Examination of the macronuclear replication band in Euplotes eurystomus with monoclonal antibodies.

A panel of eight monoclonal antibodies (MAbs) was prepared from spleen cells of mice immunized with macronuclear replication bands (RBs) isolated from Euplotes eurystomus. Antibodies were investigated with a solid phase radioimmunoassay (RIA) using either soluble chromatin from isolated RBs or from total macronuclei as antigen. The RIA showed that several MAbs recognized antigens present only in the RB or macronucleus, whereas others recognized antigens present in both structures. Specificity of the MAbs was also examined by indirect immunofluorescence. Antibody C10 recognized an antigen in the rear zone of the RB, whereas MAbs G6 and B2 appeared to stain both the forward and rear zones of the RB. Antibody A7 recognized an epitope distributed throughout the macronucleus except in the RB. Cytochemical studies with degradative enzymes suggested that antigens localized by immunofluorescence were composed of proteins. Immunoblots of SDS PAGE permitted identification of a few proteins that reacted with three of the RB-specific MAbs. Monoclonal antibodies that identify the presence or absence of reactivity of specific proteins in the RB could prove useful in the study of chromatin structure and the mechanism of chromatin replication.

Download Full-text

A competitive solid-phase radioimmunoassay for translational factors employing monoclonal antibodies

Journal of Immunological Methods ◽

10.1016/0022-1759(84)90409-5 ◽

1984 ◽

Vol 73 (2) ◽

pp. 337-345

Author(s):

James S. Hutchison ◽

Barry Feinberg ◽

Kivie Moldave

Keyword(s):

Monoclonal Antibodies ◽

Solid Phase ◽

Solid Phase Radioimmunoassay

Download Full-text

Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes.

Journal of Experimental Medicine ◽

10.1084/jem.156.6.1739 ◽

1982 ◽

Vol 156 (6) ◽

pp. 1739-1754 ◽

Cited By ~ 205

Author(s):

M E Medof ◽

K Iida ◽

C Mold ◽

V Nussenzweig

Keyword(s):

Monoclonal Antibodies ◽

Immune Complexes ◽

Solid Phase ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Alpha Chain ◽

Sds Page ◽

High Concentrations ◽

Polypeptide Chains

The main finding of this paper is that CR1, the membrane receptor for C3b and C4b, together with C3b/C4b-inactivator (I), degrades C3b bound to immune complexes (C3b*). Two fragments are generated: C3c, which is released from the immune complexes, and C3d*. The C3c fragment released from the cell intermediate EAC1423b prepared with 125I-C3 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioautography. It has a 135,000 mol wt and contains disulfide bonded labeled polypeptide chains of 75,000 and 31,000 mol wt, which presumably represent the beta and a fragment of the alpha-chain of C3b*. Silver staining of the SDS-PAGE gels revealed other C3-derived bands with 39-42,000 mol wt. Human erythrocytes + I also cleave C3b* into C3c and C3d*. The activity of the erythrocytes is CR1 mediated because it can be totally inhibited by monoclonal antibodies to CR1. In contrast with these results, I together with the serum protein beta 1H (H) transform EAC1423b into hemolytically inactive EAC1423bi and cleave the alpha' chain of C3b* into fragments of 70,000 and 40,000 mol wt. Small amounts of C3c are also released at relatively high concentrations of H. On a molar basis, the efficiency of CR1 in the generation of C3c and C3d is 10(4)-10(5) greater than H. An additional observation was that C3c could be released by treating EAC1423bi with CR1 + I and that this reaction was also inhibited by monoclonal antibodies to CR1. Therefore, it is likely that CR1 has binding affinity for iC3b and that the degradation of C3b* proceeds as follows: C3b (formula, see text) C3c + C3d*. Taken together, our findings argue that the processing of C3b* in vivo occurs in solid phase, that is, on the surface of cells bearing CR1.

Download Full-text

Identification of a phosphoprotein in the nuclear matrix by monoclonal antibodies

Biochemical Journal ◽

10.1042/bj2410693 ◽

1987 ◽

Vol 241 (3) ◽

pp. 693-697 ◽

Cited By ~ 6

Author(s):

M J Halikowski ◽

C C Liew

Keyword(s):

Monoclonal Antibodies ◽

Nuclear Matrix ◽

Matrix Protein ◽

Solid Phase ◽

Present Report ◽

Nuclear Organization ◽

Micrococcal Nuclease ◽

Nuclear Matrix Protein ◽

Solid Phase Radioimmunoassay ◽

Nuclear Phosphoprotein

Previous work in this laboratory has established that a rat liver nuclear phosphoprotein (B2:Mr 68,000, pI 6.5-8.2) is associated with actively transcribed nucleosomes, as demonstrated by its preferential release after mild treatment with micrococcal nuclease. In the present report we provide further immunological evidence (‘Western Blot’ analysis, solid-phase radioimmunoassay and indirect immunofluorescence) that in addition establishes the presence of this phosphoprotein in the nuclear-matrix protein fraction. This paradoxical localization suggests that this phosphoprotein may function in two separate and distinct roles within the realm of nuclear organization.

Download Full-text

IMMUNOAFFINITY PURIFICATION OF PLASMA INHIBITOR FOR ACTIVATED PROTEIN C

10.1055/s-0038-1643814 ◽

1987 ◽

Author(s):

M Laurell ◽

T Carlsson ◽

J Stenflo

Keyword(s):

Monoclonal Antibodies ◽

Protein C ◽

Solid Phase ◽

Gel Filtration ◽

Activated Protein C ◽

Specific Inhibitor ◽

Sds Page ◽

Fresh Plasma ◽

Barbital Buffer

Activated protein C (APC) is an important regulator of blood coagulation in vivo. In plasma this serine protease is slowly inhibited by a specific inhibitor, activated protein C inhibitor (PCI), (Suzuki et al. (1983) J.Biol.Chem. 258 , 163-168). We have now made monoclonal antibodies against PCI by immunizing with the APC-PCI complex. Positive clones were identified by solid phase immunoassay with 125I labelled partially purified inhibitor. After subcloning and expansion in mice, one of the monoclonal antibodies was immobilized on Sepharose 4B and used in the purification of the inhibitor. A two step purification procedure was deviced starting with passage of fresh human plasma over the column. Following extensive washing the inhibitor was eluted with 50 mM triethylamine- HCI,0.5 M NaCl, pH 11.0, from the column together with a small amount of high molecular weight material. After gel filtration on a column packed with AcA 44 the inhibitor appeared homogenous on SDS - PAGE. Approximatly 0.5 mg inhibitor was obtained from 200 ml of fresh plasma. The apparent Mr of the inhibitor was 57000 kDa on SDS -PAGE. The purified protein formed a complex (Mr =110000 kDa) with human APC. At the same time a band (Mr = 54000 kDa ) appeared that represented the modified inhibitor. When analyzed on agarose gel electrophoresis (75mM barbital buffer, 2mM EDTA at pH 8.7 ) the PCI migrated to the β2- region, whereas the modified inhibitor had a slightly more anodal mobility. The APC-PCI komplex migrated to the α2- region.Two immunoradiometric assays were constructed with the monoclonal antibodies. One measured the complexes between APC and PCI while the other one measured the total amount of PCI present. These assays were used to study complex formation in buffer and plasma.

Download Full-text

Solid-phase iodothyronine-5′-deiodinase (5′-D) assays applied in production of monoclonal antibodies against 5′-D

Journal of Endocrinology ◽

10.1677/joe.0.1180439 ◽

1988 ◽

Vol 118 (3) ◽

pp. 439-445

Author(s):

N. Boye ◽

H. Frøkiaer ◽

K. Kaltoftt ◽

P. Laurberg

Keyword(s):

Monoclonal Antibodies ◽

Microsomal Fraction ◽

Solid Phase ◽

Rat Kidney ◽

Enzyme Linked Immunosorbent Assay ◽

Cortical Tissue ◽

Spleen Cells ◽

Hybridoma Cell Lines ◽

Mouse Myeloma

ABSTRACT Characterization of iodothyronine-deiodinating enzymes has been difficult due to loss of enzyme activity during purification. To obtain a new tool for studying these enzymes we investigated the possibility of developing monoclonal antibodies (MAbs) against iodothyronine-5′-deiodinase (5′-D). Two specific and sensitive solid-phase microassays were developed for screening hybridoma supernatants for the presence of antibodies inhibiting rat kidney 5′-D. and antibodies binding to but not inhibiting the enzyme. BALB/c mice were immunized with a 3-((3-cholamidopropyl) -dimethylammonio) -1- propanesulphonate (CHAPS)-solubilized 5′-D-rich membrane preparation from rat kidney cortical tissue. Spleen cells were fused with NSI-Ag 4/1 mouse myeloma cells by means of polyethylene glycol. Two hybridoma cell lines (AF5 and BE8) secreting MAbs specifically binding to without inhibiting 5′-D were produced. The AF5 antibody was of the IgG2a subclass and the BE8 antibody of the IgG2b subclass. Binding of one of the antibodies to the enzyme inhibited binding of the other in both an enzyme-linked immunosorbent assay (ELISA) and a specific enzymebinding assay. CHAPS-solubilized kidney microsomal fraction was chromatographed on a Sepharose 6B column. Elution profiles of 5′-D activity and MAb-binding antigens, as measured by ELISA with both AF5 and BE8, were identical. Monoclonal antibodies should be valuable probes in the further elucidation of the nature of the iodothyronine-deiodinating activity in various tissues. J. Endocr. (1988) 118, 439–445

Download Full-text

Production of monoclonal antibodies against the N-terminal glycopeptide of porcine pro-opiomelanocortin: their use for solid-phase radioimmunoassay, western blotting, and immunogold cytochemistry

Biochemistry and Cell Biology ◽

10.1139/o91-008 ◽

1991 ◽

Vol 69 (1) ◽

pp. 58-65 ◽

Cited By ~ 6

Author(s):

Daniel Chevrier ◽

Edith Lemieux ◽

Mireille Fyfe ◽

N. Labonté ◽

Max Zollinger ◽

...

Keyword(s):

High Pressure ◽

Monoclonal Antibodies ◽

Liquid Chromatography ◽

Solid Phase ◽

Pituitary Hormones ◽

Reversed Phase ◽

Thin Sections ◽

Gold Particles ◽

Solid Phase Radioimmunoassay ◽

Mouse Antibody

We have prepared two monoclonal antibodies for the N-terminal glycopeptide of pro-opiomelanocortin 1–77 (N-POMC(1–77)) purified from porcine pituitaries. Antibody 1–244 recognizes an epitope located within the γ3-melanotropin (γ3-MSH or POMC(51–77)) sequence, whereas antibody 2–197 binds specifically to a determinant in the 1–49 region of N-POMC. These monoclonal antibodies were used to construct a two-site solid-phase radioimmunoassay that can detect as little as 50 pg of N-POMC(1–77). The assay is linear between 0.5 and 5 ng of porcine peptide and recognizes equally well the homologous peptides purified from human and bovine pituitaries. The assay has been used to analyze reversed-phase high pressure liquid chromatography fractions of crude bovine pituitary extracts and detected a peptide with chromatographic properties identical to those of N-POMC(1–77). When used to stain immunoblots of bovine intermediate pituitary extracts, both the 2–197 and 1–244 antibodies could recognize a major peptide comigrating with purified N-POMC(1–77). In addition, antibody 2–197 also detected a peptide with a mobility similar to that of standard N-POMC(1–49). When used in conjunction with a second anti-mouse antibody coupled to colloidal gold particles, antibody 2–197 stained N-POMC immunoreactive material located in granules in thin sections of pituitary.Key words: monoclonal antibodies, γ-melanotropin, pituitary hormones.

Download Full-text

Isoforms of C-protein in adult chicken skeletal muscle: detection with monoclonal antibodies.

The Journal of Cell Biology ◽

10.1083/jcb.95.1.78 ◽

1982 ◽

Vol 95 (1) ◽

pp. 78-84 ◽

Cited By ~ 82

Author(s):

F C Reinach ◽

T Masaki ◽

S Shafiq ◽

T Obinata ◽

D A Fischman

Keyword(s):

Monoclonal Antibodies ◽

Latissimus Dorsi ◽

Solid Phase ◽

Adult Chicken ◽

C Protein ◽

Variant Isoforms ◽

Anterior Latissimus Dorsi ◽

Solid Phase Radioimmunoassay ◽

Chicken Skeletal Muscle ◽

Chromatographic Elution

Monoclonal antibodies (McAbs) specific for the C-proteins of chicken pectoralis major and anterior latissimus dorsi (ALD) muscles have been produced and characterized. Antibody specificity was demonstrated by solid phase radioimmunoassay (RIA), immunoblots, and immunofluorescence cytochemistry. Both McAbs MF-1 (or MF-21) and ALD-66 bound to myofibrillar proteins of approximately 150,000 daltons; the former antibody reacted with pectoralis but not ALD myofibrils, whereas the latter recognized ALD but not pectoralis myofibrils. Chromatographic elution of the antigens from DEAE-Sephadex, and their distribution in the A-band, support the conclusion that both of these antibodies recognize variant isoforms of C-protein. Since both McAbs react with a protein of similar molecular weight in the A-band of all myofibrils of the posterior latissimus dorsi (PLD) muscle, we suggest that either another isoform of C-protein exists in the PLD muscle or both pectoralis and ALD-like isoforms coexist in the A-bands of PLD muscle.

Download Full-text