scholarly journals Generation and Characterization of Monoclonal Antibodies against the Transcription Factor Nkx6.1

2006 ◽  
Vol 54 (5) ◽  
pp. 567-574 ◽  
Author(s):  
Inger L. Pedersen ◽  
Rasmus Klinck ◽  
Jacob Hecksher-Sorensen ◽  
Stefan Zahn ◽  
Ole D. Madsen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Monoclonal Antibodies ◽  
Rabbit Serum ◽  
Β Cells ◽  
Double Labeling ◽  
Polyclonal Rabbit ◽  
Pancreatic Epithelium ◽  
Hybridoma Cell Lines ◽  
And Function

We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine β-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing β-cells, making this transcription factor a unique β-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/κ subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the β-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies. (J Histochem Cytochem 54:567-574, 2006)

Characterization of monoclonal antibodies specific to the transcription factor ETS-2 protein

2003 ◽  
Vol 86 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Elaine Sanij ◽  
Bernadette Scott ◽  
Trevor Wilson ◽  
Dakang Xu ◽  
Paul Hertzog ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Monoclonal Antibodies

Combination of Anti-CD4 with Anti-LFA-1 and Anti-CD154 Monoclonal Antibodies Promotes Long-Term Survival and Function of Neonatal Porcine Islet Xenografts in Spontaneously Diabetic NOD Mice

2007 ◽  
Vol 16 (8) ◽  
pp. 787-798 ◽  
Author(s):  
Hossein Arefanian ◽  
Eric B. Tredget ◽  
Ray V. Rajotte ◽  
Gregory S. Korbutt ◽  
Ron G. Gill ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Nod Mice ◽  
Β Cells ◽  
Short Term ◽  
Term Survival ◽  
Long Term Survival ◽  
Porcine Islet ◽  
Term Administration ◽  
And Function

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of pancreatic islet β-cells, which are required for the production of insulin. Islet transplantation has been shown to be an effective treatment option for T1DM; however, the current shortage of human islet donors limits the application of this treatment to patients with brittle T1DM. Xenotransplantation of pig islets is a potential solution to the shortage of human donor islets provided xenograft rejection is prevented. We demonstrated that a short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) was highly effective in preventing rejection of neonatal porcine islet (NPI) xenografts in non-autoimmune-prone B6 mice. However, the efficacy of this therapy in preventing rejection of NPI xenografts in autoimmune-prone nonobese diabetic (NOD) mice is not known. Given that the current application of islet transplantation is for the treatment of T1DM, we set out to determine whether a combination of anti-LFA-1 and anti-CD154 mAbs could promote long-term survival of NPI xenografts in NOD mice. Short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs, which we found highly effective in preventing rejection of NPI xenografts in B6 mice, failed to promote long-term survival of NPI xenografts in NOD mice. However, addition of anti-CD4 mAb to short-term treatment of a combination of anti-LFA-1 and anti-CD154 mAbs resulted in xenograft function in 9/12 animals and long-term graft (>100 days) survival in 2/12 mice. Immunohistochemical analysis of islet grafts from these mice identified numerous insulin-producing β-cells. Moreover, the anti-porcine antibody as well as autoreactive antibody responses in these mice was reduced similar to those observed in naive nontransplanted mice. These data demonstrate that simultaneous targeting of LFA-1, CD154, and CD4 molecules can be effective in inducing long-term islet xenograft survival and function in autoimmune-prone NOD mice.

Characterization of two porcine proinsulin reactive monoclonal antibodies by immunostaining of β-cells in pancreatic sections of different species

1992 ◽  
Vol 93 (2) ◽  
pp. 433-440 ◽  
Author(s):  
Petra Augstein ◽  
Birgitt Braun ◽  
Brigitte Ziegler ◽  
Klaus-Peter Woltanski ◽  
Manfred Ziegler
Keyword(s):  
Monoclonal Antibodies ◽  
Β Cells

scholarly journals Identification of osteoclast-specific monoclonal antibodies.

1985 ◽  
Vol 100 (5) ◽  
pp. 1592-1600 ◽  
Author(s):  
M J Oursler ◽  
L V Bell ◽  
B Clevinger ◽  
P Osdoby
Keyword(s):  
Monoclonal Antibodies ◽  
Giant Cells ◽  
Mononuclear Phagocyte ◽  
Antibody Binding ◽  
Enzyme Linked Immunosorbent Assay ◽  
Form And Function ◽  
Titration Curves ◽  
Specific Antigens ◽  
And Function

Studies on the origin, identification, and characterization of osteoclasts have been difficult. This is in part due to a lack of definitive osteoclast markers and the similarity of these cells in form and function to cells of the mononuclear phagocyte system. To solve this problem, we inoculated isolated chick osteoclasts into mice to generate osteoclast-specific monoclonal antibodies. Supernatants from growth-positive hybridomas were screened by indirect immunofluorescent methods against cultured osteoclasts, monocyte-derived multinucleated giant cells, cultured monocytes, fibroblasts, and limb mesenchyme. Select hybridomas were cloned to produce 375 clones, which were analyzed as described above. Antibody from select clones was also reacted with paraffin sections of bone. In addition, two clones have been analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Antibody binding from an osteoclast-specific clone and a clone reactive with osteoclasts, giant cells, and cultured monocytes (as determined by immunohistochemical assay) was confirmed by antibody-binding and titration curves quantitated by ELISA. The above studies demonstrate that osteoclast specific antigens exist, and that osteoclasts, giant cells, and cultured monocytes share common determinants not found on other cells screened.

scholarly journals Recent Advances in the Discovery and Function of Antimicrobial Molecules in Platelets

2021 ◽  
Vol 22 (19) ◽  
pp. 10230
Author(s):  
Alba S. Aquino-Domínguez ◽  
María de los A. Romero-Tlalolini ◽  
Honorio Torres-Aguilar ◽  
Sergio R. Aguilar-Ruiz
Keyword(s):  
Antimicrobial Properties ◽  
Human Platelets ◽  
Rabbit Serum ◽  
Functional Evaluation ◽  
Host Defense Peptides ◽  
Vascular Integrity ◽  
Activated Platelets ◽  
And Function ◽  
Identification And Characterization

The conventional function described for platelets is maintaining vascular integrity. Nevertheless, increasing evidence reveals that platelets can additionally play a crucial role in responding against microorganisms. Activated platelets release molecules with antimicrobial activity. This ability was first demonstrated in rabbit serum after coagulation and later in rabbit platelets stimulated with thrombin. Currently, multiple discoveries have allowed the identification and characterization of PMPs (platelet microbicidal proteins) and opened the way to identify kinocidins and CHDPs (cationic host defense peptides) in human platelets. These molecules are endowed with microbicidal activity through different mechanisms that broaden the platelet participation in normal and pathologic conditions. Therefore, this review aims to integrate the currently described platelet molecules with antimicrobial properties by summarizing the pathways towards their identification, characterization, and functional evaluation that have promoted new avenues for studying platelets based on kinocidins and CHDPs secretion.

scholarly journals Production and Characterization of Novel Anti-Prostate-specific Antigen (PSA) Monoclonal Antibodies That Do Not Detect Internally Cleaved Lys145-Lys146 Inactive PSA

2000 ◽  
Vol 46 (10) ◽  
pp. 1610-1618 ◽  
Author(s):  
Pauliina Nurmikko ◽  
Ville Väisänen ◽  
Timo Piironen ◽  
Sari Lindgren ◽  
Hans Lilja ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Line ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Cancer Cell Line ◽  
Peptide Sequence ◽  
Free Psa ◽  
Hybridoma Cell Lines ◽  
First Time

Abstract Background: The nature of free, uncomplexed prostate-specific antigen (PSA) in the circulation is still unknown. In this study, we developed novel anti-PSA antibodies using PSA produced by a metastasized cancer cell line, LNCaP, as an immunogen. Methods: Hybridoma cell lines were screened with different methods that aimed at finding antibodies specific for the forms of free PSA produced by LNCaP cell line. Obtained antibodies were further studied for their characteristics related to previously characterized monoclonal antibodies. Results: Numerous anti-PSA antibodies were obtained, of which four represented unique epitopes previously unrecognized by us. One free-PSA-specific antibody was bound to PSA on two distinct epitopes, and one antibody was bound to the carboxyl-terminal peptide of PSA. Two antibodies were found to bind to the peptide sequence adjacent to the internal cleavage site Lys145-Lys146. These antibodies failed to recognize internally cleaved PSA at Lys145-Lys146. We could not find anti-proPSA antibodies despite the fact that LNCaP PSA contained more than one-half of the zymogen form of PSA. Conclusions: We report, for the first time, novel anti-PSA antibodies that do not recognize internally cleaved PSA at Lys145-Lys146 and thus are specific for intact, unclipped PSA.

Characterization of the growth hormone-binding protein of human serum using a panel of monoclonal antibodies

1989 ◽  
Vol 123 (2) ◽  
pp. 327-332 ◽  
Author(s):  
R. Barnard ◽  
P. Quirk ◽  
M. J. Waters
Keyword(s):  
Monoclonal Antibodies ◽  
Human Serum ◽  
Binding Capacity ◽  
Binding Protein ◽  
Cross Reactivity ◽  
Rabbit Liver ◽  
Rabbit Serum ◽  
Gh Receptor ◽  
Hormone Binding Protein

ABSTRACT A panel of monoclonal antibodies (MAbs) reactive with distinct epitopes on the rabbit liver GH receptor and rabbit serum GH-binding protein (GHBP) were tested for cross-reactivity with the GHBP from human serum. Four of seven MAbs reacted with the human serum GHBP. Immunoprecipitation of the human binding protein enabled hormonal specificity identical to that previously reported for human GH receptors to be demonstrated. Scatchard analyses of 125I-labelled human GH binding to the serum GHBP were carried out with correction made for endogenous human GH which was measured by radioimmunoassay of each serum sample. This approach yielded the first reliable estimates of the affinity and capacity of the human GHBP. The binding capacity (mean ± s.e.m.) of female sera (804±126 pmol/l; n= 6) was greater than that of male sera (505 ± 36 pmol/l; n=9; P < 0·02). The affinity of the GHBP was 0·91 ±0·10 litres/nmol (n= 15). The presence of multiple epitopes common to the human serum GHBP and the rabbit liver GH receptor is consistent with identity between the extracellular domains of the human GHBP and the human GH receptor, as is the case for the rabbit GHBP and GH receptor. Journal of Endocrinology (1989) 123, 327–332

scholarly journals Development and Characterization of Monoclonal Antibodies Specific to the Serotonin 5-HT2A Receptor

1998 ◽  
Vol 46 (7) ◽  
pp. 811-824 ◽  
Author(s):  
Chun Wu ◽  
Elizabeth J. Yoder ◽  
Jean Shih ◽  
Kevin Chen ◽  
Peter Dias ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Rat Brain ◽  
Pyramidal Neurons ◽  
Expression System ◽  
Molecular Size ◽  
Baculovirus Expression System ◽  
Differential Staining ◽  
Receptor Subtypes ◽  
And Function

Serotonin (5-hydroxytryptamine, 5-HT) mediates many functions of the central and peripheral nervous systems by its interaction with specific neuronal and glial receptors. Fourteen serotonin receptors belonging to seven families have been identified through physiological, pharmacological, and molecular cloning studies. Monoclonal antibodies (MAbs) specific for each of these receptor subtypes are needed to characterize their expression, distribution, and function in embryonic, adult, and pathological tissues. In this article we report the development and characterization of MAbs specific to the serotonin 5-HT2A receptor. To generate MAbs against 5-HT2AR, mice were immunized with the N-terminal domain of the receptor. The antigens were produced as glutathionine S-transferase (GST) fusion proteins in insect cells using a Baculovirus expression system. The hybridomas were initially screened by ELISA against the GST-5-HT2AR recombinant proteins and subsequently against GST control proteins to eliminate clones with unwanted reactivity. They were further tested by Western blotting against recombinant GST-5-HT2AR, rat and human brain lysate, and lysate from cell lines transfected with 5-HT2AR cDNA. One of the MAbs G186-1117, which recognizes a portion of the 5-HT2AR N-terminus, was selected for further characterization. G186-1117 reacted with a band of molecular size 55 kD corresponding to the predicted size of 5-HT2AR in lysates from rat brain and a 5-HT2AR-transfected cell line. Its specificity was further confirmed by adsorption of immunoreactivity with recombinant 5-HT2AR but not with recombinant 5-HT2BR and 5-HT2CR. Rat brain sections and Schwann cell cultures were immunohistochemically labeled with this MAb. G186-1117 showed differential staining in various regions of the rat brain, varying from regions with no staining to regions of intense reactivity. In particular, staining of cell bodies and dendrites of the pyramidal neurons in the cortex was observed, which is in agreement with observations of electrophysiological studies.

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