ELISA, western blotting, immunocytolocalization and immunoaffinity column for naturally occuring bioactive compounds using monoclonal antibodies

2020 ◽  
Vol 8 (4) ◽  
Author(s):  
S.-J. Shan ◽  
W. Putalun ◽  
N. Fukuda ◽  
O. Morinaga ◽  
H. Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Bioactive Compounds ◽  
Immunoaffinity Column

IMMUNOAFFINITY COLUMN FOR ISOLATION OF BIOACTIVE COMPOUNDS USING MONOCLONAL ANTIBODIES

2002 ◽  
Vol 25 (13-15) ◽  
pp. 2387-2398 ◽  
Author(s):  
Waraporn Putalun ◽  
Noriko Fukuda ◽  
Hiroyuki Tanaka ◽  
Yukihiro Shoyama
Keyword(s):  
Monoclonal Antibodies ◽  
Bioactive Compounds ◽  
Immunoaffinity Column

Cross-Linked αsγt-Chain Hybrids in Plasma Clots Studied by 1D- and 2D Electrophoresis and Western Blotting

1993 ◽  
Vol 70 (03) ◽  
pp. 438-442 ◽  
Author(s):  
B Grøn ◽  
C Filion-Myklebust ◽  
S Bjørnsen ◽  
P Haidaris ◽  
F Brosstad
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Western Blotting ◽  
Isoelectric Point ◽  
Factor Xiii ◽  
Cross Linking ◽  
2D Electrophoresis ◽  
Complex Phenomenon ◽  
Fibrinopeptide A

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

scholarly journals Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

2021 ◽  
Vol 22 (6) ◽  
pp. 3166
Author(s):  
Jwala Priyadarsini Sivaccumar ◽  
Antonio Leonardi ◽  
Emanuela Iaccarino ◽  
Giusy Corvino ◽  
Luca Sanguigno ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Cancer Biomarkers ◽  
Trypsin Digestion ◽  
Protein Fragments ◽  
Target Epitope ◽  
Potential Activity ◽  
Antibody Epitopes

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

scholarly journals Development, Characterization, and Application of Monoclonal Antibodies against Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

2010 ◽  
Vol 17 (12) ◽  
pp. 2033-2036 ◽  
Author(s):  
Dipankar Das ◽  
Sriram Kammila ◽  
Mavanur R. Suresh
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Western Blotting ◽  
Nucleocapsid Protein ◽  
Epitope Mapping ◽  
High Specificity ◽  
Diagnostic Assays ◽  
Hybridoma Technology ◽  
Recombinant Nucleocapsid Protein

ABSTRACT Five monoclonal antibodies (MAbs) against recombinant nucleocapsid protein (NP) of severe acute respiratory syndrome (SARS)-causing coronavirus (CoV) were developed by hybridoma technology. Epitope mapping by Western blotting showed that these anti-SARS-CoV NP MAbs bind to distinct domains of NP. These anti-SARS-CoV NP MAbs, with their high specificity, are potentially ideal candidates for developing early and sensitive diagnostic assays for SARS-CoV.

Development of antibodies for N-(1-deoxy-D-fructos-1-yl) fumonisin B1 and cross-reaction with modified fumonisins

2018 ◽  
Vol 11 (4) ◽  
pp. 493-502 ◽  
Author(s):  
C.M. Maragos ◽  
K.K. Sieve ◽  
M. Busman
Keyword(s):  
Monoclonal Antibodies ◽  
Indirect Elisa ◽  
Fumonisin B1 ◽  
Cross Reactivity ◽  
Cross Reaction ◽  
Immunoaffinity Column ◽  
Carbon Backbone ◽  
Matrix Components ◽  
Covalent Modifications

Fumonisins are a group of mycotoxins that are routinely found worldwide in commodities such as maize. The group, which has many members, is generally characterised by the presence of one or more tricarballylic acid groups esterified to a long carbon backbone. The diversity of this group of toxins is further augmented by their ability to interact with matrix components non-covalently and to form covalent products with matrix constituents, such as carbohydrates and proteins. Covalent modifications to the toxins make it more difficult to assess the total amounts that may be present in a commodity. We developed monoclonal antibodies (Mabs) against a known product of the reaction of fumonisin B1 (FB1) with glucose: N-(1-deoxy-D-fructos-1-yl) fumonisin B1 (NDFrc-FB1). Similar reactions were used to produce fructosyl-analogs of fumonisins B2 and B3, as well as galactose, maltose, and rhamnose analogs of FB1. These analogs were tested in a competitive indirect ELISA for cross-reactivity towards one of the developed antibodies (Mab 213221). All of the carbohydrate analogs cross-reacted with the Mab, at levels ranging from 75% (the FB3 analog derived from D-glucose) to 181% (the FB1 analog derived from maltose). These results suggested the assay was capable of binding to a wide variety of fumonisin-carbohydrate derivatives. The same antibody was incorporated into an immunoaffinity column that was used to isolate modified fumonisins from a sample of naturally contaminated maize. These results demonstrate the potential to isolate and detect modified fumonisins and will facilitate efforts to determine the frequency of the occurrence of these compounds in maize.

A Tropical Lichen, Dirinaria consimilis Selectively Induces Apoptosis in MCF-7 Cells through the Regulation of p53 and Caspase-Cascade Pathway

2020 ◽  
Vol 20 (10) ◽  
pp. 1173-1187
Author(s):  
Anil K. Shendge ◽  
Sourav Panja ◽  
Tapasree Basu ◽  
Nripendranath Mandal
Keyword(s):  
Western Blotting ◽  
Bioactive Compounds ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Phytochemical Analysis ◽  
Crude Mortality Rate ◽  
Crude Mortality ◽  
Caspase Cascade ◽  
Mcf 7

Background: Breast cancer is the most leading cause of death, with 49.9% of crude incidence rate and 12.9% of crude mortality rate. Natural resources have been extensively used throughout history for better and safer treatment against various diseases. Objectives: The present study was aimed to investigate the antioxidant and anticancer potential of a tropical lichen Dirinaria consimilis (DCME) and its phytochemical analysis. Methods: The DCME was preliminarily evaluated for ROS, and RNS scavenging potential. Furthermore, DCME was evaluated for in vitro anticancer activity through cell proliferation assay, cell cycle analysis, annexin V/PI staining, morphological analysis, and western blotting study. Finally, the HPLC and LC-MS analyses were done to identify probable bioactive compounds. Results: The in vitro antioxidant studies showed promising ROS, and RNS scavenging potential of DCME. Moreover, the in vitro antiproliferative study bared the cytotoxic nature of DCME towards MCF-7 (IC50 - 98.58 ± 6.82μg/mL) and non-toxic towards WI-38 (IC50 - 685.85 ± 19.51μg/mL). Furthermore, the flow-cytometric analysis revealed the increase in sub G1 population as well as early apoptotic populations dose-dependently. The results from confocal microscopy showed the DNA fragmentation in MCF-7 upon DCME treatment. Finally, the western blotting study revealed the induction of tumor suppressor protein, p53, which results in increasing the Bax/Bcl-2 ratio and activation of caspase-cascade pathways. Conclusion: The activation of caspase-3, -8, -9 and PARP degradation led us to conclude that DCME induces apoptosis in MCF-7 through both intrinsic and extrinsic mechanisms. The LC-MS analysis showed the presence of various bioactive compounds.

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