scholarly journals A new fluorescent detection system for identifying variant hemoglobins after gel electrophoresis using immunobinding with monoclonal antibodies.

1986 ◽  
Vol 34 (5) ◽  
pp. 585-591 ◽  
Author(s):  
R E Smith
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Gel Electrophoresis ◽  
Detection System ◽  
Great Sensitivity ◽  
Fluorescent Detection ◽  
Fluorogenic Substrate ◽  
Allelic Variants ◽  
Resolution System ◽  
Entire Procedure

This paper describes a low-resolution system for identifying variant hemoglobins with great sensitivity and specificity. After electrophoresis of the hemoglobin sample in a gel, fixation is used to entrap the hemoglobin. The gel is dried, incubated with a monoclonal antibody against the desired hemoglobin, then incubated with a second antibody against the first antibody which is conjugated with the enzyme beta-d-galactosidase. An enzyme overlay membrane containing a fluorogenic substrate is then placed on the gel surface, incubated, and removed, yielding an immunofluorescent print. The entire procedure takes only two hours, and by virtue of fluorescent detection gives sharper band resolution and greater sensitivity than conventional dye methods. The system clearly distinguishes SS sickle-cell hemoglobin from heterozygous and "S-like" hemoglobins. The technique therefore holds promise as a powerful probe for allelic variants.

scholarly journals Quantum dots nanoparticle-based lateral flow assay for rapid detection of Mycobacterium species using anti-FprA antibodies

2012 ◽  
Vol 2 (1) ◽  
pp. 5 ◽  
Author(s):  
Fabio Cimaglia ◽  
Alessandro Aliverti ◽  
Maurizio Chiesa ◽  
Palmiro Poltronieri ◽  
Enrico De Lorenzis ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Quantum Dots ◽  
Monoclonal Antibodies ◽  
Rapid Detection ◽  
Detection System ◽  
Lateral Flow ◽  
Fluorescence Signal ◽  
Human Pathogens ◽  
Detection Zone ◽  
Diagnostic Investigation

A lateral flow (LF) device combined with quantum dots (QDs) technology was developed for rapid detection of a specific mycobacterial flavoprotein reductase (<em>fprA</em>). In order to develop the LF assay based on a double-antibody sandwich format, two monoclonal antibodies recognizing different epitopes located in separated <em>fprA</em> domains were identified. The first monoclonal antibody was immobilized onto the detection zone of a porous nitrocellulose membrane, whereas another monoclonal antibody was conjugated to QDs nanoparticles as a detection system. Using these monoclonal antibodies we recorded a good fluorescence signal, the intensity of which was directly proportional to the concentration of <em>fprA</em> protein. The use of antibodies conjugated with fluorescent semiconductor QDs via biotin-streptavidin bridge, allowed the detection of <em>fprA</em> protein at concentrations as low as 12.5 pg/μL in less than 10 min. The reported technology could be useful in the diagnostic investigation of <em>Mycobacterium tuberculosis</em> and other human pathogens in clinical specimens.

Monoclonal antibody-based solid-phase immunoenzymometric assays for quantifying human immunoglobulin G and its subclasses in serum.

1985 ◽  
Vol 31 (12) ◽  
pp. 1940-1945 ◽  
Author(s):  
C Papadea ◽  
I J Check ◽  
C B Reimer
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Solid Phase ◽  
Detection System ◽  
Reference Intervals ◽  
Microtiter Plates ◽  
Myeloma Proteins ◽  
Human Proteins ◽  
Reference Preparation ◽  
Apparently Healthy

Abstract We developed quantitative immunoenzymometric assays for human IgG and its subclasses by using monoclonal antibodies, an avidin-biotin detection system and, as the calibrant, the U.S. National Reference Preparation for Specific Human Proteins. The assays are sensitive (detecting as little as 6 micrograms/L), precise (average inter-assay CV less than 11%), and vary linearly with concentrations over a five- to 10-fold range, depending on the monoclonal antibody. We evaluated 22 different monoclonal antibodies, many of which remained highly reactive when immobilized in wells of microtiter plates coated with bovine serum albumin-glutaraldehyde to "capture" total IgG or subclasses of IgG in the sample. We demonstrated the specificity of the most reactive antibodies by using a panel of 20 purified myeloma proteins. The sum of IgG subclass concentrations correlated well (r = 0.84, p less than 0.001) with the total IgG measured in sera from 63 apparently healthy adults (26 men, 37 women). We estimated 95 percentile reference intervals for the immunoglobulins in these subjects and determined the following mean percentage distributions of IgG subclasses: IgG1 49, IgG2 33, IgG3 9, and IgG4 7. The availability of these assays should facilitate studies of the clinical significance of the subclasses.

scholarly journals Anti-Lipid A Monoclonal Antibody Centoxin (HA-1A) Binds to a Wide Variety of Hydrophobic Ligands

1998 ◽  
Vol 66 (2) ◽  
pp. 870-873 ◽  
Author(s):  
E. J. Helmerhorst ◽  
J. J. Maaskant ◽  
B. J. Appelmelk
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Bovine Serum Albumin ◽  
Gel Electrophoresis ◽  
Lipid A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Properties ◽  
Antibody Epitopes

ABSTRACT This note describes the binding specificities of four lipid A monoclonal antibodies (MAbs) including Centoxin (HA-1A); these MAbs display similar binding properties. MAbs reacted with lipid A and heat-killed smooth bacteria, whereas no reactivity was observed with smooth lipopolysaccharide (LPS). Immunoblotting of bacterial extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the MAbs bound to many polypeptide bands including the molecular weight markers. Denaturation of bovine serum albumin (BSA) by boiling or dithiothreitol treatment unmasked antibody epitopes. In addition, binding both to a hydrophobic aliphatic C12 chain covalently coupled to BSA and to single-stranded DNA was observed. The polyreactivity of these clones is most likely mediated by a preferential reactivity with hydrophobic molecular patches.

Isolation And Characterization Of Platelet Membrane Glycoprotein IIB-IIIa With A Monoclonal Antibody

1981 ◽  
Author(s):  
R P McEver ◽  
N L Baenziger ◽  
P W Majerus
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Single Molecule ◽  
Gel Electrophoresis ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Membrane ◽  
Normal Platelet ◽  
Isolation And Characterization ◽  
And Function

We have developed hybridoma cell lines that produce monoclonal antibodies directed to different components on the human platelet membrane. One of these lines produces an antibody, named Tab, that binds to a protein on normal platelets that is deficient in platelets from patients with Glanzmann’s thrombasthenia. We isolated this protein from Triton X-100 solubilized normal platelet membranes by affinity chromatography on Tab-agarose. As determined by SDS polyacrylamide gel electrophoresis, the isolated protein consists of two polypeptides previously designated as glycoproteins IIb and IIIa. The isolation of two polypeptides by a single monoclonal antibody suggests that IIb and IIIa are subunits of a single molecule. We prepared 125I-Tab to determine the number of glycoprotein IIb-IIIa molecules on normal and thrombasthenic platelets by a direct binding assay. Platelets from 17 normal individuals bound 39,000 ± 4,600 Tab molecules/platelet. Platelets from four patients with thrombasthenia lacked Tab binding sites (<5%) . Nine heterozygotes for thrombasthenia bound 24,500 ± 5,800 Tab molecules/platelet. We separated the IIb and IIIa subunits by preparative gel electrophoresis and analyzed their structures. Both polypeptides contain ∼15% carbohydrate, but IIIa has a higher percentage of mannose residues. The amino acid compositions are similar, suggesting some sequence homology. However, tryptic peptide maps of 125I IIb and 131I IIIa, analyzed simultaneously by high performance liquid chromatography, are completely different. This indicates that neither subunit is derived from the other and suggests that IIb and IIIa may be products of separate genes. The Tab antibody should prove useful in further elucidation of the structure and function of glycoprotein IIb-IIIa. Other monoclonal antibodies may serve as valuable probes of additional platelet membrane proteins.

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.

Therapeutic Monoclonal Antibodies in Clinical Practice against Cancer

2020 ◽  
Vol 20 (16) ◽  
pp. 1895-1907
Author(s):  
Navgeet Kaur ◽  
Anju Goyal ◽  
Rakesh K. Sindhu
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Clinical Practice ◽  
Therapeutic Agent ◽  
Hematologic Malignancies ◽  
Immune Checkpoints ◽  
Malignant Cells ◽  
Main Target ◽  
Therapeutic Monoclonal Antibodies ◽  
Cancerous Cells

The importance of monoclonal antibodies in oncology has increased drastically following the discovery of Milstein and Kohler. Since the first approval of the monoclonal antibody, i.e. Rituximab in 1997 by the FDA, there was a decline in further applications but this number has significantly increased over the last three decades for various therapeutic applications due to the lesser side effects in comparison to the traditional chemotherapy methods. Presently, numerous monoclonal antibodies have been approved and many are in queue for approval as a strong therapeutic agent for treating hematologic malignancies and solid tumors. The main target checkpoints for the monoclonal antibodies against cancer cells include EGFR, VEGF, CD and tyrosine kinase which are overexpressed in malignant cells. Other immune checkpoints like CTLA-4, PD-1 and PD-1 receptors targeted by the recently developed antibodies increase the capability of the immune system in destroying the cancerous cells. Here, in this review, the mechanism of action, uses and target points of the approved mAbs against cancer have been summarized.

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
High Yield ◽  
Staphylococcal Protein A ◽  
Purification Step ◽  
Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

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.

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