scholarly journals Distribution of the Na(+)-Ca2+ exchange protein in mammalian cardiac myocytes: an immunofluorescence and immunocolloidal gold-labeling study

1992 ◽  
Vol 117 (2) ◽  
pp. 337-345 ◽  
Author(s):  
JS Frank ◽  
G Mottino ◽  
D Reid ◽  
RS Molday ◽  
KD Philipson
Keyword(s):  
Monoclonal Antibodies ◽  
Guinea Pig ◽  
Immunoelectron Microscopy ◽  
Polyclonal Antibodies ◽  
Heart Cells ◽  
Spleen Cells ◽  
Cardiac Sarcolemma ◽  
Immunofluorescence Studies ◽  
Tubular System ◽  
Mouse Myeloma

The present study reports on the location of the Na(+)-Ca2+ exchanger in cardiac sarcolemma with immunofluorescence and immunoelectron microscopy. Both polyclonal and monoclonal antibodies to the Na(+)-Ca2+ exchanger were used. The mAb was produced from a hybridoma cell line generated by the fusion of mouse myeloma NS-1 cells with spleen cells from a mouse repeatedly immunized with isolated reconstituted canine cardiac Na(+)-Ca2+ exchanger (Philipson, K. D. S. Longoni, and R. Ward. 1988. Biochim. Biophys. Acta. 945:298-306). The polyclonal antibody has been described previously and reacts with three proteins (70, 120, 160 kD) in cardiac sarcolemma associated with the Na(+)-Ca2+ exchanger (Nicoll, D. A., S. Longoni, and K. D. Philipson. 1990. Science (Wash. DC). 250:562-565). Both the monoclonal and the polyclonal antibodies appear to react with extracellular facing epitopes in the cardiac sarcolemma. Immunofluorescence studies showed labeling of the transverse tubular membrane and patchy labeling of the peripheral sarcolemma. The immunofluorescent labeling clearly delineates the highly interconnected T-tubular system of guinea pig myocytes. This localization of the exchanger to the sarcolemma, with an apparent high density in the transverse tubules, was also seen with immunoelectron microscopy. It is of great interest that the Na(+)-Ca2+ exchanger, as the main efflux route for Ca2+ in heart cells, would be abundantly located in sarcolemma closest to the release of Ca2+.

scholarly journals Production and Characterization of Monoclonal Antibodies to Psittacine Beak and Feather Disease Virus

1992 ◽  
Vol 4 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Branson W. Ritchie ◽  
Frank D. Niagro ◽  
Kenneth S. Latimer ◽  
W. L. Steffens ◽  
Denise Pesti ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Myeloma Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Elisa System ◽  
Mouse Myeloma Cell Line ◽  
Feather Disease Virus ◽  
Mouse Myeloma

Monoclonal antibodies specific for the virus that causes psittacine beak and feather disease (PBFD) were produced by fusing spleen cells from mice immunized with purified concentrated PBFD virus with mouse myeloma cell line Sp2/0. The resulting hybridomas were tested for reactivity against whole purified virus by an enzyme-linked immunosorbent assay (ELISA) system. Four clones, designated 15H8, 8E3, 11G12, and 2C3, were subcloned by limiting dilution. Isotyping indicated that clone 15H8 was secreting IgG, whereas the remaining clones secreted IgM. The secreted immunoglobulins were characterized by reactivity against purified PBFD virus using immunoblotting procedures, by immunohistochemical staining of virus-induced lesions in infected tissues, and by inhibition of PBFD virus agglutination of cockatoo erythrocytes. Antibodies secreted by clones 15H8 and 8E3 had the strongest activity against purified whole virus. Only immunoglobulin secreted by the clone 15H8 could be used to detect viral antigen in infected tissues. None of the monoclonal antibodies had hemagglutination-inhibition activity.

Monoclonal Antibodies Towards the Fast Inhibitor of Tissue Plasminogen Activator from Human Plasma and Serum

1986 ◽  
Vol 55 (03) ◽  
pp. 383-387 ◽  
Author(s):  
G Urdén ◽  
Ulla Johansson ◽  
Joanna Chmielewska ◽  
J Brandt ◽  
B Wiman
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Hybridoma Cells ◽  
Spleen Cells ◽  
Inhibitor Complex ◽  
Tissue Plasminogen ◽  
Different Levels ◽  
Mouse Myeloma

SummaryHybridoma cells were produced by fusing mouse myeloma cells (SP 2/0 - Ag 14) with spleen cells from a Balb/c mouse, previously immunized with the partially purified complex between tissue plasminogen activator (t-PA) and its fast inhibitor from human plasma (serum). Screening with a radioimmunoassay revealed a number of hybridomas secreting antibodies directed towards the complex. Of these, about 1/3 reacted both with the complex and t-PA, whereas about 2/3 reacted only with the complex. Three of the latter hybridomas, producing antibodies directed towards the inhibitor-moiety in the complex have been cloned and the antibodies were studied in detail. PA-inhibitor activity in plasma or serum and t-PA/PA-inhibitor complex could be specifically adsorbed on all three insolubilized monoclonal antibodies (MCI, MC2 and MC3). None of the antibodies seems to be directed against structures of vital importance for the functional activity of the PA-inhibitor. In accordance with this finding the antibody with the highest avidity (MCI) reacts equally well with the PA-inhibitor alone or in complex with t-PA. A radioimmunoassay was devloped with this antibody and significant displacement was obtained with samples with PA-inhibitor concentrations above 2 AU/mL. In 13 plasma samples with different levels of PA-inhibitory activity a significant correlation was obtained when comparing this activity with the PA-inhibitor antigen as measured with the radioimmunoassay (r = 0.88).

Development of the ELISAs for detection of hormone-disrupting chemicals

2000 ◽  
Vol 42 (7-8) ◽  
pp. 81-88 ◽  
Author(s):  
Y. Goda ◽  
A. Kobayashi ◽  
K. Fukuda ◽  
S. Fujimoto ◽  
M. Ike ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Phthalate Esters ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Myeloma Cells ◽  
Structural Resemblance ◽  
Reaction Test ◽  
Mouse Myeloma

Six kinds of enzyme-linked immunosorbent assay (ELISA) systems were developed for the quantitative analysis of hormone-disrupting chemicals (HDCs), such as estrogen (ES: the total amount of estrone (E1), 17 β-estra (E2) and estriol (E3)), E2, bisphenol A (BPA), alkylphenol (AP), phthalate esters (PE) and chlorophenols (CP). To generate specific monoclonal antibodies against BPA, AP, PE, CP, hybridoma cells were produced by the fusion of mouse myeloma cells and spleen cells from mice immunized with carboxylated derivatives, while anti E2 monoclonal antibody was selected from those available on the market, and anti ES monoclonal antibody was purchased from Teikoku Hormone Mfg Co. Ltd. The detection limits of ES, E2, BPA, AP, PE and CP ELISAs were 0.1, 0.1, 5, 10, 200, 10 μg/L, when E2, E2, BPA, Nonylphenol (NP), Dibutylphthalate (DBP), 2,4-CP were used as standard, respectively, and the specificity of each ELISA was confirmed with the cross-reaction test using several compounds which have structural resemblance to the compounds of interest.

scholarly journals Monoclonal antibodies to human platelet glycoprotein IIb beta that initiate distinct platelet responses

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1112-1119 ◽  
Author(s):  
LK Jennings ◽  
DR Phillips ◽  
WS Walker
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Membrane Glycoprotein ◽  
Spleen Cells ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Mouse Myeloma

Abstract Hybridomas secreting monoclonal antibodies (MoAbs) to human platelet membrane glycoprotein IIb (GPIIb) were prepared by fusing cells of a mouse myeloma line to spleen cells from a BALB/c mouse immunized with purified GPIIb. Six of the hybridomas secreted MoAbs that recognized epitopes on the 23,000-dalton, disulfide-linked subunit of GPIIb, GPIIb beta. All six of these MoAbs agglutinated platelets in the absence of calcium. The agglutination titers of three of the MoAbs, however, were enhanced between 2 and 6 log2 dilutions when titrated in the presence of mmol/L of calcium. The enhancement in titer was the result of MoAb- induced platelet activation followed by platelet aggregation, a reaction that could also be initiated by the monovalent Fab fragments prepared from one of the MoAbs. The MoAbs did not significantly agglutinate platelets from patients with Glanzmann's thrombasthenia, confirming biochemical evidence that there is a paucity of GPIIb beta in the membranes of these cells. Our results show that MoAbs to epitopes on GPIIb beta initiate distinct platelet responses; therefore, they should be useful for studying the ways in which regions of surface glycoproteins are involved in platelet-platelet interactions. In addition, these reagents may prove of value in diagnosing and typing patients with Glanzmann's thrombasthenia.

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

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

scholarly journals Monoclonal antibodies to human platelet glycoprotein IIb beta that initiate distinct platelet responses

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1112-1119
Author(s):  
LK Jennings ◽  
DR Phillips ◽  
WS Walker
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Membrane Glycoprotein ◽  
Spleen Cells ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Mouse Myeloma

Hybridomas secreting monoclonal antibodies (MoAbs) to human platelet membrane glycoprotein IIb (GPIIb) were prepared by fusing cells of a mouse myeloma line to spleen cells from a BALB/c mouse immunized with purified GPIIb. Six of the hybridomas secreted MoAbs that recognized epitopes on the 23,000-dalton, disulfide-linked subunit of GPIIb, GPIIb beta. All six of these MoAbs agglutinated platelets in the absence of calcium. The agglutination titers of three of the MoAbs, however, were enhanced between 2 and 6 log2 dilutions when titrated in the presence of mmol/L of calcium. The enhancement in titer was the result of MoAb- induced platelet activation followed by platelet aggregation, a reaction that could also be initiated by the monovalent Fab fragments prepared from one of the MoAbs. The MoAbs did not significantly agglutinate platelets from patients with Glanzmann's thrombasthenia, confirming biochemical evidence that there is a paucity of GPIIb beta in the membranes of these cells. Our results show that MoAbs to epitopes on GPIIb beta initiate distinct platelet responses; therefore, they should be useful for studying the ways in which regions of surface glycoproteins are involved in platelet-platelet interactions. In addition, these reagents may prove of value in diagnosing and typing patients with Glanzmann's thrombasthenia.

scholarly journals Characterization of two monoclonal antibodies obtained after immunization with human liver mitochondrial membrane preparations

1984 ◽  
Vol 221 (3) ◽  
pp. 765-776 ◽  
Author(s):  
E E Billett ◽  
B Gunn ◽  
R J Mayer
Keyword(s):  
Monoclonal Antibodies ◽  
Human Liver ◽  
Portal Tract ◽  
Immunoperoxidase Staining ◽  
Cell Type ◽  
Spleen Cells ◽  
Mitochondrial Membranes ◽  
Particulate Distribution ◽  
Mouse Myeloma

Two monoclonal antibodies have been generated by fusion of mouse myeloma cells with spleen cells from mice immunized with human liver mitochondrial membranes. One antibody, 1H6/C12, an immunoglobulin G2a (IgG2a), binds to the inner membrane of rat hepatocyte mitochondria, and immunoperoxidase staining demonstrates that its epitope has an intracellular particulate distribution within rat and human hepatocytes and human brain neurons. The epitope reactive with 1H6/C12 is partially sensitive to proteinase digestion. The second antibody, 3F12/F2, an IgG1, binds to a contaminating cell type, namely the granulocyte, but it does not bind to monocytes, lymphocytes and red cells in human blood. This antibody reacts with cells in the portal tract and sinusoids of rat and human liver, as shown by immunoperoxidase staining. The epitope for 3F12/F2 is extremely sensitive to proteinase digestion and is only exposed when granulocytes are fixed in acetone, indicating an internal localization.

The production and characterization of monoclonal antibodies to the human thyroid microsome

1985 ◽  
Vol 105 (1) ◽  
pp. 47-NP ◽  
Author(s):  
A. P. Weetman ◽  
C. A. Gunn ◽  
D. P. Rennie ◽  
R. Hall ◽  
A. M. McGregor
Keyword(s):  
Monoclonal Antibodies ◽  
Liver Microsomes ◽  
Cross Reactivity ◽  
Immunoglobulin M ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Spleen Cells ◽  
Cell Monolayers ◽  
Mouse Myeloma

ABSTRACT By suitable immunization of mice and fusion of their spleen cells with a non-secretor mouse myeloma line, monoclonal antibodies have been produced which react with the human thyroid microsomal (M) antigen. These monoclonal antibodies showed no reactivity by enzyme-linked immunoassay with liver microsomes or thyroglobulin and their specificity was confirmed by immunolocalization studies, in which they showed the staining characteristics of human M antibodies. All four monoclonal antibodies tested were immunoglobulin M; three were cytotoxic to thyroid cell monolayers. The lack of cytotoxicity with the fourth monoclonal supports the concept that certain epitopes of the M antigen may be partially or completely absent at the thyroid cell surface. These monoclonal antibodies should permit further characterization of the thyroid M antigen in view of their absence of cross-reactivity with thyroglobulin. J. Endocr. (1985) 105, 47–52

Use of monoclonal antibodies for radioimmunoassay of water buffalo milk progesterone

1987 ◽  
Vol 54 (4) ◽  
pp. 471-477 ◽  
Author(s):  
Rosanna Capparelli ◽  
Domenico Iannelli ◽  
Aldo Bordi
Keyword(s):  
Monoclonal Antibodies ◽  
Water Buffalo ◽  
Myeloma Cell ◽  
Buffalo Milk ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Mouse Myeloma Cell ◽  
Mouse Myeloma Cell Line ◽  
The Mean ◽  
Mouse Myeloma

SummaryIn order to standardize a radioimmunoassay of milk progesterone as a routine method for confirmation of oestrus and diagnosis of pregnancy in water buffalo, monoclonal antibodies against progesterone were produced. Hybridomas were prepared by fusing spleen cells from a Balb/c mouse immunized with progesterone 11α-hemisuccinate–bovine serum albumin conjugate with the mouse myeloma cell line NS-1. Thirty wells out of 94 secreted anti-progesterone antibodies. Of the ten independent hybridomas derived, one (AF65) was suitable for the quantification of milk progesterone by radioimmunoassay. The tracer used in the assay was progesterone-11α-hemisuccinate ([2-125I]iodohistamine). The sensitivity of the assay was 50 pg/tube. The mean progesterone concentration at oestrus was 0·8±0·2 ng/ml increasing to 8·5±0·8 ng/ml 24 d later in pregnant animals.

scholarly journals Protein G-gold complex: comparative evaluation with protein A-gold for high-resolution immunocytochemistry.

1988 ◽  
Vol 36 (6) ◽  
pp. 597-607 ◽  
Author(s):  
M Bendayan ◽  
S Garzon
Keyword(s):  
Monoclonal Antibodies ◽  
High Resolution ◽  
Guinea Pig ◽  
Comparative Evaluation ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Gold Complex ◽  
Protein G ◽  
Specific Antibodies ◽  
Antigenic Sites

We combined the protein G-gold complex with several polyclonal and monoclonal antibodies for localization of various antigenic sites. The labelings were compared with those obtained using the protein A-gold complex. The results from either the immunodot experiment or immunoelectron microscopy have demonstrated that, for rabbit and guinea pig antibodies, both protein G-gold and protein A-gold complexes label several different specific antibodies with similar efficiency. However, with antibodies raised in goats or in mice, and particularly with mouse monoclonal antibodies, protein G-gold yielded intense and specific labeling, whereas protein A-gold yielded intense and specific labeling, whereas protein A-gold was very variable; it either gave weaker signals or failed to reveal any specific site or, as with one monoclonal, both protein G and protein A gave similar results. The higher affinity and versatility of protein G over protein A, established by the immunochemical approach, was confirmed by immunocytochemistry. Because of its enhanced reactivity with monoclonal antibodies and its broader affinity for polyclonal antibodies, protein G-gold complex appears to be a better and more versatile probe for high-resolution immunocytochemistry.

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