sodium-dodecyl-sulfate and Multiple-Myeloma

High resolution two-dimensional (2D)-electrophoresis has been developed to the stage that it is possible to resolve several thousand proteins in cells and tissue, and over six-hundred proteins are separated in human serum. The 2D-technique has been applied to analyses of serum from patients with multiple myeloma, macroglobulinemia and other gammopathies, and to separate apolipoproteins and study abnormalities and polymorphism of these proteins. Cerebrospinal fluid from patients with various neurological diseases has been studied by 2D-electrophoresis and seems to yield information on multiple sclerosis. The 2D-technique has been applied to normal and malignant cells and biopsies, and offers a possibility to detect disease-related proteins. Protein spots from 2D-gels may be used to raise monoclonal antibodies which subsequently can be used to develop simple clinical chemical tests for disease markers. The 2D-electrophoretic method is, however, not yet suitable as a typical routine analysis in the clinical chemistry laboratory, but is primarily a research tool of considerable potential.

Topics: Blood Proteins; Cerebrospinal Fluid Proteins; Electrophoresis, Polyacrylamide Gel; Humans; Hypergammaglobulinemia; Isoelectric Focusing; Multiple Myeloma; Nervous System Diseases; Proteins; Sodium Dodecyl Sulfate; Waldenstrom Macroglobulinemia

We evaluated a new sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) for urinary protein analysis in patients with multiple myeloma (MM; n = 47; ages, 62 +/- 2 years, mean +/- SE). Abnormal proteinuria (mean = 1872 +/- 360 mg/24 h) was present in 95% of the samples; 75% of the patients had some sign of renal dysfunction (glomerular and/or tubular) according to their SDS-AGE pattern. A band suggesting Bence Jones proteinuria (BJP) was detected in 40 vs 33 specimens by routine AGE. Immunofixation identified BJP in 38 patients; the calculated sensitivity of SDS-AGE for BJP was 97%. Excellent correlation (P <0.0001) was obtained with routine AGE (r = 0.994) and immunonephelometry (r = 0.963) for light chain quantification. SDS-AGE allows easy evaluation of renal dysfunction and shows high sensitivity for BJP detection. In a specialized laboratory, it is useful for following the progress of MM patients through the semiquantification of BJP.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bence Jones Protein; Electrophoresis, Agar Gel; Female; Humans; Immunoelectrophoresis; Immunoglobulin Light Chains; Male; Middle Aged; Multiple Myeloma; Nephelometry and Turbidimetry; Proteinuria; Reproducibility of Results; Sensitivity and Specificity; Sodium Dodecyl Sulfate

Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. On the basis of the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins.

Topics: Cell Line, Tumor; Cell Membrane; Computational Biology; Detergents; Gels; Humans; Hydrophobic and Hydrophilic Interactions; Mass Spectrometry; Membrane Proteins; Molecular Weight; Multiple Myeloma; Nanoparticles; Proteolysis; Proteome; Proteomics; Reproducibility of Results; Sensitivity and Specificity; Sodium Dodecyl Sulfate; Solubility; Solutions; Trypsin

Influence of protein composition on total urinary protein assays was evaluated for pyrogallol red-molybdate both with and without sodium dodecyl sulfate (SDS) and pyrocatechol violet-molybdate complex (UPRO) techniques. Using mixtures of albumin and gamma-globulins (n = 8; albumin/globulin ratio, 0 to 10), mean recoveries were 79, 77, and 81% for pyrogallol red, pyrogallol red-SDS, and UPRO, respectively. Using diluted myeloma sera (n = 26; A/G ratio, 0.39 to 2.35), mean recovery by the UPRO method was 115% (vs. 63% for pyrogallol red and 83% pyrogallol red-SDS). Results positively correlated with A/G ratio for UPRO (r = 0.69; P < 0.001), pyrogallol red (r = 0.48; P < 0.05), but not pyrogallol red-SDS (r = 0.191; NS). The difference between UPRO and pyrogallol red assays correlated with the A/G ratio (r = 0.82; P < 0.001). In light chain proteinuria (n = 10), no significant difference (< 15%) was observed between techniques, whereas in glomerular selective proteinuria (n = 10), values were significantly higher with the UPRO assay (2.20 +/- 1.61 vs. 1.43 +/- 1.10 g/L; P < 0.02). Our results support the idea that screening for renal diseases can be performed with UPRO or pyrogallol red assays. However, since A/G ratios may vary with renal disease evolution, follow-up of patients with positive proteinuria should be performed using the same assay, preferably the pyrogallol red-SDS.

Topics: Bence Jones Protein; Benzenesulfonates; Coloring Agents; gamma-Globulins; Humans; Molybdenum; Multiple Myeloma; Proteinuria; Pyrogallol; Sensitivity and Specificity; Serum Albumin; Serum Albumin, Bovine; Sodium Dodecyl Sulfate; Solutions

Monoclonal antibodies (mAbs) of IgG1 class produced by hybridomas raised with NS-1 myelomas, which were purified homogeneously by anion-exchange high-performance liquid chromatography (HPLC), contained two types of immunoglobulin light (kappa) chain. Since NS-1 myeloma synthesizes the light (kappa) chain, the mAb was presumed to be the mixture of hybrid mAbs formed by the random association of heavy (gamma l) and light chains from antigen-immunized spleen cells and light chain from NS-1 cells. Hydrophobic interaction HPLC using TSKgel Phenyl-5PW was applicable to separate 3 species of hybrid mAb from mAb fractions obtained by anion-exchange HPLC. mAbs in the fractions were adsorbed onto the gel equilibrated with phosphate-buffered saline containing 1 M ammonium sulfate and eluted by reducing the concentration to 0 M. The hybrid mAbs were purified separately. The hydrophobic interaction HPLC could discriminate a small difference in hydrophobicity between kappa chains from spleen and NS-1 cells. The immunoreactivities of hybrid mAb bearing light chains only from spleen cells and that bearing those from both spleen and NS-1 cells were almost the same, and hybrid mAb bearing light chains derived only from NS-1 cells showed a relatively lower immunoreactivity than the others. The method described here could be useful for purification of hybrid mAbs.

Topics: Animals; Antibodies, Monoclonal; Antigens; Chemical Phenomena; Chemistry, Physical; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Epitopes; Humans; Hybridomas; Immunoblotting; Mice; Multiple Myeloma; Sodium Dodecyl Sulfate

We have developed a mouse-human chimeric antibody MH171, in which the antigen-recognizing variable regions of the mouse monoclonal antibody MRK17 are joined with the constant regions of human IgG1 antibodies. The MRK17 recognizes specifically the multidrug transporter P-glycoprotein and inhibits the growth of human multidrug resistant (MDR) tumor cells in vitro and in the xenograft nude mouse model system. The established chimeric MH171 antibody forms an apparently intact IgG composed of heavy and light chains covalently assembled via disulfide bonds in sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and is specific to MDR cell lines with a similar affinity to the original mouse MRK17. MH171 also displays strong antibody-dependent cell-mediated cytotoxicity to the target cells in vitro, when human mononuclear cells are used as effector cells. The chimeric antibody against P-glycoprotein, MH171, should be a useful agent in the treatment of human drug-resistant tumors.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Antigens; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carrier Proteins; Chimera; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Genes, Immunoglobulin; Humans; Membrane Glycoproteins; Mice; Multiple Myeloma; Recombinant Proteins; Sodium Dodecyl Sulfate; Transfection

The Coomassie Brilliant Blue G-250 method for urinary proteins underestimates urinary immunoglobulin light chains when albumin or pooled serum is used as the protein standard. The specific color yields of these and other proteins can be brought closer together by adding sodium dodecyl sulfate to the reagent; however, there is some loss of sensitivity. We found such a reagent to be satisfactory for assaying urinary proteins on studying 43 patients with light-chain proteinuria, 19 of whom had multiple myeloma and six possible multiple myeloma.

Topics: Adult; Aged; Aged, 80 and over; Coloring Agents; Female; Humans; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Male; Middle Aged; Multiple Myeloma; Proteinuria; Rosaniline Dyes; Sodium Dodecyl Sulfate; Spectrophotometry

Hybridoma antibodies against bovine collagenase inhibitor were produced by fusion of myeloma cells NS-1 (P3-NS1-1) with spleen cells from mice hyperimmunized with collagenase inhibitor purified from the explant medium of bovine dental pulps. Hybridomas positive by an enzyme-linked immunosorbent assay (ELISA) for bovine collagenase inhibitor were cloned by the dilution method. Seventeen hybridomas producing antibodies were isolated, four of which also recognized purified human collagenase inhibitor in the ELISA. Using a monoclonal antibody-Sepharose affinity column, we easily purified both bovine and human collagenase inhibitors to homogeneity. They showed the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 32,000 daltons.

Topics: Animals; Antibodies, Monoclonal; Aorta, Thoracic; Cattle; Dental Pulp; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Female; Hybridomas; Immunoenzyme Techniques; Immunohistochemistry; Immunosorbent Techniques; Mice; Multiple Myeloma; Sodium Dodecyl Sulfate; Spleen; Tissue Inhibitor of Metalloproteinases

A new human B lymphocyte membrane antigen, CB2, has been detected by a mouse monoclonal IgM antibody. CB2 appears to be predominantly expressed on normal and malignant cells expressing surface membrane immunoglobulin (SmIg). By indirect immunofluorescence, the number of CB2-positive cells in normal peripheral blood correlated well with the number of SmIg-positive cells. Cytotoxicity studies on isolated cell populations showed that CB2 was present on normal B cells isolated from the spleens of 52 donors and on peripheral blood B cells from 8 donors. Monocytes, T cells, granulocytes, platelets, and red cells were CB2 negative. Only malignant cells expressing SmIg were positive. These included B-CLL, B lymphoma, prolymphocytic leukemia, and B lymphoma cell lines Daudi, Raji, and Conception. SmIg-negative leukemia cells, such as common acute lymphoblastic leukemia, acute and chronic myelogenous leukemia, and T cell leukemias, were negative. Blocking studies with human immunoglobulin suggests that the CB2 antigen is not directed against immunoglobulin determinants. Immunoperoxidase studies on normal lymph node sections show that CB2-positive cells are predominantly present in the mantle region of the follicle, whereas B1-positive cells are mainly in the germinal center.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Reactions; Antigens, Surface; B-Lymphocytes; Cell Line; Cytotoxicity Tests, Immunologic; Electrophoresis, Polyacrylamide Gel; Humans; Leukemia; Lymphoma; Mice; Mice, Inbred BALB C; Multiple Myeloma; Precipitin Tests; Receptors, Antigen, B-Cell; Sodium Dodecyl Sulfate; Spleen

Human serum proteins were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis after protein denaturation in the presence or absence of 2-mercaptoethanol. Both electrophoretic techniques give characteristic and reproducible banding patterns and achieve a high degree of resolution within the limits of a one-dimensional separation. The major protein bands have been identified, and the merits of the two techniques are compared. Protein denaturation in the absence of 2-mercaptoethanol gives more discrete bands for the purpose of protein identification by maintaining the disulfide-bridge-dependent polypeptide associations characteristic of many serum proteins. However, simultaneous use of both electrophoretic techniques enhances identification by exploiting the response of an individual protein to mercaptoethanol treatment. The clinical potential of this approach for detecting protein disorders is discussed.

Topics: Animals; Blood Protein Electrophoresis; Blood Proteins; Electrophoresis, Polyacrylamide Gel; Humans; Immunoglobulin A; Immunoglobulin M; Indicators and Reagents; Mercaptoethanol; Multiple Myeloma; Protein Denaturation; Rabbits; Sodium Dodecyl Sulfate

Circular dichroism (CD) of serum alpha1-acid glycoprotein, urinary Bence Jones protein, human carbonic anhydrase B, deoxyribonuclease from bovine pancreas, porcine pepsinogen, and plasminogen from human serum was tested in the absence and presence of 0.005-0.05 M sodium dodecyl sulfate. It was found that in all cases the CD spectra of these proteins were modified by the dodecyl sulfate into spectra indicating the presence of a moderate content of alpha-helix. The transitions were enhanced by addition of acid (pH 2.1-4.4) in all cases tested. Comparison of the various proteins with respect to the amount of reconstruction of the main chain conformation showed that the amount of helix formed depended on the amino acid composition of the protein. Rigidity due to cross-linking by disulfide bridges is the strongest deterrant to the conformational change of the main chain. The CD bands of the native proteins in the 250-350 nm spectral zone were extinguished by sodium dodecyl sulfate, and new weak bands were observed the positions of which corresponded approximately to those of the native proteins. In all cases, except the carbonic anhydrase B, the bands of thus denatured proteins were negative.

Topics: alpha-Macroglobulins; Animals; Bence Jones Protein; Binding Sites; Carbonic Anhydrases; Circular Dichroism; Deoxyribonucleases; Multiple Myeloma; Pepsinogens; Plasminogen; Protein Binding; Protein Conformation; Sodium Dodecyl Sulfate; Spectrophotometry, Ultraviolet

Sera from normal persons and patients with IgA, IgD, IgG, and IgM monoclonal gammopathies were electrophoresed in polyacrylamide gels containing sodium dodecyl sulfate. The gels were stained with Coomassie blue or were used for immunodiffusion. By this method IgG multiple myeloma and IgM Waldenström's macroglobulinemia sera were readily distinguished by electrophoresis alone, whereas IgA and IgD myeloma sera were distinguished by further immunodiffusion against anti-alpha-chain antibody and anti-delta-chain antibody.

Topics: Electrophoresis, Polyacrylamide Gel; Humans; Immunodiffusion; Immunoglobulin A; Immunoglobulin D; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Multiple Myeloma; Sodium Dodecyl Sulfate; Waldenstrom Macroglobulinemia

The human myeloma protein Boh (gamma 2, lambda) was isolated and completely reduced and aminoethylated. The light chain was obtained by chromatography on Sephadex G-100 in 4 M guanidine HC1. The amino-terminal sequence on the blocked light chain could be determined by automatic sequence degradation after PCAase treatment. Twenty-one peptides were isolated from a tryptic digest and 12 peptides from a chymotryptic digest. The sequence determination on these peptides was performed by automatic sequencing methods. The light chain of Boh protein belongs to the lambda II subgroup. Unique substitutions have been found at position 8 (Arg) and position 62 (Tyr). Furthermore, the Boh light chain has six cysteine residues, the additional (sixth) cysteine being adjacent to the invariable intrachain-S-S linking cysteine at position 91. Sequence comparison of lambda II proteins reveals a high degree of homology emphasizing the biologic significance of the hypervariable region sequences;

Topics: Amino Acid Sequence; Amino Acids; Ammonium Sulfate; Carboxypeptidases; Chromatography, DEAE-Cellulose; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Freeze Drying; Humans; Immunoglobulin Fragments; Immunoglobulin lambda-Chains; Multiple Myeloma; Myeloma Proteins; Peptide Chain Termination, Translational; Peptides; Protein Conformation; Pyrrolidonecarboxylic Acid; Sodium Dodecyl Sulfate; Subtilisins; Trypsin

Topics: Animals; Blood Protein Disorders; Dog Diseases; Dogs; Electrophoresis, Polyacrylamide Gel; Female; Immunoelectrophoresis; Immunoglobulin A; Multiple Myeloma; Myeloma Proteins; Sodium Dodecyl Sulfate

Lymphoid cells from A/J mice were iodinated (125I) by the lactoperoxidase lysed with the non-ionic detergent NP-40. The plasma membrane glycolipid receptor for cholera toxin and cell surface immunoglobulin were utilized in immune precipitation systems to characterize the degree of dissociation of the plasma membrane under various conditions. It was found that at 0.1% NP-40 and at cell concentration from 5 to 10 times 10(7) cells/ml, lipid-protein and protein-lipid-protein complexes formed in NP-40 which were soluble after centrifugation at 10(5) times G. Column chromatography of 125I-cell lysates on agarose A-0.5 M in 0.1% or 0.5% NP-40/PBS indicated that the majority of iodinated cell surface material existed as aggregates in detergent micelles. The availability of the oligosaccharide moiety of the glycolipid to interact with the cholera toxin was dependent on both the detergent concentration and the cell concentration used for cell lysis. However, the cell surface immunoglobulin was immunoprecipitable under all conditions of lysis tested.

Topics: Animals; Binding Sites, Antibody; Cell Membrane; Cholera; Chromatography; Detergents; Electrophoresis, Polyacrylamide Gel; Glycolipids; Goats; Immune Sera; Iodine Radioisotopes; Lymphocytes; Mice; Mice, Inbred A; Multiple Myeloma; Myeloma Proteins; Precipitin Tests; Rabbits; Receptors, Antigen, B-Cell; Sodium Dodecyl Sulfate; Spleen; Toxins, Biological

Topics: Alkylation; Amino Acid Sequence; Amino Acids; Chemical Phenomena; Chemistry; Chromatography, Gel; Chromatography, Ion Exchange; Colostrum; Cyanogen Bromide; Electrophoresis, Disc; Epitopes; Humans; Immune Sera; Immunoelectrophoresis; Immunoglobulin A; Immunoglobulin Fragments; Immunoglobulin Heavy Chains; Immunoglobulin J-Chains; Methods; Molecular Weight; Multiple Myeloma; Myeloma Proteins; Sodium Dodecyl Sulfate; Ultracentrifugation

Fusion of human peripheral blood lymphocytes, not forming detectable immunoglobulins, with mouse myeloma cells (TEPC-15), secreting mouse immunoglobulin A with known antibody activity, yielded a somatic cell hybrid clone that secreted both human and mouse immunoglobulins. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that human gamma, alpha, and light chains, as well as mouse alpha and light chains, were formed by the hybrid cells. To determine whether individual antibody molecules with both human and mouse components were secreted, medium from the hybrid cells was precipitated first with antibody against mouse immunoglobulin produced in rabbit, reduced, and alkylated, and then reprecipitated with antibody against human immunoglobulin produced in rabbit. Human gamma, alpha, and light chains were detected after electrophoresis of the immunoprecipitates on sodium dodecyl sulfate-polyacrylamide gels, indicating that antibody molecules containing both human and mouse components were secreted by the hybrid cells. These data indicate that this somatic cell hybrid clone synthesized human gamma and alpha heavy chains and human light chains, as well as mouse alpha heavy chains and mouse light chains. Some of these immunoglobulin components were assembled as hybrid antibody molecules.

Topics: Animals; Cell Fusion; Cell-Free System; Clone Cells; Electrophoresis, Polyacrylamide Gel; Humans; Hybrid Cells; Immune Sera; Immunoglobulin Fragments; Immunoglobulins; Iodine Radioisotopes; Lymphocytes; Mice; Mice, Inbred BALB C; Multiple Myeloma; Myeloma Proteins; Parainfluenza Virus 1, Human; Precipitin Tests; Rabbits; Sodium Dodecyl Sulfate; Species Specificity

Topics: Bone Marrow; Bone Marrow Cells; Cytoplasm; Humans; Immune Sera; Immunoglobulin A; Immunoglobulin Fragments; Molecular Weight; Multiple Myeloma; Plasma Cells; Precipitin Tests; Sodium Dodecyl Sulfate

Topics: Animals; Autoradiography; Electrophoresis, Polyacrylamide Gel; Methods; Mice; Multiple Myeloma; Myeloma Proteins; Neoplasm Proteins; Neoplasms, Experimental; Phosphorus Isotopes; Polyribosomes; Protein Biosynthesis; Protein Conformation; RNA, Messenger; RNA, Neoplasm; Sodium Dodecyl Sulfate

Topics: Animals; Carbon Isotopes; Cell-Free System; Chromatography, DEAE-Cellulose; Cytoplasm; Electrophoresis, Polyacrylamide Gel; Glutamine; Hydrolysis; Immunoglobulin Fragments; Mice; Microsomes; Molecular Weight; Multiple Myeloma; Myeloma Proteins; Neoplasms, Experimental; Peptides; Plasmacytoma; Ribosomes; Sodium Dodecyl Sulfate; Tritium; Trypsin

A cell-free system derived from Krebs II ascites tumor has been used to assay biologically active mRNA for myeloma (MOPC-41) light chain during its purification by oligothymidylate-cellulose chromatography and sucrose gradient centrifugation. The purified mRNA directs the synthesis of a product that yields tryptic peptides corresponding to those derived from authentic myeloma protein and that forms a specific immunoprecipitate with antibody directed against the MOPC-41 protein. The fact that the light-chain mRNA anneals to oligothymidylic acid-cellulose suggests that it, like several other eukaryotic mRNAs, contains a region rich in adenylic acid residues. The most active fractions of light-chain mRNA, representing about 0.1% of the RNA originally extracted from membrane-bound myeloma polysomes, sediment as a discrete peak with an s(20,w) of about 13, roughly corresponding to an RNA molecule containing 850 bases. The results suggest that the light-chain mRNA is monocistronic and that it contains about 200 more bases than would be necessary to encode the variable and constant regions of a single light-chain molecule.

Topics: Adenine Nucleotides; Animals; Carbon Isotopes; Carcinoma, Krebs 2; Centrifugation, Density Gradient; Chromatography, Affinity; Chromatography, Ion Exchange; Goats; Leucine; Multiple Myeloma; Myeloma Proteins; Peptides; Precipitin Tests; Ribosomes; RNA, Messenger; RNA, Neoplasm; Sodium Dodecyl Sulfate; Thymine Nucleotides; Tritium; Trypsin