Sequence of the full-length immunoglobulin kappa-chain of mouse myeloma MPC 11.

MPC 11 mouse myeloma cells synthesize two immunoglobulin kappa light chains, coded by two separate genes. One of these Kappa-chains has no variable region and is degraded intracellularly. The other is a full-length kappa-chain contaning both variable and constant regions: this chain is secreted, both by itself and combined with heavy chains in molecules of immunoglobulin G. This paper reports the amino acid sequence of the myeloma MPC 11 full-length kappa-chain. The chain is unusual in having 12 extra residues at its N-terminus when its sequence is aligned with those of other mouse kappa-chains; no other anomalies were found in its sequence.     

Novel human light chain V kappa segment: serologic and structural analyses of the kappa III-like Bence Jones protein and IgG kappa light chain REE

Immunochemical and sequence analyses of kappa light chain REE (Bence Jones protein REE and the light chain isolated from IgG kappa myeloma protein REE) revealed antigenic and structural features not previously described for human kappa-chains. Although closely related to proteins of the V kappa III subgroup, light chain REE is readily distinguished from light chains classified serologically as members of the kappa IIIa or kappa IIIb sub-subgroups. Light chains REE (Bence Jones protein REE and light chain REE) are identical in sequence and differ from kappa III proteins by at least 10 uncommon amino acid substitutions in the first three framework regions. Further, kappa-chain REE is unique by virtue of a four-residue deletion in the third complementarity-determining region. The deletion encompasses the three carboxyl-terminal residues in the V kappa-encoded segment and the first residue at the site of V-J recombination. Urine specimens from patient REE also contained a light chain fragment that lacked the first (amino-terminal) 85 residues of the native light chain but otherwise was identical in sequence to the light chain REE. The extensive amino acid differences and unique length of the V kappa segment in light chain REE indicate that this kappa-chain is the product of an unusual V kappa III gene or, alternatively, represents a rarely expressed and novel human V kappa gene.     

The specificity of chain interactions among immunoglobulins. Combinations of γ chains with κ chains of the same subgroup as in the parent immunoglobulin G

1. The specificity of combination of heavy and light chains from selected human immunoglobulins was examined in the light of greater structural information than in previous studies. Heavy (gamma) chains from immunoglobulin G (kappa) myeloma proteins were allowed to combine with their homologous light (kappa) chains or with other kappa chains of the same variable-region subgroup. The affinity of each such pairing was assessed by having the test kappa chain compete with a standard population of normal light chains. 2. There was a spread of affinities among the heavy-light pairings with the homologous pairings having an average affinity significantly higher than the heterologous pairings. 3. It follows that (a) the preference shown for homologous heavy-light pairings is not explicable simply in terms of the known subdivisions of the variable and constant regions of the chains, and (b) it is unlikely that those residues specifying the subgroups of kappa-chain variable regions have a predominant role in the formation of interchain bonds with the gamma-chain variable regions.     

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase. Applications in immunoassays

The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP2.Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG1 and IgG2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase

Summary The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP₂·Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG₁ and IgG_2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin. This study demonstrates some of the potential advantages of using an epitope specific monoclonal bi-specific developing reagent like McC10 in an immunobased technique like ICC. Its potential use in a variety of other immunobased procedures is discussed.     

Immunoglobulin chain recombination among antidigoxin antibodies by hybridoma-hybridoma fusion

Conditions necessary for in vitro chain recombination of high affinity (10(9) to 10(12) M-1) antidigoxin monoclonal antibodies resulted in decreased affinity for both intact "native" and chain recombinant molecules. Chain recombination by somatic cell fusion was used instead to study the effects on antigen specificity and idiotypy of recombinants in which an homologous light (L) chain substituted for the parental L chain. The antidigoxin antibody 26-10 utilizes a VL sequence highly homologous to that of antibody 40-20, an antidigoxin antibody which uses a different VH gene than does 26-10 and lacks significant reactivity with an anti-26-10 idiotypic serum. The drug-marked antidigoxin cell line 26-10 (gamma 2a, kappa) and a drug-marked light chain producing variant of antidigoxin hybridoma 45-20 (lambda 1) which lacks both digoxin binding and idiotypy were fused. The fusion progeny (gamma 2a, kappa, lambda 1) which binds digoxin and is idiotype-positive, was selected for kappa loss (resulting in loss of digoxin and idiotype binding) and then fused with a heavy (H) chain loss variant of antidigoxin hybridoma 40-20 (kappa, digoxin nonbinding, idiotype negative). The resultant cell line CR-57 (gamma 2a, kappa, lambda) secretes antibodies which assemble the 26-10 H chain with both the 40-20 kappa-chain and the 45-20 lambda 1-chain. The affinity purified recombinant species consisting of 26-10 H chain and 40-20 kappa-chain expresses complete 26-10 idiotypic determinants. However, this recombinant antibody binds digoxin with decreased affinity and altered specificity relative to native 26-10. The binding specificity pattern nonetheless is most similar to the H chain donor. Amino acid and nucleotide sequence analyses of the respective light chains demonstrate six variable region differences between them, two of which are in complementarity-determining regions and the remainder in the framework. Hybridoma-hybridoma fusion provides an alternative to in vitro chain recombination for studying the contribution of chain combinational diversity to antibody diversity, antigen binding, and idiotypy.     

A conditional secretory mutant in an Ig L chain is caused by replacement of tyrosine/phenylalanine 87 with histidine.

The kappa-chain of the myeloma MOPC 21 is an unusual L chain, in that it is not secreted unless complexed with a H chain. This nonsecreted kappa-chain seems to be retained in the endoplasmic reticulum in association with the protein BiP/GRP78, both in myeloma cells and when expressed in COS-1 fibroblasts. By assaying the fate of the MOPC 21 kappa-chain and its mutated derivatives in COS-1 cells, we show that the cause of the nonsecreted phenotype is the presence of histidine in position 87 of the variable domain. When this amino acid is changed back to the tyrosine that usually occupies position 87, secretion of the unassembled kappa-chain is restored. As in B lymphoid cells, co-expression of gamma H chains in COS-1 cells complements the mutation in the L chain, and rescues secretion of the arrested kappa. Thus, the presence of histidine at position 87 creates a conditional L chain secretory mutant: it is not compatible with normal transport of free L chain, but can be rescued in the presence of H chain.     

Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.     

The primary structure of human gamma-glutamyl transpeptidase

A cDNA hybridizable to that of rat gamma-glutamyl transpeptidase (GGT) was cloned from a cDNA library of human fetal liver. The insert of the cDNA clone contained 1866 bp consisting of an open reading frame (ORF) of 1709 bp (569 amino acids (aa), N-terminal portion truncated) and a 135-bp 3'-untranslated region followed by a polyadenylated tail. In parallel, amino acid sequences of N-terminal portions of heavy and light chains of a purified human GGT were determined. Two stretches of amino acid sequences identical to the N-terminal sequences of heavy and light chains were found in the ORF. We therefore concluded that the clone is a cDNA for human GGT. From the amino acid sequence deduced from cDNA, the heavy and the light chains of the purified enzyme are estimated to be composed of 351 aa (Mr 38,336) and of 189 aa (Mr 20,000), respectively. The heavy chain is preceded by a signal peptide of at least 29 aa presumed to be cleaved by bromelain treatment. Six putative N-glycosylation sites are present in the heavy subunit region and one in the light subunit region. Primary structure and hydrophobicity profile are closely similar to those of rat GGT.     

A double monoclonal IgG1 kappa and IgG2 kappa in a single myeloma patient. Variation in clonal products and therapeutic responses

Two electrophoretically homogeneous immunoglobulins were detected in the serum of a patient with multiple myeloma. The heavy chains were of different classes (gamma 1 and gamma 2). The light chains of both were kappa, but had different electrophoretic mobilities on polyacrylamide gels. Amino acid sequence analysis, carbohydrate determinations, and biosynthetic experiments indicated that the difference seen in their electrophoretic mobility was due to the glycosylation of one but not the other kappa-chain. The primary structure of both chains demonstrated that they both used a V kappa 1 and a J kappa 2 gene segment and the same C kappa allele, Km(1,2), and that both contained the same junctional three amino acid deletion. However, they varied by 19 amino acids in the first 94 amino terminal residues encoded for by the V kappa gene, with some of the substitutions requiring two base changes in the appropriate codons. Moreover, the malignant "clones" producing the two proteins differed in their responses to chemotherapy. These data indicate that, although the two clones producing the serum proteins were different at the time of study, they may have arisen from the same precursor clone.     

Three M-components IgA lambda + IgG kappa n + IgG kappa h in one patient (DA): lack of shared idiotypic determinants between IgA and IgG, and the presence of an unusual kappa h chain of 30,000 M.W

Two apparently homogeneous electrophoretic bands were found in the serum of a patient (DA) with multiple myeloma. These M-components were identified as IgA-lambda and IgG-kappa paraproteins bearing different idiotypic determinants. Further analysis of the L chains showed that the lambda-chain was homogeneous but the kappa-chain could be separated by SDS-polyacrylamide gel electrophoresis into two different bands. Both of them were associated with gamma-chains but one (termed kappa n) had normal m.w. (24,500) whereas the other (termed kappa h) was larger (m.w. 30,000). Sugar content of the two DA IgG, as determined by anthrone reaction, was similar in DA IgG kappa n (0.73%) and in DA IgG kappa h (1.1%), clearly demonstrating that the difference in m.w. was not due to a large sugar chain. Furthermore, the peptide map of the kappa h chain included nine peptides absent in those of four other control kappa-chains. Sequence analysis showed that the first 25 N-terminal amino acids of the kappa n differed from those of the kappa h chain at positions 4, 5, 15, 18, and 21. Thus the two kappa-chains had different framework regions.     

Establishment and validation of an immunoenzymatic assay to determine mouse IgG levels based on a rat monoclonal antibody

In this paper we describe an immunoenzymatic assay based on a rat monoclonal antibody (Ram kappa) developed to determine mouse IgG concentration, which is widely used for samples obtained on purification processes, like supernatant waste and the content of IgG in the vaccine (rHBsAg). This assay involves the use of a rat antibody-horseradish peroxidase-conjugated for the revealing of the antigen-antibody reaction. The rat antibody was produced in cell culture using a dialysis tube (DT). The immunoassay was standardized following several concepts, such as specificity, precision, and linearity. The result obtained permitted us to replace the use of polyclonal antibodies to determine the kappa light chain mouse antibodies by a rat monoclonal antibody of high sensibility and reproducibility. The assay permitted a reliable measurement of murine kappa Ig up to 0.68 ng/ml and was capable of quantifying 6.25 ng/ml. Due to the high frequency of the kappa light chain in mouse antibodies this system acquires a great application.     

Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A)+RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S17 protein (pRS17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid (aa) sequence of S17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an Mr of 15 377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an Mr of 12 652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3'-noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].     

Cloning and characterization of cDNAs coding for heavy and light chains of a monoclonal antibody specific for pre-S2 antigen of hepatitis B virus.

Binding specificity of a monoclonal antibody (mAb) (kappa, gamma 2b) H8 which can react with the pre-S2 peptide of hepatitis B virus (HBV) was determined by Western blot analyses. From the hybridoma cell line secreting mAb H8, poly(A)+ RNA was prepared and used as a template for cDNA synthesis and cloning. Full-length cDNAs coding for the heavy and kappa light chains of the mAb were cloned from the cDNA library and characterized by nucleotide (nt) sequence analyses and N-terminal amino acid sequencing. The sequence analyses revealed that both heavy and light chain-specific cDNAs are functional, and the variable regions of the heavy and light chains are members of mouse heavy chain subgroup III(c) and light chain group I, respectively. Comparison of the nt sequences with mouse immunoglobulin genes listed in the GenBank data base show that the cDNAs have not been previously reported. The cDNAs will be used for the construction of a therapeutic antibody for HBV infection.     

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.     

The rheumatoid factor reactivity of a human IgG monoclonal autoantibody is encoded by a variant V kappa II L chain gene

To determine the genetic and molecular basis for rheumatoid factor (RF) autoantibody reactivity in patients with destructive, erosive arthritis, we established a human lymphoblastoid cell line (hRF-1) from a patient with polyarthritis that produced an IgG RF mAb, mAb hRF-1. Studies of isolated H and L chains showed that the specificity of RF reactivity is conferred by mAb hRF-1 L chains. The L chain gene was cloned from a cDNA library prepared from hRF-1 cells. The nucleotide sequence was similar to known V kappa II L chains except for a two nucleotide change corresponding to a change of two amino acids in an invariable region of FR3. A germ-line gene with one of the nucleotide changes was identified by polymerase chain reaction in multiple cell lines, including K562 that does not rearrange Ig genes, but the other nucleotide change appeared to be due to mutation. Either or both of these amino acid changes may contribute to the RF reactivity, because an antibody with the same V kappa II L chain except for these two amino acid changes in FR3 did not have RF reactivity. The RF reactivity of isolated L chains from mAb hRF-1 was confirmed by transfecting COS cells with an expression vector encoding the hRF-1 kappa-chain and showing that the secreted k-chains had RF reactivity. Expression of this variant V kappa II L chain gene may form the basis for RF autoantibody reactivity in some patients.     

Relationship between the rearrangement of immunoglobulin genes, the appearance of a B lymphocyte antigen, and immunoglobulin synthesis in murine pre-B cell lines

Eighteen Abelson virus-transformed immature B cell lines were established and immunoglobulin biosynthesis, expression of a B lymphocyte antigen detected by a monoclonal antibody, and rearrangement of immunoglobulin genes in these cell lines were studied. Only one cell line (A1) synthesized micro-chains but no light chains, and the other cell lines synthesized no detectable immunoglobulins. None of the cell lines established had detectable membrane-associated IgM. Fifteen cell lines expressed a B lymphocyte antigen on their cell surfaces. In three cell lines, however, the majority (greater than 99%) of cells did not express this antigen. Heavy chain genes were rearranged on both chromosomes in all the cell lines, although one heavy chain gene was deleted in three cell lines. In 12 of 18 cell lines, one or both kappa-chain genes were rearranged. In six cell lines, however, both kappa-chain genes remained in embryonic form; lambda-chain genes were in embryonic form in all the cell lines. These results suggested the hierarchy of Ig gene rearrangements, beginning with mu and proceeding to kappa and then to lambda. JH rearrangement was also shown to precede the appearance of a B lymphocyte antigen. In three cell lines (A1-A3), which were considered subclones derived from a single common precursor, it was suggested that one rearranged JH gene was functional, and the other was nonfunctional, indicating that allelic exclusion already operated in pre-B cells.