Immunological and structural properties of human monoclonal IgG cryoglobulins.

Structural and immunological properties were determined for sixteen IgG and one Bence-Jones, human monoclonal cryoglobulins. The heavy chain subclass percentages were 47% IgG1, 14% IgG2 and 29% IgG3, and were different from previously reported distributions of myeloma proteins. In addition, 69% (eleven out of fifteen) of the cryoglobulins and 100% (seven out of seven) of the IgG1 cryos contained type lambda light chains. Electrofocussing of the cryoproteins by analytical liquid gradient column showed the isoelectric points to be included in the range of pH 6.3--8.9. The pI of six light chains and five out of six heavy chains were at acidic and slightly basic pH, respectively. The pI of the intact cryoglobulins were thus close to those of their constituent heavy chains. Six out of seven of the heavy chains were subjected to automated Edman degradation and were classified as containing vH-i or vH-ii variable region subgroups on the basis of their blocked amino termini. One type lambda light chain was unusual in that it contained an amino terminal sequence initially described in an amyloid fibril protein and is the first instance in which light chains with this sequence have been isolated from IgG. The data support the notion that the cryoglobulins are IgGs with unique structural and immunological properties which separate them from non-cryoprecipitable IgGs.     

Determination of cryoglobulins as lipoprotein-autoantibody immune complexes and antigenic determinants against antilipoprotein autoantibody.

Serum IgG-antilipoprotein-autoantibody activity (at 4 degrees C) of a plane xanthoma patient was shown by double-immunodiffusion method. Cryoglobulins in the serum were dissociated to polyclonal IgG and alpha- and beta-lipoproteins by acidification and were reconstructed by neutralization. IgG fraction of the cryoglobulins precipitated with lipoproteins. The cryoglobulins were thus demonstrated to be immune complexes of polyclonal IgG-antilipoprotein-autoantibody and both alpha- and beta-lipoproteins. A part of the lipoprotein-autoantibody immune complexes was not cryoprecipitable. Antigenic determinants for the autoantibody existed in the lipid moieties of lipoproteins, in contrast to the apoproteins which determined the specificity to heteroimmune antilipoprotein antibody. The presence of more than nine different antigenic determinants against the autoantibody indicated that lipoproteins were immunologically heterogeneous depending upon the lipid moieties. Lipoproteins reactive with the autoantibody varied quantitatively in normal individuals and were not detected in a primary hyper-beta-lipoproteinaemia patient and in a primary biliary liver cirrhosis patient with much lipoprotein-X. The absence of antigenicity in the two patients' sera is most likely caused by abnormal lipid moieties of lipoproteins.     

Serological heterogeneity of the IgM components of mixed (monoclonal IgM–polyclonal IgG) cryoglobulins

Mixed IgG–IgM cryoglobulins were isolated from the sera of seven patients with macroglobulinaemia or cryoglobulinaemia. The IgM components of all seven cryoproteins were monoclonal, containing κ light chains only, whereas the IgG components were polyclonal, containing both κ and λ light chains. Despite their apparent immunological homogeneity, the IgM components showed a wide range of antiglobulin activity. The data indicate that serological specificity may vary from one mixed cryoglobulin to another and that the monoclonal IgM components of different mixed cryoglobulins represent a heterologous group of antiglobulins.     

Isolation of t cell membrane proteins (IgT) with antisera to non-isotypic determinants of immunoglobulins: Evidence thaT IgT ‘Light’ chains are not identical to B cell kappa chains

Radiolabelled cell membrane proteins obtained by detergent lysis of murine T cells labelled by lactoperoxidase-catalysed radioiodination were specifically immunoprecipitated with an antiserum to immunoglobulin Fab determinants. Absorption of the antiserum by myeloma proteins or normal immunoglobulins coupled to Sepharose and the use of antibody proteins eluted from these absorbents indicated that the antibody activity in the serum responsible for binding detergent-soluble T cell membrane proteins was directed towards variable region and not to the constant region determinants of kappa light chains. Immunoglobulin light chain sized polypeptides isolated from T cells with this antiserum were found to possess serological characteristics which differed from serum or B cell membrane immunoglobulin kappa chains. Evidence is presented which suggests that a portion of the light chain sized polypeptides isolated from T cells may be proteolytic fragments of a larger polypeptide chain. The results suggest that immunoglobulin-like detergent-soluble T cell membrane proteins isolated with antisera to immunoglobulins (IgT) possess neither immunoglobulin heavy nor light chain constant regions determinants but do carry determinants associated with immunoglobulin variable regions.     

Immunoglobulin epitopes defined by synthetic peptides corresponding to joining region sequence: conservation of determinants and dependence upon the presence of an arginyl or lysyl residue for cross-reaction between light chains and T-cell receptor chains

Joining or J region sequences of rearranging immunoglobulins and T-cell receptors show considerable sequence homology, particularly in their C-terminal portion corresponding to the fourth framework region of immunoglobulin variable regions. In order to test the question of whether serological cross-reactions between immunoglobulin variable regions and T-cell receptors were due to antigenic similarities in their J regions, we synthesized synthetic peptides corresponding to immunoglobulin J regions and to J regions predicted from gene sequence of the T-cell receptor beta chain. We found that antibodies produced against a synthetic 16-mer J beta sequence reacted with T-cell receptor chains and also with immunoglobulin light chains. The cross-reactivity was dependent upon the J signature sequence FG()GT(R or K)L where the presence of a positively charged lysyl or arginyl residue was essential for cross-reactivity. We were able to classify J region determinants into two distinct antigenic sets; one corresponding to JH and the other corresponding to J kappa, J lambda, J beta and J alpha. Although considerable homology occurs between JH and JL (or J beta) sequences, little cross-reactivity was observed between these two J subsets. Antibodies raised against polyclonal murine IgG immunoglobulins contained antibody subpopulations specifically reactive with either JH or J beta peptides. The serological data derived here using antipeptide antibodies are consistent with computer modeling studies that indicate that the conformations of T-cell receptor variable regions resemble those of classical immunoglobulins. Our data comparing cross-reactivities restricted to the J region indicate that the expression of the J region by intact T-cell receptor beta chains is probably more similar to that of light chains than it is to the corresponding region of heavy chains.     

Immune complex cryoglobulinaemia in lepromatous leprosy: a pathogenetic approach to some clinical features of leprosy

Cryoglobulins isolated from the sera of six patients with lepromatous leprosy were extensively studied by various immunochemical techniques.

Immunoelectrophoresis and analytical ultracentrifugation revealed in all cases the presence of two components only, which were identified as IgG and IgM respectively. The IgG component of all six cryoprecipitates and the IgM component of five of them were polyclonal, whereas the remaining IgM component was monoclonal as it produced a narrow-banded electrophoretic spike and contained kappa light chains only.

Anti-γ-globulin activity, detected in all sera and isolated cryoglobulins, was consistently found to be associated with the IgM fraction of the mixed cryoglobulins and was present at very high titres in the IgM monoclonal component. β_1A serum levels were decreased in all cases.

The cryoglobulins that appear to be immune complexes of the IgG/anti-IgG type may play a role in the pathogenesis of some clinical features of leprosy patients.

     

Lupus anti-ribosomal P peptide antibodies show limited heterogeneity and are predominantly of the IgG1 and IgG2 subclasses

A 22-amino acid synthetic peptide (P peptide) containing the conserved, shared autoantigenic determinants of the ribosomal P proteins was conjugated to rabbit serum albumin and used to analyze anti-P heterogeneity in 17 lupus sera. Anti-P peptide antibodies demonstrated moderate restriction in isotype (IgM and IgG, but not IgA), subclass (predominantly IgG1 and IgG2), light chain type (predominantly kappa) and spectrotype. In one serum, almost exclusive use of IgG2 and the kappa light chain was observed. These findings indicate that there is a nonrandom selection of heavy and light chain constant region genes as well as limited variable region diversity in the anti-P peptide response.     

A study of possible reciprocal influences within the respective allotypy of the polypeptidic chains consituting the immunoglobulin molecule--I. The rabbit a series allotypic specificities

Anti-allotypic sera prepared against IgG or anti-allotypic sera prepared against heavy chains did not allow us to reveal any sign of an influence of allotypically differen κ light chains on the expression of rabbit a series allotypic patterns carried by the variable region of the heavy chains. The a series allotypic determinants seem to be relatively far from the association zone of the heavy and light chains in the IgG molecule. These allotypic determinants also seem to have a configuration or a molecular distribution which is different on the isolated heavy chains from that on the part of these chains which is accessible on the entire IgG.Antisera prepared against heavy chains contain two kinds of antibodies: (1) anti-allotypic antibodies which recognize only the homologous heavy chains, and the corresponding IgG and (2) anti-isotypic antibodies which recognize all the allotypically different heavy chains we studied (even those of the rabbit subjected to the anti-allotypic immunization), but do not recognize the corresponding IgG. Thus, the isotypic determinants recognized on the heavy chains are hidden in the intact IgG. These anti-isotypie antibodies were obtained during isoimmunizations.     

DNA-anti-DNA circulating complexes in the nephritis of systemic lupus erythematosus.

In order to determine whether circulating antigen-antibody complexes in systemic lupus erythematosus (SLE) consist of DNA and anti-DNA, cryoglobulins were isolated from the sera of 38 patients with SLE nephritis and analysed for DNA and anti-DNA. Cryoglobulins were detected in 36 of the 38 sera, and DNA was found in 30 of 33 examined by either fluorescence of ethidium bromide or a radioimmunoassay. Anti-DNA activity was not detectable in any of the whole cryoglobulins but anti-IgG activity was found in 17. Twenty cryoglobulins were therefore treated by acid dissociation and ultracentrifugation to obtain isolated immunoglobulins; IgG was isolated from all, and IgM from eight. Using a modified Farr assay to detect anti-dsDNA and an enzyme-linked immunoadsorption assay to detect anti-ssDNA, anti-dsDNA activity alone was found in five IgG fractions, anti-ssDNA activity alone in five, and both anti-ds- and anti-ssDNA activity in four. Anti-dsDNA activity was found in three of the IgM fractions. In all, anti-dsDNA activity was found in nine of these 20 cryoglobulins, and anti-DNA in 14. Analysis of these 14 cryoglobulins with anti-DNA Ig fractions showed that there was enrichment of the IgG anti-DNA activity in the cryoglobulin compared to the patient's serum in all but two cases. In six of the 20 cryoglobulins studied there was no detectable anti-DNA activity in isolated IgG or IgM fractions. We therefore concluded that DNA-anti-DNA complexes were present in most of our patients with SLE nephritis, but there was clearly a substantial minority in whom they were undetectable. In some of the latter who also had active disease the cryoglobulins had anti-IgG activity. Thus it would seem that SLE can occur in the absence of DNA-anti-DNA complexes, but with other complexes present. DNA was also found in cryoglobulins isolated from patients with idiopathic glomerulonephritis, but, in contrast to recent reports, anti-DNA activity was not detectable in immunoglobulins isolated from their cryoglobulins.     

Cell-diffusion precipitin analysis of mixed IgM-IgG cryoglobulins. An unusual kappa-chain determinant of the IgM component

Antigenic features of monoclonal IgM kappa components of three mixed IgM-IgG cryoglobulins were studied by gel-diffusion precipitin analysis. An unusual kappa-chain determinant was revealed in the IgM components but not in other monoclonal immunoglobulins (IgM kappa, BJ kappa) or normal IgG. Antibodies to this determinant were found not only in anti-kappa, but also in anti-lambda sera, and in antisera to hidden determinants of L-chains.     

Monozygotic Rheumatoid Arthritis Twin Pairs Express Similar Levels of Conserved Immunoglobulin V Gene in Polyclonal Rheumatoid Factors Irrespective of Disease Status

The degree of polyclonal RF heterogeneity was assessed in diseased and non-diseased twins with rheumatoid arthritis (RA). The distribution of variable region determinants encoded by a set of immunoglobulin germline, or minimally mutated germline, genes within IgM RF, IgG RF and IgA RF isotypes was determined by ELISA using specific mouse monoclonal antibodies (MoAb) in fractionated plasma from 12 members of six monozygotic twin pairs with RA, The results reveal that at least 40% (range ∼ 18–87%) of IgM RF are encoded by a small set of ∼ 10 genes from the V_H1, 3 and 4 families. Furthermore, a significant proportion of IgG RF and IgA RF (∼ 30%) are also encoded by these same genes. Comparison with RF-negative fractions of immunoglobulins showed that the examined variable region determinants were overrepresented in the RF fractions.
The level of expression of the variable region determinants in RF were generally similar within twins but different between unrelated twin pairs irrespective of disease status. The variability of V_H gene usage between unrelated individuals suggests that the level of expression and regulation of the variable region determinants may be genetically regulated or influenced by common environmental factors.     

Conversion of a mouse Fab into a whole humanized IgG antibody for detecting botulinum toxin

Antibodies serve as the gold standard in most immunodiagnostic assays. Recent advances in recombinant DNA technology have offered the production of antibody fragments or Fabs as promising alternatives. However, the lack of the Fc region of the antibody can be difficult in many standard diagnostic platforms. Therefore we sought to convert a murine Fab into a whole humanized IgG. The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified humanized IgG demonstrated conversion to human IgG with no traces of mouse Fab as determined by Western blot analysis. In addition, the humanized IgG performed better as both a detection and capture reagent in an ELISA format and detected the botulinum toxoid at a lower concentration than the parental murine Fab. This technique offers the ability to convert various species of antibodies or antibody fragments into humanized antibodies with improved characteristics in immunodiagnostic assays, for use as human controls in serological assays, or for possible therapeutic benefit.     

Antibody Responses to λ1J558 and λ2315 Light Chains

Specificity of BALB/c antibody responses to lambda chains of isologous myeloma proteins 315 and J558 was explored by enzyme-linked immunosorbent assay. lambda-chain binding antibodies were not detected when immunizing with assembled (H + L) myeloma proteins. However, relatively high titred IgG antibodies were elicited by free lambda 2(315) immunization. Antibodies were directed to 'hidden' determinants since binding was abrogated upon H + L assembly of chains. At least a portion of antibodies bound antigenic determinants in the variable region and cross-reacted with lambda 1 land lambda 3 chains. Free lambda 1J558 immunization induced low-titred, predominantly IgM antibodies that also only reacted with 'hidden' determinants. These determinants were most probably located in the constant (C) region and no cross-reaction to lambda 2 or lambda 3 was observed. An artefact of technical importance was noted: myeloma proteins exposed 'hidden' determinants on their lambda chains when coated directly to polystyrene walls. This artefactual exposition was lost when anti C-region antibody spacer molecules were inserted between the wall and the myeloma proteins. Antibody and T helper cell (Th) responses to free lambda 2(315) covaried significantly in various strains while antibody and Th responses to free lambda 1J558 did not. In some strains, weak antibody responses were detected without detectable Th.     

A study of possible reciprocal influences within the respective allotypy of the polypeptidic chains constituting the immunoglobulin molecule--II. The rabbit b series allotypic specificities

We did not reveal any difference in the expression of the b series allotypic patterns (carried by the constant region of the κ light chains) either with anti-allotypic sera prepared against IgG or with anti-allotypic sera prepared against light chains when the light chains carrying a given allotypic pattern were respectively associated with allotypically different heavy chains to form an IgG molecule. Very probably, the b series allotypic determinants are relatively far away from the association region of the light and the heavy chains in the IgG molecule. These two kinds of antisera reveal the same set of determinants on IgG and on their isolated light chains, but this set seems to have a different configuration on the isolated light chains and on the same light chain included in the IgG.     

The Effect of Interchain Disulfide Bond Cleavage on the Cold Induced Precipitation of Cryoimmunoglobulins

Selective cleavage of the interchain disulfide bonds present in the two IgG₁-k monoclonal cryoglobulins Ger and Muk results in a partial loss of cryoprecipitability of the parent proteins at 0®C. The progressive loss of cryoprecipitability which occurs as a function of increasing reductant concentration parallels the successive cleavage of interheavy-light and interheavy-heavy chain disulfides. Circular dichroism shows that reduction and alkylation of hinge region disulfides induces small conformational changes in the IgG molecules that could alter cryoprecipitability. The N-terminal amino acid sequence of the Fc component derived by restricted proteolysis with trypsin of protein Muk was found to be completely homologous with N-terminal Fc sequences of noncryoglobulin IgG reference proteins, indicating identical hinge regions. Reduction and alkylation of two monoclonal IgM cryoglobulins also reduces cryoprecipitability. After reduction and alkylation of either the monoclonal IgM rheumatoid factor or the polyclonal IgG component of two mixed cryoglobulins recombination results in decreased cryoprecipitation of the intact cryoglobulin complex. In all cases inhibition of cryoprecipitation is greater when iodoacetic acid rather than iodoacetamide is employed as the S-alkylating group. These results do not support a direct role for the hinge region in the precipitation of cryoimmunoglobulins.     

Marsupial immunoglobulins: the distribution and evolution of macropod IgG2, IgG1, IgM and light chain antigenic markers within the sub-class Metatheria.

The distribution within Australian and American marsupials of the heavy and light chain antigenic markers identified by antisera to purified quokka (Setonix brachyurus) immunoglobulins is described. Markers for IgM and IgG2 constant region determinants as well as for light chains were widely distributed in Australian species and were also detected in Didelphis, the American opossum, thus indicating a long-term structural conservatism of some immunoglobulins within the marsupials. More detailed analysis of the distribution of quokka IgG2 determinants by quantitative precipitation and sequential absorption procedures suggested that there had been a gradual and cumulative acquisition of these markers with time. The presence of IgG2 markers in species separated for 130 million years (quokka and opossum) suggested that IgG2 was the ancestral IgG present before the divergence of these separate lines. The origin of IgG1 remains obscure as it appears to be limited to a small group of closely related diprotodont marsupials suggesting a recent origin.     

Loss of cryoprecipitability following proteolytic eleavage of the VH domains from a human IgG cryoglobulin

A human IgG3/κ cryoglobulin, IgG Pav, which had been stored at 4°C for several months was found to have lost its property of gelling when cooled. Electrophoresis in starch gel in the presence of 8 M urea showed that IgG Pav had broken down during storage into two fragments. Digestion of freshly prepared, intact IgG Pav with plasmin or trypsin produced identical fragments. Electrophoresis, immunodiffusion and sequence analysis were used to identify these fragments as the variable regions of the heavy chains and the remainder of the molecule. IgG Pav is only the third human immunoglobulin described in the literature from which V_Hdomains have been isolated by enzymatic digestion of the intact molecule; all three are cryoglobulins. Proteolytic cleavage in the fourth hypervariable region of the heavy chains completely removes the ability of IgG Pav to gel when cooled.     

Incidence,character and clinical relevance of mixed cryoglobulinaemia in patients with chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection has been implicated in the pathogenesis of mixed cryoglobulinaemia. Several studies have shown the presence of anti-HCV antibodies and HCV-RNA in both sera and cryoglobulins of such patients. However, the prevalence and clinical significance of cryoglobulins remain uncertain in patients with chronic HCV infection. We have studied 113 consecutive patients referred for assessment because of the presence of anti-HCV antibody in serum for the presence of cryoglobulinaemia and ascertained their clinical relevance and immunochemical properties. Twenty-one of 113 (19%) had detectable cryoglobulins with a mean protein concentration of 0.38 g/l (range 0.15–3.34 g/l). Most of these patients were asymptomatic. The cryoglobulins were of type III in 19 (91%) and of type II in two patients (9%). The latter two patients had the highest concentration of cryoglobulins, subnormal C4 and C1q levels suggesting classical pathway activation and vasculitis with renal impairment. The cryoglobulin IgG subclasses were mainly IgG1 and IgG3. HCV-RNA was detected more frequently in the sera of cryoglobulin-positive patients than in cryoglobulin-negative patients. This study showed that mixed cryoglobulinaemia is common in chronic HCV infection, and is predominantly type III. Evidence of systemic or renal disease was rare except in those with type II cryoglobulinaemia, and this may reflect either the concentration of the cryoprecipitate or the presence of a monoclonal complement-activating IgM paraprotein. The detection of HCV-RNA in the majority of the cryoprecipitates further supports the important role of HCV in the etiopathogenesis of essential mixed cryoglobulinaemia, although the mechanism is at present unclear.     

Selective pathogenicity of murine rheumatoid factors of the cryoprecipitable IgG3 subclass.

To analyze the involvement of rheumatoid factors (RF) in the generation of cryoglobulins and the development of related tissue injuries, we have established a panel of anti-IgG2a RF mAbs derived from MRL/MpJ-lpr/lpr (MRL-lpr), C3H/HeJ-lpr/lpr, and 129/Sv mice. After injection of hybridoma cells to normal mice, all four IgG3 RF mAbs induced cryoglobulinemia, and various degrees of glomerulonephritis and skin leukocytoclastic vasculitis. In contrast, none of the RF mAbs of the other isotypes generated cryoglobulins or tissue lesions. Since the same observation was obtained with another panel of five clonally related anti-IgG2a RF mAbs of MRL-lpr origin with almost identical heavy and light chain variable (V) regions but five different isotypes, it seems unlikely that the absence of pathogenicity of non-IgG3 RF mAbs was due to differences in fine specificity or V framework regions. In addition, the analysis of serum RF in MRL-lpr mice has demonstrated that a majority of 4 month old MRL-lpr mice produced substantial amounts of IgG3 RF with cryoglobulin activity. Because the cryoglobulin activity is associated with the murine IgG3 heavy chain constant region, RF of this subclass may play a significant role in the development of autoimmune-related tissue injuries, especially in MRL-lpr mice.     

Use of enzyme-linked immunosorbent assay for the determination of serological relationship between two comoviruses

ELISA was used to determine the degree of serological relationship between red clover mottle (RCMV) and broad bean stain (BBSV) viruses and the optimal conditions for the differentiation of the two viruses by ELISA were established. The titres of homologous and heterologous antibodies determined by ELISA were 100- to 200-fold higher as compared with the ring precipitin test. Antigen concentrations of 0.15 and 0.075 ng/ml, respectively, could be detected. The reactions in ELISA depended on the concentration of antigen, of IgG used for coating and of conjugated IgG. In anti-RCMV IgG, the ratio of group-specific to species-specific antibody as determined by ELISA was from 1:1 to 1:2, while in anti-BBSV IgG this ratio was from 1:4--1:8. The differences between homologous and heterologous reactions in ELISA suggested a similar ratio of antigenic determinants in the two viruses.