Fluorescent nanosensor for in situ detection of phosphate and alkaline phosphatase in mice with parathyroid dysfunction

2020 ◽  
Vol 56 (16) ◽  
pp. 2431-2434
Author(s):  
Jiheng Xu ◽  
Hui Zhang ◽  
Wei Zhang ◽  
Ping Li ◽  
Wen Zhang ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Fluorescence Imaging ◽  
Parathyroid Dysfunction ◽  
In Situ Detection

A composite nanosensor based on Zr(iv)-MOFs and PNPP was developed, which successfully applied for the in situ fluorescence imaging of phosphate and ALP levels in mice with parathyroid dysfunction.

scholarly journals Conjugate formation in urea: coupling of insoluble peptides to alkaline phosphatase for ELISA and in situ detection of antibody-forming cells.

1991 ◽  
Vol 39 (7) ◽  
pp. 987-992 ◽  
Author(s):  
K Gerritse ◽  
M Fasbender ◽  
W Boersma ◽  
E Claassen
Keyword(s):  
Alkaline Phosphatase ◽  
Synthetic Peptide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sufficient Degree ◽  
Antipeptide Antibody ◽  
In Situ Detection ◽  
Antibody Secreting Cells

We report here a new method to produce synthetic peptide/alkaline phosphatase (AP) conjugates in the presence of urea. The method allows the use of peptides that are not soluble to a sufficient degree in aqueous buffers. The presence of 8 M urea during the construction of the synthetic peptide/AP conjugates does not influence enzyme activity nor the affinity of the anti-peptide antibodies for the conjugated peptide. We demonstrate that these synthetic peptide/AP conjugates can be used for detection of specific antipeptide antibody-forming cells (AFC) in vivo. This method for constructing enzyme conjugates with insoluble proteins or peptides suggest not only new possibilities for detection of specific AFC in vivo but also for applications in receptor-ligand studies, ELISA (enzyme-linked immunosorbent assay), and spot ELISA for detection of antibody-secreting cells in vitro.

scholarly journals A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ.

1993 ◽  
Vol 41 (2) ◽  
pp. 313-317 ◽  
Author(s):  
Z Huang ◽  
W You ◽  
R P Haugland ◽  
V B Paragas ◽  
N A Olson ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Line ◽  
Egf Receptor ◽  
Egf Receptors ◽  
Fluorogenic Substrate ◽  
Epidermal Growth ◽  
In Situ Detection ◽  
Apase Activity ◽  
Human Epidermoid Carcinoma

We describe here the in situ detection of alkaline phosphatase (APase) activity with a new fluorogenic substrate, 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (CPPCQ). CPPCQ is very soluble and colorless. APase converts it into a rapidly precipitating product, whose strong fluorescence marks the sites of APase activity. The detected APase was either a probing enzyme anchored to epidermal growth factor (EGF) receptors of fixed human epidermoid carcinoma cell line (A431) by biotinylated EGF and streptavidin-APase conjugates or an endogenous marker existing in a fixed canine kidney cell line (MDCK). With CPPCQ staining, the EGF receptors and the endogenous APase were both visualized by fluorescence microscopy as contrasting, photostable, and well-resolved fluorescent stains. The EGF receptor staining was specific since it could be blocked by excessive unlabeled EGF. In contrast, fluorescein-labeled EGF failed to specifically stain the EGF receptors under the same fluorescent microscope. The endogenous APase staining with CPPCQ was sensitive to heating, levamisole and L-homoarginine, showing an APase tissue specificity of the liver/bone/kidney type. Therefore, CPPCQ appears to be a novel substrate dye for sensitive fluorescence APase histochemistry.

Simultaneous in situ detection of alkaline phosphatase activity and polyphosphate in arbuscules within arbuscular mycorrhizal roots

2007 ◽  
Vol 34 (9) ◽  
pp. 803 ◽  
Author(s):  
Rintaro Funamoto ◽  
Katsuharu Saito ◽  
Hiroshi Oyaizu ◽  
Masanori Saito ◽  
Toshihiro Aono
Keyword(s):  
Alkaline Phosphatase ◽  
Inorganic Phosphate ◽  
Host Plants ◽  
Arbuscular Mycorrhizal ◽  
Am Fungi ◽  
Alp Activity ◽  
Mycorrhizal Roots ◽  
In Situ Detection ◽  
Labelling Method

Inorganic phosphate (Pi) metabolism in arbuscules of arbuscular mycorrhizal (AM) fungi is not well understood, although recent research has revealed that host plants absorb Pi around arbuscules with mycorrhiza-specific transporters. Therefore, we analysed the localisation of polyphosphate (polyP) and alkaline phosphatase (ALP) activity in arbuscules, which could be indicators of Pi metabolism. We developed a dual-labelling method for polyP and ALP activity, i.e. first labelling with fluorescent probes 4′,6-diamidino-2-phenyl-indole dihydrochloride (DAPI) and then labelling with enzyme-labelled fluorescence (ELF97). The dual-labelling method made it possible to observe polyP and ALP activity signals simultaneously in mycorrhizal roots. The dual-labelling method revealed that ALP activity was mainly observed in mature arbuscules where polyP was rarely observed. The expression of the AM fungal ALP gene was suppressed in the knockdown plants of an AM-inducible Pi-transporter, and there was much polyP in arbuscules that showed low ALP activity. These topological observations suggest that there may be some relationships between polyP metabolism and ALP activity in arbuscules, and that these are, in part, controlled by Pi uptake by plants via the AM-inducible Pi-transporter.

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

Rapid quantification and in situ detection of nitrifying bacteria in biofilms by monoclonal antibody method

2000 ◽  
Vol 41 (4-5) ◽  
pp. 301-308 ◽  
Author(s):  
N. Noda ◽  
H. Ikuta ◽  
Y. Ebie ◽  
A. Hirata ◽  
S. Tsuneda ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Fluorescent Antibody Technique ◽  
Antibody Technique ◽  
Antibody Method ◽  
In Situ Detection ◽  
Rapid Quantification

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

scholarly journals Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

2021 ◽  
Vol 10 (2) ◽  
pp. 319
Author(s):  
Hee Cheol Yang ◽  
Won Jong Rhee
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Molecular Beacon ◽  
Disease Diagnosis ◽  
Single Step ◽  
Flow Cytometer ◽  
Surface Proteins ◽  
Biomarker Detection ◽  
In Situ Detection

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer.

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