeled the lectins. Lectin-enzyme assay, in which Fab and Fc fragments of IgG were used, showed that lectin of Pisum sativum possesses a higher affinity for Fab regions. These findings and the glycosylation analysis of paraproteins and Bence-Jones proteins of multiple myeloma patients help to understand the details of interaction of immunoglobulins and lectins.

A new concept for detection of Bence Jones proteinuria in patients with monoclonal gammopathy.

Bence Jones (free light chain-FLC) proteinuria is usually detected by immunofixation electrophoresis (IFE) of urine. With a new, automated immunoassay, it is now possible to quantify FLC in serum and urine. In the present study, we compared different routine methods for monoclonal FLC screening. From our results we conclude that the measurement of FLC in serum is highly sensitive and should replace urine tests. Additionally, possible kidney dysfunction, caused by nephrotoxicity of FLC, should be assessed by urine protein analysis and cystatin C measurements.

Does catalytic activity of Bence-Jones proteins contribute to the pathogenesis of multiple myeloma?

Some Bence-Jones proteins have been found to be capable of hydrolyzing DNA, chromogenic amide substrates, such as benzoylarginine p-nitroanilide, and natural oligopeptides, such as arginine vasopressin. Patients who excrete Bence-Jones protein with the DNA-nicking activity have shown moderately severe symptoms. When incubated with LLC-PK1 (porcine kidney proximal tubule) cells, some Bence Jones proteins penetrated the cytoplasm, and entered the nucleus with little or no degradation of epitopes. Intranuclear Bence Jones proteins ultimately induced DNA fragmentation in situ and cell death. This cytocidal activity was not directly associated with the DNA-nicking activity, since Bence Jones proteins with no detectable DNase activity also produced cell death. These results, however, suggest that the biological activities of Bence Jones proteins described here makes a significant contribution to the development and/or deterioration of multiple myeloma.

Constant region of a kappa III immunoglobulin light chain as a major AL-amyloid protein.

AL-amyloidoses are generally described as a group of disorders in which N-terminal fragments of monoclonal immunoglobulin light chains are transferred into amyloid fibrils. We have, by amino acid sequence analyses and immunological methods, characterized the Bence-Jones protein and the corresponding AL protein as a kappa III immunoglobulin light chain from material of a patient with systemic AL-amyloidosis presenting as a local inguinal tumour. The two proteins showed some unique features. The major part of the AL amyloid fibril protein consisted of C-terminal fragments of the Bence-Jones protein. Furthermore, both the Bence-Jones protein and the AL protein were glycosylated, with possibly a glycosylation in the constant part of the light chain.

Characterization of a light chain product of the human JC lambda 7 gene complex.

The human light chain JC lambda locus is comprised of seven distinct segments, designated JC lambda 1, JC lambda 2, JC lambda 3, JC lambda 4, JC lambda 5, JC lambda 6, and JC lambda 7. Whereas three of these seven represent pseudogenes (psi Clambda 4, psi C lambda 5, and psi C lambda 6), the JC lambda 1, JC lambda 2, and JC lambda 3 complexes are functional, as demonstrated by the finding of their protein products through sequence analyses of lambda-type Bence Jones proteins and light chains derived from monoclonal Igs. Although the JC lambda 7 segment also appears functional, as evidenced through analysis of lymphocyte-derived mRNA, heretofore no monoclonal JC lambda 7-containing lambda-chains have been identified. Serologically, two distinct isotypic markers, Mcg and Oz, are associated, respectively, with JC lambda 1 and JC lambda 3 proteins, in contrast to JC lambda2 components, which do not express these determinants and represent a third isotype. Although another serologic marker, Ke (Kern), considered a fourth isotype, has been assigned to the JC lambda 7 complex, this relationship has been questioned. We now report the primary structural features of a lambda-type Bence Jones protein that include the four distinctive residues encoded by the JC lambda 7 gene segment. This protein, obtained from a patient with multiple myeloma and designated MCP, represents the first example of such a molecule and provides definitive evidence that the JC lambda 7 gene complex is functional. Additionally, comparison of the C lambda sequences of Mcg-/Oz- Bence Jones proteins MCP and KERN supports the contention that the Ke-associated one-residue amino acid variation at position 152 reflects a C lambda A2 polymorphism and that yet another isotypic marker, provisionally designated Mcp, is encoded by the JC lambda 7 gene segment. Thus, we posit that there are four human JC lambda isotypes, Mcg, Ke-Oz-/Ke+Oz-, Ke-Oz+, and Mcp, that represent, respectively, products of the JC lambda 1, JC lambda 2, JC lambda 3, and JC lambda 7 gene complexes.

Serologic crossreactions among primate immunoglobulins.

We have generated and characterized 50 murine monoclonal antibodies (mAb) specific for baboon IgG. We examined crossreactivity of these mAb to baboon IgM and immunoglobulin (Ig) of various other primates including human, chimpanzee, rhesus monkey, cynomolgus monkey, and African green monkey. Those mAB that crossreacted with human IgG were further examined using myeloma proteins for specificity to human Ig subclasses. One mAB crossreacted with all four human IgG subclasses and with human IgM. We further analyzed this reactivity utilizing Bence Jones proteins representative of various light (L) chain germline gene family products. This mAB reacted with Bence Jones proteins indicating the recognition of a kappa (k) L chain specificity associated with the kappa I, kappa III, and kappa IV subgroups, but not with kappa II. Based on the differences between kappa II germ line gene encoded L chains and the other kappa L chain subgroups, we ascribe this reactivity to six amino acids that define a discontinuous epitope.

Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light-chain amyloid proteins.

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient-derived proteins. This well-defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.

Chemical and serologic characterization of human lambda VIII light chains.

The primary structural features and serologic properties of a newly recognized human lambda light (L) chain V region subgroup (V lambda VIII) were elucidated through study of two monoclonal L chains, Bence Jones proteins HAG and BIV. The V region amino acid sequences of these components were highly homologous to each other and to that deduced from the prototypic V lambda VIII cDNA, Humla8f10, which encodes the L chains of the IgM lambda rheumatoid factor HAF10. Proteins HAG and BIV could be classified as members of the V lambda VIII subgroup and distinguished from L chains of the V lambda I, V lambda II, V lambda III, V lambda IV, and V lambda VI subgroups on the basis of amino acid sequence. In addition to distinctive residues found within the V lambda gene-encoded portion of the molecules, L chains HAG, BIV, and HAF10 contained remarkably different second complementarity-determining regions (CDR2) that consisted of 11 residues, rather than the seven typically found among members of the other five V lambda subgroups. This elongated structure would presumably impart to the ligand-binding site of lambda VIII molecules a markedly different canonical structure compared with those of lambda I, lambda II, lambda III, lambda IV, and lambda VI L chains. By using Bence Jones protein HAG as an immunogen, we obtained polyclonal and monoclonal anti-V lambda VIII subgroup-specific Abs that were used to identify and quantify lambda VIII-related molecules in normal and pathologic states. Among the Ig lambda components present in the serum of normal individuals, approximately 3% had lambda VIII L chains, a frequency comparable to that found among monoclonal Ig lambda proteins or surface(s) Ig lambda+ cells obtained from patients with malignant plasma cell- or B cell-related disorders, respectively. In contrast, lambda VIII L chains were detected on approximately 19% of monoclonal IgM lambda rheumatoid factors produced by B cell lines established from PBLs or synovial cells from patients with rheumatoid arthritis. The results of our studies provide new information on the structural and immunochemical features of lambda VIII L chains and the possible functional importance of the human V lambda VIII subgroup.

Cross-reactions of anti-immunoglobulin sera with synthetic T-cell receptor beta peptides: mapping on a 3-dimension model.

The derived amino acid sequences of human T-cell receptor beta chain shows significant homology to lambda light chains of immunoglobulins in its variable, joining, and constant regions. We assessed the cross-reactivity between Tcr beta chains and immunoglobulin light chains by determining the capacity of rabbit antisera to human or murine immunoglobulins to react to a synthesized set of nested, overlapping 16-mer peptides corresponding to the VDJC sequence of the Tcr beta chain YT35. The observed reactivities were consistent with homologies to lambda and kappa light chains, the strongest reactivity being with a peptide that corresponds to the "switch peptide" of light chains, as assessed by ELISA binding and competitive inhibitions assays. Other regions reactive with anti-light chain sera corresponded to CDR1 and Fr3 segments of the variable domain and a segment of the constant region predicted to loop out of the tight globular structure. The peptide immunochemical results, together with the identification of specific regions of sequence correspondence between Tcr beta and the characterized lambda light chain Mcg, allowed us to develop a 3-dimensional model of the beta chain consistent with its role in antigen recognition.

Novel immunization protocol and ELISA screening methods used to obtain and characterize monoclonal antibodies specific for human light chain variable-region subgroups.

We have developed a novel immunization protocol for the production of a panel of high-affinity murine monoclonal antibodies (MoAbs) that are specific for each of the major human kappa and lambda light chain variable-region (VL) subgroups. Mice were injected with heat-precipitated human Bence Jones proteins or VL-related fragments emulsified in monophosphoryl lipid A (MPL) and trehalose dimycolate (TDM) at two- to four-week intervals over a seven-month period. A unique direct capturing enzyme-linked immunosorbent assay (ELISA) employing biotinylated monoclonal light chains was designed to select optimally immunized animals for hybridoma preparation and to screen culture supernatants for high-affinity anti-VL MoAbs. These methods have led to the generation of MoAbs that by ELISA react specifically with each of the four V kappa subgroups--V kappa I, V kappa II, V kappa III, and V kappa IV or five V lambda subgroups--V lambda I, V lambda II/V, V lambda III, V lambda IV, and V lambda VI. These reagents have been used successfully to establish, on the basis of VL subgroup, the monoclonal nature of serum or urinary immunoglobulins as well as those found in the cytoplasm or on the cell surface of monoclonal plasma cell or B-lymphocyte populations, respectively. The availability of anti-VL subgroup-specific MoAbs will facilitate the immunodiagnosis and study of patients with multiple myeloma, AL amyloidosis, and related B-cell proliferative disorders.