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We applied two-dimensional gel electrophoresis (2-DE) to study the repertoire of tetanus toxoid (TT)-specific antibodies produced after TT immunization in healthy humans and in severe combined immunodeficient mice xenotransplanted with either human peripheral blood leukocytes (PBLe) or with human adult tonsil (hu-ton) pieces. Specific anti-TT antibodies, as well as total immunoglobulins (Ig), were purified by affinity chromatography on TT-Sepharose or Protein G-Sepharose, respectively. 2-DE unambiguously allowed us to differentiate between the specific humoral responses produced either by humans or by the two xenochimeric mouse models. Anti-TT antibodies produced by humans were polyclonal with a superimposed oligoclonality that was donor-dependent and that did not change upon time. By contrast, immunized hu-PBLe-SCID mice exhibited an evident clonal restriction of the Ig, which increased with time after boosting. Hu-ton-SCID mice showed a clonal diversity which was intermediate between those observed in humans and in hu-PBLe-SCID mice, and which was stable over time. In addition, information was gained by 2-DE, correlating with data obtained by enzyme-linked immunosorbent assay (ELISA), on the isotype composition of the anti-TT IgM response. Altogether, our results clearly demonstrated that the clonal diversity of monospecific antibodies can be appreciated by 2-DE, and that the largest diversity was found in humans when compared to that in xenochimeric models. In addition, mice implanted with pieces of lymphoid organs had the broadest anti-TT Ig diversity, an observation supporting the use of this model for the generation of antibodies with restricted specificity.

Cryoproteins are defined as proteins precipitating at low temperature. Most frequently, the precipitate contains immunoglobulins (Igs), and are therefore called cryoglobulins. Three types of cryoglobulins have been described: type I contains a single monoclonal Ig, whereas type II is a mixture of a monoclonal Ig with polyclonal Igs, and type III is a mixture of polyclonal Igs of different isotypes, most frequently IgG and IgM. Type II and type III are also called mixed cryoglobulins. A new type of cryoglobulins, containing polyclonal IgG associated with a mixture of polyclonal and monoclonal IgM has recently been described after two-dimensional polyacrylamide gel electrophoresis (2-DE). This type of cryoglobulin has been called type II-III cryoglobulin. In this study, we report on 2-DE analysis of 335 cryoproteins from patients with heterogeneous clinical conditions. In 69 out of 335 samples (20.7%), 2-DE revealed patterns that were inadequate to characterize the cryoproteins. Out of 335 (79.3%) cryoproteins, 266 were identified according to their two-dimensional patterns: 265 samples contained Igs and were diagnosed as cryoglobulins, and one sample consisted of fibrinogen, and was identified as cryofibrinogen. Among the 265 cryoglobulins, types I, II, and III were observed in 9 (3.4%), 69 (26%), and 116 (43.8%) cases, respectively, whereas type II-III was detected in 71 (26.8%) cases. Eleven of the latter consisted of oligoclonal Igs (IgM in 10 cases, IgA in 1 case) mixed with traces of polyclonal IgG. These cryoproteins were tentatively named type II-IIIvariant cryoglobulins. Taken together, our result clearly show that 2-DE is a suitable technique to analyze cryoproteins.

Abnormal low molecular weight immunoglobulin heavy chain can be detected in serum samples from patients with heavy chain disease. In this paper, we report the characterization of a gamma heavy chain in a patient suffering from a lymphoplasmocytic disorder, using immunofixation analysis and two-dimensional polyacrylamide gel electrophoresis. We demonstrate that the combination of serum protein agarose electrophoresis and two-dimensional electrophoresis (three-dimensional electrophoresis) can be used to further characterize abnormal protein bands detected by immunofixation.

In this paper, we summarize our five-year observation of two-dimensional polyacrylamide gel electrophoretic (2-D PAGE) analysis of immunoglobulin (Ig) light (L) chain patterns on serum/plasma and/or purified human Ig, and compare this technique with agarose electrophoresis and/or immunofixation examination. Polyclonal Ig L chains were seen as large "fuzzy" areas with several zones of high density. The majority (71%) of the monoclonal Ig L chains of monoclonal gammopathy detected by conventional electrophoresis appeared as a single large and well-defined spot on 2-D PAGE analysis, with the remaining appearing as multiple spots. The presence of oligoclonal Ig, reflected by multiple spots in 2-D PAGE, and several bands in immunofixation, was observed in 5 of 26 patients after allogeneic bone marrow transplantation and in 5 patients with tumors. In the majority (77%) of hypergammaglobulinemia, L chains appeared as a wide spread of small and well-defined spots in 2-D PAGE analysis. This pattern suggested oligoclonal Ig-secreting B cell clone expansion, and corresponding abnormalities were not detected with immunofixation. 2-D PAGE analysis also detected oligoclonal Ig expansions whereas conventional electrophoretic examination was normal in 10 additional patients after bone marrow transplantation, and in 6 of 10 immunocompetent patients with acute severe infections. Analysis of the Ig L chain pattern of a severe combined immune-deficient mouse populated with human peripheral blood leukocytes confirmed the skewed human Ig production in the model. In summary, 2-D PAGE appears to be a sensitive tool for the analysis of Ig diversity in various clinical situations.

This publication updates the reference human liver protein map. By microsequencing, 27 spots or 34 polypeptide chains were identified. The most abundant polypeptides detected on the silver stained liver map were key elements in major hepatic biochemical pathways. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing the 1993 reference human liver SWISS-2DPAGE database. SWISS-2DPAGE and the SWISS-PROT protein sequence databases are closely linked together through the use of common accession numbers.

This publication updates the reference plasma and red blood cell protein maps obtained with immobilized pH gradients. Seventeen polypeptide spots or chains were partially characterized by direct N-terminal sequencing or by sequencing of peptides obtained from enzymatic digestion. Additional new polypeptides and previously known proteins are listed in a table and/or labeled on the protein maps, thus providing the 1993 update of the human plasma and red blood cell two-dimensional gel SWISS-2DPAGE database. SWISS-2DPAGE and the SWISS-PROT protein sequence databases are closely linked together through the use of common accession numbers.

High-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to analyze serum samples and purified immunoglobulins (Ig) obtained from "normal" individuals and from patients diagnosed with monoclonal gammopathies (MG) (n = 47; 5 IgA, 15 IgM, 15 IgG, 4 biclonal IgG, 1 IgD, 7 Bence Jones proteins). Polyclonal and monoclonal heavy (H) chains were located at different restricted gel positions according to their isotype. Monoclonal H chains appeared as sets of spots characterized by charge (pI) and size (M(r)) microheterogeneity. Most of the monoclonal gamma chains were not seen on the gels (12/15). Supplementary polypeptides of 45-48 kDa were detected in serum samples containing monoclonal IgM, but were not seen in MG of other isotypes. However, these polypeptides were not specifically associated with monoclonal IgM because they were also found on protein maps of purified polyclonal IgM. Polyclonal light (L) chains appeared as cloudy bands containing several zones of higher density, whereas monoclonal L chains were usually resolved as single sharp spots. In 6 samples, monoclonal L chains were not seen, and in 9 samples, they appeared as two or more spots, characterized by different pI and/or M(r). In one sample obtained from a patient with a biclonal gammopathy, the L chains were resolved as 4 different spots. Our results confirm that 2-D PAGE is an excellent tool to study Ig. Analysis of the L chain region of the gels was particularly informative. Several monoclonal L chains exhibited heterogeneous two-dimensional spot patterns, suggesting that "subtile" clonal mutations of B-cell lineage and/or posttranslational modifications were involved in their production.

To study the clonal events occurring during ontogeny of the humoral immune system, we evaluated plasma immunoglobulin (Ig) production in term newborns and young children by high-resolution two-dimensional gel electrophoresis. The clonality pattern of Ig light (L) chains from healthy newborns (n = 19) was similar to that observed on protein maps of their mothers or of normal adults (n > 100), that is, rare distinguishable small spots in a cloud-like large band of indiscrete Ig L chain spots (polyclonal pattern; maternal Igs). Analysis of plasma samples obtained from infants between 1 month and 5 years of age (n = 55) revealed discrete but evident alterations of the clonality of Ig production. Between the 2nd and 4th months of life, transient attenuation of the "polyclonal background" was observed in association with the appearance of an increasing number of well-resolved Ig L chain spots (corresponding to plasma Ig concentrations between 0.5 and 1 g/L per spot). This "restricted" clonal pattern was progressively less apparent on protein maps of infants older than 2 year and evolved towards a "normal" adult polyclonal pattern at the age of 5. These results suggest that the development of the B-cell clones is heterogeneous, either through limited outgrowth of precursor cells or through selective antigenic pressures.

This publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two-dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.

The reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two-dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two-dimensional gel database.