sepharose and Multiple-Myeloma

Monoclonal immunoglobulin produced by clonal plasma cells is the main cause in multiple myeloma and monoclonal gammopathy of renal significance. Because of the complicated purification method and the low stoichiometry of purified protein and glycans, site-specific N-glycosylation characterization for monoclonal immunoglobulin is still challenging. To profile the site-specific N-glycosylation of monoclonal immunoglobulins is of great interest. Therefore, in this study, we presented an integrated workflow for micro monoclonal IgA and IgG purification from patients with multiple myeloma in the HYDRASYS system, in-agarose-gel digestion, LC-MS/MS analysis without intact N-glycopeptide enrichment, and compared the identification performance of different mass spectrometry dissociation methods (EThcD-sceHCD, sceHCD, EThcD and sceHCD-pd-ETD). The results showed that EThcD-sceHCD was a better choice for site-specific N-glycosylation characterization of micro in-agarose-gel immunoglobulins (~2 μg) because it can cover more unique intact N-glycopeptides (37 and 50 intact N-glycopeptides from IgA1 and IgG2, respectively) and provide more high-quality spectra than sceHCD, EThcD and sceHCD-pd-ETD. We demonstrated the benefits of the alternative strategy in site-specific N-glycosylation characterizing micro monoclonal immunoglobulins obtained from bands separated by electrophoresis. This work could promote the development of clinical N-glycoproteomics and related immunology.

Topics: Chromatography, Liquid; Glycopeptides; Glycosylation; Humans; Immunoglobulin A; Immunoglobulin G; Multiple Myeloma; Polysaccharides; Sepharose; Tandem Mass Spectrometry

Cleavage of IGFBPs by proteases results in IGFBP fragments that have altered IGF-binding affinity, and IGF-independent roles. We have previously purified a specific IGFBP-1 protease activity from the urine of an individual with multiple myeloma and dermatitis. The aim of this study was to determine whether IGFBP-1 protease activity and/or IGFBP-1 fragments were present in the circulation of this patient.. The size of immunoreactive IGFBP-1 in serum samples was determined after Superose 12 chromatography. Intact IGFBP-1 and IGFBP-1 fragments were characterized in four RIAs and after SDS-PAGE.. Specific proteolysis of IGFBP-1 generated an N-terminal fragment (IGFBP-1(1-130)) with a predicted molecular mass of 13kDa but an apparent mass of 21kDa on SDS-PAGE. A C-terminal fragment (IGFBP-1(131-234)) produced in vitro migrated at 11.4kDa, close to its predicted size. However a C-terminal fragment of cleaved IGFBP-1 (IGFBP-1(142-234)) migrated at 14kDa on SDS-PAGE. Serum from the patient inhibited IGFBP-1 protease activity. Immunoreactive IGFBP-1 in patient serum was present at molecular masses consistent with IGFBP-1 fragments, in addition to intact IGFBP-1.. Specific cleavage of IGFBP-1 occurs at the tissue level and not in the circulation in a patient with multiple myeloma and dermatitis. The fragments that are generated may have endocrine roles.

Topics: Aged; Antimicrobial Cationic Peptides; Biotinylation; Blood Proteins; Carrier Proteins; Chromatography; Dermatitis; Female; Humans; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor I; Multiple Myeloma; Protein Isoforms; Radioimmunoassay; Recombinant Proteins; Sepharose

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.

Topics: Actins; Animals; Biotechnology; Blotting, Western; Cattle; Cell Line; Chickens; CHO Cells; Chromatography, Affinity; Cricetinae; DNA, Complementary; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Enzyme Activation; Enzyme Precursors; Genetic Vectors; Humans; Kinetics; Methotrexate; Mice; Multiple Myeloma; Mutation; Plasmids; Platelet Aggregation; Promoter Regions, Genetic; Prothrombin; Recombinant Proteins; Sepharose; Tetrahydrofolate Dehydrogenase; Thrombin; Time Factors; Transfection

In a woman suffering from IgE myeloma, hay fever and polyvalent respiratory and skin allergy the IgE monoclonal protein VL was isolated and investigated with respect to structural and functional properties. The amino acid sequence of 22 isolated peptides--especially of the biologically significant C2-C3 part--corresponded with that originally described by Bennich et al. (Immunol Rev 1978;41:3-23; Prog Immunol 1974;13:49-58). However, in mass spectrometry the sugar residues on ASN 99 (219) and 252 (371) were deficient in sialic acids. The native IgE VL protein precipitated with high intensity all mannose-specific lectins as concanavalin A (Con A) and was able to release histamine after triggering by these lectins. The same lectins also elicited more histamine release and more positive skin reactions in atopic than in healthy persons. In sera from atopic patients the binding of IgE on Con A Sepharose 4B column was stronger than in normal persons. It is suggested that changes in the IgE glycosylation state may contribute to IgE-mediated pictures of clinical allergy by the nonimmunological pathway.

Topics: Allergens; Amino Acid Sequence; Binding Sites, Antibody; Female; Glycosylation; Humans; Hypersensitivity, Immediate; Immunoglobulin E; Immunoglobulin kappa-Chains; Immunoglobulin Variable Region; Lectins; Molecular Sequence Data; Molecular Weight; Multiple Myeloma; Myeloma Proteins; Oligosaccharides; Precipitin Tests; Sepharose; Structure-Activity Relationship

Starting from two IgA1 myeloma sera, the isolation of monoclonal monomeric, dimeric, trimeric and tetrameric IgA in a high state of purity and size homogeneity for each serum is described. The method combined repetitive gel filtrations on Ultrogel AcA22 with affinity chromatography on Jacalin-Sepharose. These various forms of pure polymeric IgA obtained from the same monoclonal IgA should allow a precise comparison of their respective structure and reactivity with different IgA-binding proteins, such as IgA Fc-receptors, the polymeric Ig receptor, and lectins.

Topics: Antibodies, Monoclonal; Chromatography, Affinity; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Humans; Immunoglobulin A; Immunoglobulin J-Chains; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Lectins; Multiple Myeloma; Myeloma Proteins; Plant Lectins; Protein Conformation; Sepharose

A novel experimental model was established in normal rats for studying the localisation and tissue distribution of a murine monoclonal antibody directed against kappa light chain B cell malignancies. The antibody, K-1-21 was raised against human kappa Bence Jones Proteins and reacts with a cell membrane antigen KMA which is restricted to some kappa myeloma and lymphoma cells. In the rat model, kappa or lambda Bence Jones protein-conjugated sepharose was implanted subcutaneously on either flank 24 h before the injection of 131I-labelled K-1-21 or its F(ab')2 fragment. Gamma camera imaging and tissue distribution studies showed specific localisation of the K-1-21 antibody in the kappa sepharose. Injection of F(ab')2 antibody fragments resulted in faster background clearance, earlier delineation of the specific image and significantly higher target to blood ratios than those obtained with the intact antibody. These results suggest that the model may provide an alternative system to tumour xenograft bearing nude mice for studying localisation of antibodies with therapeutic potential.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Bence Jones Protein; Female; Gastric Mucosa; Immunoglobulin kappa-Chains; Iodine Radioisotopes; Kidney; Male; Models, Biological; Multiple Myeloma; Radionuclide Imaging; Rats; Rats, Inbred F344; Sepharose; Thyroid Gland; Tissue Distribution

Monoclonal antibodies to bovine alpha-crystallin have been produced using hybridoma technology. Five selected hybridoma clones were maintained as ascites tumours in mice and gram quantities of the antibodies were purified from the ascites fluids by affinity chromatography on alpha m-crystallin covalently bound to Sepharose CL-2B. Preliminary characterization studies suggest that the antibodies are pure and monospecific. The antibodies could be divided into two groups on the basis of their reactivities towards iodinated alpha-crystallin, their ability to bind to Protein A-Sepharose, their immunoglobulin subclass, their immunoelectrophoretic patterns and their abilities to react with chicken and opossum alpha-crystallin. The availability of these monoclonal antibodies will greatly facilitate studies on the surface topography of alpha-crystallin.

Topics: Amino Acids; Animals; Antibodies, Monoclonal; Antibody Specificity; Carcinoma, Ehrlich Tumor; Cattle; Chromatography, Affinity; Crystallins; Electrophoresis, Agar Gel; Female; Hybridomas; Immunodiffusion; Immunoglobulin G; Isoelectric Focusing; Mice; Mice, Inbred BALB C; Multiple Myeloma; Neoplasms, Experimental; Pregnancy; Rabbits; Rats; Sepharose; Staphylococcal Protein A

A method for the determination and removal of circulating immune complexes in pathological sera was developed using human secretory or dimeric myeloma IgA covalently bound to Sepharose 4B. IgA-Sepharose 4B was able to selectively bind heat or antigen-aggregated human IgG (circulating immune complexes) but not monomeric IgG. The absorbent was also able to remove a very high proportion (95%) of circulating immune complexes from pathological sera as determined by a turbidimetric technique. An immunoradiometric assay for the direct measurement of circulating immune complexes is described. The assay uses IgA-Sepharose 4B as an absorbent (for the binding of IgG immune complexes from sera) and 125I-rabbit anti-IgG antibody (for the quantitation of IgG immune complexes bound to IgA-Sepharose 4B). The mean value obtained for pathological sera (27.1 +/- 0.9) was significantly higher than that of normal sera (4.8 +/- 0.5).

Topics: Antigen-Antibody Complex; Chromatography, Affinity; Colostrum; Female; Humans; Immunoglobulin A; Immunoglobulin A, Secretory; Immunoglobulin G; Multiple Myeloma; Nephelometry and Turbidimetry; Pregnancy; Radioimmunoassay; Reference Values; Sepharose

Immobilized Concanavalin A-Sepharose 4B (CSB) binds myeloma immunoglobulins (IgG through IgE) in larger proportions than it binds normal immunoglobulins. Some possible explanations for this are discussed. Also, CSB shows differential affinity among immunoglobulins, normal or myelomatous; its greatest affinity is for IgM and IgE. This property can be used to separate a large percentage of the IgM and IgE from both normal and myelomatous sera.

Topics: Humans; Immunoglobulin E; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Immunosorbents; Multiple Myeloma; Polysaccharides; Radioimmunoassay; Sepharose

Topics: Cell Line; Chromatography, Affinity; HeLa Cells; Humans; Multiple Myeloma; Neoplasms, Experimental; Poly A; Poly U; Polysaccharides; RNA; RNA, Messenger; Sepharose; Subcellular Fractions

Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanvalin A (Con A)--Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains: (abstract:see text). Such glycopeptides have only a single mannose residue capable of interacting with Con A--Sepharose; an interacting mannose residue is either an alpha-linked nonreducing terminal residue or an alpha-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl alpha-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures: (abstract: see text) where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A--Sepharose and elution as a sharp peak with 0.1 M methyl alpha-D-glucopyranoside; glycopeptides giving this pattern had the structures: (abstract: see text) where R2 is either H, glcNAc, Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc. These glycopeptides all have two interacting mannose residues, the mimimum required for binding to the column; one of these mannose residues must, however, be a terminal residue to obtain tight binding and sharp elution. The fourth profile type showed tight binding of glycopeptide to the column but elution with 0.1 M methyl alpha-D-glucopyranoside resulted in a broad peak indicating very tight binding; glycopeptides showing this behaviour had the structures: (abstract: see text) where R3 is either GlcNAc,Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc. Therefore it can be concluded that although a minimum of two interacting mannose residues is required for binding to Con A--Sepharose, the residues linked to these mannoses can either strengthen or weaken binding to the column.

Topics: Carbohydrates; Chromatography, Affinity; Concanavalin A; Glycopeptides; Humans; Immunoglobulin G; Molecular Conformation; Multiple Myeloma; Sepharose

Six fluorescent antihuman Ig preparations were tested for their Ig class specificity by reacting them with highly purified IgG, IgM, IgA, and OVA coupled covalently to Sepharose beads. OVA was used as a measure for nonimmunologic binding. Bead fluorescence was determined by microfluorometry. The amounts of USS and NSS were expressed quantitatively. These data were compared with the performance of these particular conjugates in a biologic system, namely, monoclonal bone marrow cells. Five of the six conjugates satisfied the requirement of monospecific activity; one did not. At a dilution of 1 : 8, the five monospecific conjugates reacted between five and 50 times stronger with their appropriate antigens than with OVA-coupled beads. Cross reactivity with other Ig classes, after correction for OVA staining was maximally 6%. The conjugate that was nonspecific in the bone marrow system gave very high cross reactivity with the Ig-coupled beads. A good correlation was found between OVA bead staining and nonimmunologic binding of conjugates in bone marrow slides. In this respect, conjugates prepared from antibody preparations isolated by solid immunoadsorbents proved to be superior to globulin or whole IgG fractions. Ig coupled to Sepharose beads seems to represent a very promising substrate for conjugate specificity testing.

Topics: Adsorption; Animals; Bone Marrow; Bone Marrow Cells; Epitopes; Fluorescent Antibody Technique; Goats; Humans; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Multiple Myeloma; Ovalbumin; Polysaccharides; Rabbits; Sepharose; Sheep; Staining and Labeling; Swine; Waldenstrom Macroglobulinemia

Topics: Agar; Animals; Blood; Immunoelectrophoresis; Multiple Myeloma; Polysaccharides; Precipitin Tests; Rabbits; Sepharose; Waldenstrom Macroglobulinemia